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9,178,475
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The bovine parainfluenza virus type-3 (BPIV-3) hemagglutinin/neuraminidase glycoprotein expressed in baculovirus protects calves against experimental BPIV-3 challenge.
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Despite the availability of numerous vaccine schedules, "shipping fever", an acute bronchopneumonia brought on in part by a complex of bovine respiratory viruses, remains a major source of economic loss in the beef and dairy industries. We are exploring new strategies of bovine vaccine design which we hope may provide more effective and more cost-efficient control of these pathogens. In this report, we examined the possible use of subunit vaccines, using as an example the hemagglutinin/neuraminidase (HN) protein of bovine parainfluenza virus type-3 (BPIV-3) expressed in the baculovirus expression system. We showed that the protein was expressed at high levels, and was modified to a similar, but not identical size as the native HN protein expressed from BPIV-3 infected bovine cells. We further demonstrated antigenicity and biological activity of the expressed HN protein. Finally, we vaccinated colostrum deprived sera-negative calves with the baculo HN recombinant protein and challenged with BPIV-3. Vaccination induced excellent serum neutralizing antibody responses, and surprisingly, good mucosal antibody responses, even though the vaccine was administered parenterally. The vaccinated animals were well protected against challenge.
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9,178,473
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Induction of neutralizing antibody and T-cell responses to varicella-zoster virus (VZV) using Ty-virus-like particles carrying fragments of glycoprotein E (gE).
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During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.
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9,178,468
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Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) L1 gene protects against virus challenge .
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A DNA vaccine encoding the major capsid protein L1 of cottontail rabbit papillomavirus (CRPV) was constructed and administered intracutaneously (i.c.) to rabbits as supercoiled plasmids bound to gold beads using a specialized delivery device ("gene gun"). L1 DNA-vaccinated rabbits developed cellular proliferative responses to CRPV virus-like particles and developed high titered antibodies with neutralizing activity to CRPV. Following experimental challenge with CRPV, all of the L1 DNA-vaccinated rabbits, vs none of the controls, were protected from papilloma formation. These results demonstrate that i.c. vaccination of rabbits with the L1 papillomavirus capsid gene can induce antibodies that protect against subsequent papillomavirus infection.
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9,178,476
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Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi.
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The lipoprotein outer surface protein A (OspA) of the Lyme disease agent. Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette. Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.
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9,178,474
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Canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease.
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Inflammatory changes following infection are central to the clinical manifestation of disease. However, information regarding such changes in animal disease is limited. In canine parvovirus infected puppies we measured the levels of acute phase proteins and changes in leukocyte phenotypes and cell trafficking by flow cytometry. These parameters correlated with conventional assessment of clinical disease in a vaccine efficacy study. Seropositive (CPV-2) 6-week-old puppies given three doses of a CPV-2 containing vaccine developed significant antibody titers and remained healthy after experimental infection with CPV-2b. Unvaccinated controls developed clinical signs and shed virus. Importantly, acute phase proteins became elevated, and lymphopenia, neutropenia and modulation of neutrophil-CD4 were detected in controls but not in vaccinates.
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9,178,469
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Determination of protein loading in biodegradable polymer microspheres containing tetanus toxoid.
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Various methods to determine loading of vaccine in biodegradable polymer microspheres encapsulating tetanus toxoid were evaluated. The microspheres were composed of poly (D-lactic acid) (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA). Dissolution of microspheres in organic solvents such as methylene chloride, chloroform, or dimethyl sulfoxide and extraction of vaccine antigen or total protein with phosphate buffered saline gave variable results which depended upon the characteristics of the microspheres, such as type of polymer, excipients used in the microspheres and formulation conditions. Microspheres made from low molecular weight PLGA polymer and showing a large burst release exhibited up to 25% extraction of antigen whereas microspheres made from PLA microspheres with low burst release showed < 1% extraction. Extraction of total protein with 0.1 N NaOH and 5% sodium dodecyl sulfate showed results similar to those obtained with organic solvent extraction method. Partial digestion of microspheres with 6 N HCl at 60 degrees C for 20 h resulted in approximately 30% loss in TT protein by micro-bicinchoninic acid (BCA) assay. The major problem with this method was strong reactions in the micro-BCA assay of stabilizers, particularly sugars (glucose, sucrose) used in the microsphere formulations. Complete digestion of microspheres with 6 N HCl at 110 degrees C for 20 h or with 13.5 N NaOH at 121 degrees C for 1 h and quantitation of amino acids by a modified ninhydrin assay showed reproducible results on the protein loading in the microspheres. However, this method was affected by the presence of stabilizers, such as gelatin, which contain amino acids. Further, sucrose concentrations higher than 10% caused interference in the ninhydrin assay on samples hydrolyzed with 6 N HCl. In contrast, hydrolysis with 13.5 N NaOH did not show any interference by sucrose. Stabilizers used outside the microspheres for lyophilization purposes may be removed by washing the microspheres before loading determination or by dialysis but stabilizers used inside the microspheres would still cause interference. For reliable determination of total protein in the microspheres containing vaccines, we suggest complete digestion of microspheres with acid or base followed by amino acid analysis by colorimeteric assays such as ninhydrin method or using amino acid analyzers. The method needs to be optimized for each type of formulation to eliminate interference by the excipients. Alternatively, total protein nitrogen in the microspheres may be determined by the Kjel-dahl method if no amino acids or other nitrogen containing stabilizer is used inside the microspheres.
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9,178,471
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Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra.
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Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H37Ra using different column chromatography techniques. These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins. Out of six proteins, three proteins showed good reactivity. However, the activity was at a maximum with 30 kDa antigen. The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent. The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and gamma-interferon (IFN-gamma). The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M. tuberculosis H37Rv. The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD50 of M. tuberculosis H37Rv was significantly higher in comparison to BCG vaccinated animals. However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG. These findings suggest that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.
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9,178,478
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Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunization.
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The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.
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9,178,479
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Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance T-cell immunity and antitumor responses.
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Recombinant vaccinia viruses containing tumor associated genes represent an attractive vector to induce immune responses to weak immunogens in cancer immunotherapy protocols. The property of intense immunogenicity of vaccinia proteins, however, also serves to limit the number of inoculations of recombinant vaccinia viruses. Host immune responses to the first immunization have been shown to limit the replication of subsequent vaccinations and thus reduce effectiveness of boost inoculations. The use of recombinant avian pox viruses (avipox) such as the canarypox (ALVAC) or fowlpox are potential candidates for immunization protocols in that they can infect mammalian cells and express the inserted transgene, but do not replicate in mammalian cells. We report here the construction and characterization of a canarypox (ALVAC) recombinant expressing the human carcinoembryonic antigen (CEA) gene (designated ALVAC-CEA). Antibody, lymphoproliferative and cytolytic T-cell responses as well as tumor inhibition were shown to be elicited by the ALVAC-CEA recombinant in a murine model. The utilization of a diversified immunization scheme using a recombinant vaccinia virus followed by recombinant avian pox virus was shown to be far superior than the use of either one alone in eliciting CEA-specific T-cell responses. Experiments were conducted to determine if the use of a diversified immunization scheme using a recombinant vaccinia virus (rV-CEA) and ALVAC-CEA would be superior to the use of either one alone in eliciting CEA-specific T-cell responses. When mice were immunized with rV-CEA and then ALVAC-CEA. CEA-specific T-cell responses were at least four times greater, and for superior to those achieved with three immunizations of ALVAC-CEA. Multiple boosts of ALVAC-CEA following rV-CEA immunization further potentiated anti-tumor effects and CEA specific T-cell responses. These studies demonstrate the proof of concept of the advantage of diversified immunization protocols employing both recombinant vaccinia and recombinant avipox vectors.
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9,178,482
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Chronic and recovered cases of sheep-associated malignant catarrhal fever in cattle.
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Malignant catarrhal fever (MCF) is traditionally regarded as a disease with a short clinical course, low morbidity and high case fatality rate. Owing to the limitations of the assays used for laboratory diagnosis. It was difficult in characterise the clinical spectrum of sheep-associated MCF, particularly when the cattle recovered from an MCF-like clinical syndrome. Over a period of three years, 11 cattle that survived MCF for up to two-and-a-half years were identified on four premises. A clinical diagnosis of MCF was confirmed by the detection of ovine herpesvirus-2 DNA in peripheral blood leucocytes using a polymerase chain reaction (PCR) assay that detects a specific 238 base-pair fragment of viral genomic DNA. Of the 11 cattle examined, six recovered clinically with the exception of bilateral corneal oedema with stromal keratitis (four animals) and unilateral perforating keratitis (one animal). The 10 animals available for postmortem examination had disseminated subacute to chronic arteriopathy. Recovery was associated with the resolution of the acute lymphoid panarteritis that characterises the acute phase of MCF, and with the development of generalised chronic obliterative arteriosclerosis. Bilateral leucomata were due in part to the focal destruction of corneal endothelium secondary to acute endothelialitis. Formalin-fixed tissues and/or unfixed lymphoid cells from all 11 cattle were positive for sheep-associated MCF by PCR. These observations indicate that recovery and chronic disease are a significant part of the clinical spectrum of MCF and that such cases occur with some frequency in the area studied. The affected cattle remain persistently infected by the putative sheep-associated MCF gammaherpesvirus.
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9,178,480
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Integration of hepatitis B vaccination into the expanded programme on immunization in Chonburi and Chiangmai provinces, Thailand.
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Hepatitis B (HB) immunization was introduced as part of the expanded programme on immunization (EPI) in two provinces in Thailand and evaluated over a four year period. Three doses of HB vaccine were offered to 60,980 newborns at birth, 2 and 6 months of age. The overall coverage for complete HB immunization was 90.4%. Serosurveys of randomly selected children under the age of 5 years were undertaken before and at the end of the project. Levels of HBsAg positivity were reduced by 85%, and there was a corresponding 70% increase in protective immunity. These findings demonstrate that HB immunization can be successfully integrated into EPI without adverse effect on coverage rates of other antigens, and results in a marked reduction in the rate of chronic carriage of HB virus in preschool age children.
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9,178,483
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Comparison of standardbred trotters exercising on a treadmill and a race track with identical draught resistances.
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The responses in heart rate, plasma lactate and rectal temperature of standardbred trotters to draught loaded interval exercise on a treadmill and a race track were studied. The horses were exercised with incrementally increasing trotting speeds for two-minute intervals with draught loads of 10, 20 and 30 kilopond (kp) in three different tests. Each trotting interval was followed by two-minute periods at a walk without a draught load. Measurements of heart rate and plasma lactate were made at the end of each interval and the rectal temperature was taken at the end of the exercise. The heart rate and plasma lactate levels were significantly lower on the treadmill than on the track in the tests with 10 kp, but no significant differences were found between the treadmill and track exercise tests with the heavier draught resistances. No differences were observed in rectal temperature between treadmill and track conditions. From these findings it was concluded that the workload was significantly greater on the race track compared to the treadmill when the draught resistance was low (10 kp). Although the workload increased on both the race track and the treadmill as draught resistance increased, at the heavier draught resistances track exercise was no longer more demanding than exercise on the treadmill.
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9,178,477
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Immunity status against poliomyelitis in persons 13-14 years old living in Rome.
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The immunity against poliomyelitis in 1000 subjects 13-14 years old was evaluated. Neutralizing antibodies against poliovirus type 1, 2 and 3 were detected in 97.6%, 95.8% and 70% of samples, respectively. 3/1000 (0.3%) subjects were simultaneously seronegative to the three types. WHO does not suggest a protective level of International Units (IU), but our data indicate that such level is 0.45 IU for polio type 1, 0.65 IU for the type 2 and 0.138 for the type 3. A booster dose of vaccine in adolescence to ensure personal and herd immunity is recommended.
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9,178,484
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Surgical treatment of a septic dentigerous cyst in a goat.
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A slowly growing lesion of the rostral mandible of a goat was diagnosed to be a septic dentigerous cyst. The lesion was treated surgically to remove one displaced tooth and debride the cystic cavity, and systemic antibiotic therapy was applied. Thirty-four weeks later the goat was clinically and radiographically improved and the problem had not recurred.
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9,178,481
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Immunogenicity and safety of Haemophilus influenzae type b polysaccharide-Neisseria meningitidis conjugate vaccine in 7.5 micrograms liquid formulation: a comparison of three lots with the 15.0 micrograms lyophilized formulation. Study Group for 7.5 micrograms Liquid PedvaxHIB.
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We conducted a multicenter, single-blind, randomized comparisons of the immunogenicity and safety of three manufacturing-scale lots of 7.5 micrograms liquid Haemophilus influenzae type b polysaccharide- Neisseria meningitidis conjugate vaccine (PRP-OMPC) and a single lot of 15.0 micrograms lyophilized PRP OMPC. A total of 908 infants were entered into the study. Each infant received two primary injections intramuscularly 2 months apart beginning at age 2-6 months and a booster injection at 12-15 months. Blood samples for serology were obtained before each injection and 1 month after the second and the booster dose. Immune responses were measured by radioimmunoassay. Approximately 80% of the infants achieved a titer > 1.0 micrograms ml-1 after the second primary dose of all four lots tested: the geometric mean titer (GMT) was ca 3 micrograms ml-1 for each vaccine group. After the booster dose, more than 90% of infants from each vaccine group had a titer > 1.0 microgram ml-1;GMTs ranged from 8 to 10 micrograms ml-1. No serious vaccine-associated adverse reactions were reported. Thus the 7.5 liquid PRP OMPC vaccine was at least as immunogenic and well tolerated as the 15.0 micrograms lyophilized vaccine.
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9,178,491
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The TATA-less promoter of hepatitis B virus S gene contains a TBP binding site and an active initiator.
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The surface antigen (S) gene promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of a 2.1 kb mRNA which encodes the preS2 and S polypeptides. The preS2/S promoter does not contain a classical TATA box, and transcription regulation of the preS2/S gene has not been fully elucidated. We analysed two regions involved in preS2/S gene transcription of the HBV adw subtype: the diverged TATA box and a putative initiator element. We demonstrated sequence specific promoter activity of the putative TATA-like sequences in the preS2/S gene promoter (-25 to -32 bp). Using end labeled synthetic oligonucleotides we observed specific binding of nuclear extracts to the diverged TATA sequence, that was significantly reduced using a mutated oligonucleotide. Specific binding of yeast TBP to the diverged TATA sequence was shown which was increased in the mutant containing a classical TATA box. We analysed the proposed initiator (Inr) sequence of the preS2/S promoter region (-13 to -16 bp). Deletion of the inr element markedly reduced promoter activity as assessed by CAT expression. Gel shift assays showed specific binding of nuclear extracts to wild type but not to mutant Inr. Expression studies with double mutants of the diverged TATA and the Inr element established that both elements are active in transcription regulation.
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9,178,492
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Phosphorylation of tick-borne encephalitis virus NS5 protein.
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The largest tick-borne encephalitis virus (TBEV) non-structural protein NS5 (100 kDa) is believed to be involved in RNA replication. The protein is phosphorylated in infected cell extracts in the presence of [gamma-32P]ATP, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using monoclonal antibodies raised against TBEV NS5 protein. Radioactive labeling of NS5 in cellular extracts at an early stage post-infection is higher than at 24 h post-infection. Incubation of immunoprecipitates of NS5 protein with [gamma-32P]ATP in the presence of Mg2+ resulted in the phosphorylation of TBEV NS5 protein and of immunoglobulins. Phosphoamino acid analysis demonstrated that NS5 contains phosphoserine, but not phosphothreonine, or phosphotyrosine.
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9,178,493
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Towards defining a minimal functional domain for NTPase and RNA helicase activities of the hepatitis C virus NS3 protein.
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Hepatitis C virus (HCV) possesses two separate enzymatic functions in the NS3 protein: a protease and an NTPase/RNA helicase. In order to determine the minimal domain for NTPase and RNA helicase activities of the HCV NS3 protein, serial deletion mutants were constructed. The NS3H protein a fusion protein of 25 amino acids (aa) from an expression vector and the C-terminal 466 aa of the HCV NS3 protein, contains an NTPase/RNA helicase activity. We made deletion mutants of 10, 30, 50, 97, and 135 aa from the C-terminus and 16 and 32 aa from the N-terminus of the NS3H protein. The deleted protein lacking 50 aa from the C-terminus still possessed both activities, while the protein lacking 97 aa from the C-terminus lost an RNA helicase activity. The mutant lacking 16 amino acids from the N-terminus retained the enzymatic activities and the N-terminal 32 aa deleted mutant lost an NTPase/RNA helicase activity. A combinational deletion mutant lacking 16 aa the N-terminus and 50 aa from the C-terminus retained the enzymatic activities. These results show that the functional domain of the HCV NTPase/ RNA helicase is about 400 aa in length and maps between NS3 residues 1209 and 1608.
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9,178,494
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Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.
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The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.
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9,178,495
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Production of active polyomavirus large T antigen in yeast Pichia pastoris.
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The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immunoaffinity chromatography. We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.
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9,178,496
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Capsid protein composition of reference strains and wild isolates of human astroviruses.
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Astroviruses are small RNA viruses that are frequently associated with gastroenteritis in humans and animals. Despite much work on the genetic analysis of astrovirus strains, little progress has been made in the characterization of the proteins composing mature virions. We have analyzed the capsid protein composition of the reference strains and several wild isolates of human astroviruses using high-resolution polyacrylamide gradient gels. For reference strains of the seven serotypes analyzed, a consistent pattern of three infection-specific proteins--designated P1, P2, and P3 -was generally observed. The strains could be divided into two groups, based upon the reactivity of these proteins in immune precipitation assays that used homologous rabbit serum. One group included reference types 1 4 for which all three proteins were precipitated by homologous rabbit sera; for the other group, types 5 7, only proteins P2 and P3 were precipitated. When wild isolates from around the world were compared to the reference strains, a correlation between genetic type and the pattern of protein sizes and immune reactivity was observed for strains of the common types (1-4). Strains of types 2 and 4 consistently exhibited P3 proteins larger than those of types 1 and 3. Unusual patterns of proteins or immune reactivity were detected in strains of types 5-7, indicating that there may be incomplete processing of the capsid precursor during growth in cell culture.
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9,178,497
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Multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species.
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Primary cell cultures prepared from embryos of the thrips species Frankliniella occidentalis and Thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (TSWV). Using polyclonal antibodies against the viral nucleocapsid protein (N) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a F. occidentalis, and a T. tabaci population which failed to transmit the virus. The infection was found in approximately 40% of the monolayer cells. Using antibodies against a nonstructural protein (NSs) of TSWV, uniform and more diffused staining was observed throughout the cytoplasm of these cells, underlying active genome replication. The NSs protein accumulated slower than the N protein in the cells of both thrips species. No multiplication of TSWV was observed in a heterologous insect cell line, i.e. from Spodoptera frugiperda, suggesting the existence of specific host factors in the thrips-derived cells.
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9,178,498
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Genetically variable triplet repeats in a RING-finger ORF of Helicoverpa species baculoviruses.
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Nucleotide sequence analysis of the Helicoverpa zea S-type nucleopolyhedrovirus (HzSNPV) genomic interval between the polh and iel genes has revealed an open reading frame (HOAR ORF) that contains a complex A 1-T rich triplet repeat region (RAT-repeats). HOAR ORF is predicted to encode an acidic, arginine residue rich. 712 aa protein, with a C3HC4 (RING-finger) zinc binding motif. RAT-repeats, distributed over 450 bp. consist of GAT. AAT, and GTT codons, correspond to Asp, Asn and Val residues which display an extreme codon bias not seen with nine other genes of this virus. A survey of four other (field) isolates of Helicoverpa sp. NPVs confirms a high incidence of mutation in the RAT-repeat region. A 158-bp conserved block, homologous to the pe38-ien promoter of AcMNPV, was identified upstream of HOAR ORF. The sub-region of the genome in which HOAR ORF is located is susceptible to rearrangement.
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9,178,499
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Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis. Its potential use for detection of uncharacterized arenaviruses.
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Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.
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9,178,500
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Evidence for the assignment of two strains of SPLV to the genus Potyvirus based on coat protein and 3' non-coding region sequence data.
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The use of potyvirus-specific primers and subsequent application of the RACE procedure allowed the cloning of the 3' terminal 1088 nucleotides of the genomic RNA of the Taiwan isolate of sweetpotato latent virus (SPLV-T) and the 3' genomic 1085 nucleotides of a SPLV-like virus from China (SPLV-CH). The sequence of an internal part of the presumptive nuclear inclusion b gene was also determined for both isolates. Detailed sequence analyses revealed the presence of consensus motifs which indicated that SPLV-CH and SPLV-T should be regarded as members of the genus Potyvirus. Multiple sequence alignments and phylogenetic analyses were also performed and unambiguously assessed these isolates as strains of a distinct Potyvirus. SPLV was not related to other potyviruses infecting sweetpotato nor to any other sequenced virus. From the presence of the DAG box, SPLV-CH is expected to be a typical aphid transmitted Potyvirus whereas a conceivable explanation is proposed for the non-aphid transmission of SPLV-T.
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9,178,501
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Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection.
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The Pestivirus bovine viral diarrhea virus (BVDV) causes the fatal diarrheal syndrome, mucosal disease, because of mutations in the viral genome which convert the common noncytopathic (ncp) BVDV into a cytopathic (cp) biotype. We examined the nature of the cytopathic effect of cp-BVDV in cultured bovine cells in order to accurately describe the process and to gain insight into the mechanism of cp-BVDV-induced cell death. The findings demonstrate that cells infected with cp-BVDV in vitro die by apoptosis, but cells infected with ncp-BVDV do not. Analysis of nuclear morphology by staining with fluorescent DNA dye and cpi-fluorescence microscopy showed chromatin condensation and margination in cells infected with cp-BVDV. Transmission electron microscopy (TEM) confirmed the condensation of chromatin, as well as cell shrinkage and generation of apoptotic bodies. The chromosomal DNA of cells infected with cp-BVDV undergoes fragmentation, generating the typical oligonucleosomal fragments commonly noted during apoptosis. The fragmented DNA was released from the nucleus to the cytoplasm, and eventually to the culture supernatant. Infection with cp-BVDV activates cellular proteases of the ICE family leading to cleavage of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in genome maintenance. This demonstration that cp-BVDV kills cells by triggering apoptosis suggests the possibility that cp-BVDV is associated with a fatal disease by the acquisition of a new apoptosis-inducing activity. We consider BVDV to be an excellent model system for studies of the biological and medical relevance of apoptosis in viral infections.
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9,178,502
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Yarrowia lipolytica SRP54 homolog and translocation of Kar2p.
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To investigate the role of Srp54p in protein translocation, the Yarrowia lipolytica SRP54 homolog was cloned. Sequencing revealed an open reading frame of 536 amino acids coding for a 57.2 kilodalton polypeptide with 55 to 57% sequence identity to Srp54ps of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse. Like these Srp54ps, Y. lipolytica Srp54p has an N-terminal domain with a highly conserved GTP-binding site and a methionine-rich C-terminal domain. Differing results regarding the essentiality of SRP subunits were obtained. SRP54 is important but not essential for growth, but it was reconfirmed that at least one SRP RNA gene is essential. Cells with SRP54 deleted grow about six times more slowly than wild type; faster-growing colonies, still growing much slower than wild type, appeared quite frequently. In srp54 delta cells, no untranslocated alkaline extracellular protease precursor was detected. Therefore, to develop another reporter molecule the Y. lipolytica KAR2 homolog was cloned and Kar2p antibodies were produced. For Kar2p an untranslocated precursor was detected in srp54 delta but not in wild-type cells, suggesting that its translocation was defective in the srp54 delta cells. These results confirm an in vivo rule for SRP in protein translocation in Y. lipolytica, suggest that SRP RNA or an SRP core-particle has functions not shared by Srp54p, and show that, as in S. cerevisiae and Sz. pombe, reporter molecules differ in their dependency on SRP for translocation.
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9,178,503
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Mechanisms of salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae.
|
Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K+ and decreasing intracellular Na+. The effect of HAL1 on intracellular Na+ was mediated by the PMR2/ENA1 gene, corresponding to a major Na+ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K+ was independent of the TRK1 and TOK1 genes, corresponding to a major K+ uptake system and to a K+ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K+ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K+ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stressed.
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9,178,504
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The osmotic hypersensitivity of the yeast Saccharomyces cerevisiae is strain and growth media dependent: quantitative aspects of the phenomenon.
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Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth. Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 M-NaCl; most strains fell within a 300 mM range in LD10 values (lethal dose yielding 10% survival). Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e. ten-fold decreased viability for every 250 mM increase in salinity. Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations. This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses. The laboratory strain X2180-1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae.
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9,178,505
|
Role of the cytoskeleton in endocytosis of the yeast maltose transporter.
|
Certain components of the cytoskeleton play a role in yeast fluid-phase endocytosis as well as in endocytosis of the alpha-factor when this pheromone is bound to its 7-transmembrane segment receptor. The yeast maltose transporter is a 12-transmembrane segment protein that, under certain physiological conditions, is degraded in the vacuole after internalization by endocytosis. In this work, the possible role of the cytoskeleton in endocytosis of this transporter has been investigated. Using mutants defective in beta-tubulin, actin and the actin-binding proteins Sac6 and Abp85. as well as nocodazole, which inhibits formation of microtubules, we have shown that actin microfilaments are involved in endocytosis of the maltose transporter whereas microtubules are not.
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9,178,506
|
Expression cassettes for formaldehyde and fluoroacetate resistance, two dominant markers in Saccharomyces cerevisiae.
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We employed two genes in constructing yeast expression cassettes for dominant, selectable markers. The Saccharomyces cerevisiae gene SFA1, encoding formaldehyde dehydrogenase, was placed under the control of the GPD1 promoter and CYC1 terminator. The Moraxella sp. strain B gene dehH1, encoding fluoroacetate dehalogenase, was placed under the control of both the GPD1 and CYC1 promoters. With these constructs it was possible to select directly for yeast strains resistant to either formaldehyde or fluoroacetate. Both selective agents are completely metabolized and inexpensive, making them very useful in the pursuit of yeast gene functions and for industrial applications. An additional advantage of the formaldehyde dehydrogenase marker is that it is an S. cerevisiae gene, thus allowing 'all yeast' constructs.
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9,178,507
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Characterization of the Saccharomyces cerevisiae cdc42-1ts allele and new temperature-conditional-lethal cdc42 alleles.
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Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts allele and identified multiple mutations in the coding region and 5' promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42T58A and cdc42S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe: these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts.
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9,178,509
|
Sequence analysis of a 33.2 kb segment from the left arm of yeast chromosome XV reveals eight known genes and ten new open reading frames including homologues of ABC transporters, inositol phosphatases and human expressed sequence tags.
|
The complete nucleotide sequence of a 33221 bp segment, contained in cosmid pEOA1044, derived from the left arm of chromosome XV of Saccharomyces cerevisiae, appears in public databases between coordinates 177013 and 210234 (http://speedy.mips.biochem.mpg.de/). Computer analysis of that sequence revealed the presence of the previously known genes IRA2, DEC1, NUF2, HST1, RTG1, RIB2 and HAL2, one previously partially sequenced open reading frame (ORF) of unknown function (SCORFAC) and ten newly identified ORFs. One of the new ORFs is similar to the Drosophila melanogaster white gene and other transmembrane ABC transporters, another one has similarities to inositol phosphatases and others are similar to ORFs of unknown function from various organisms, including human Expressed Sequence Tags (ESTs). Potential transmembrane regions, ATP/GTP-binding and WD motifs have also been identified. The existence of yeast ESTs for two of the newly identified ORFs indicates that they are transcribed.
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9,178,508
|
Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae.
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The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the 'positive inside rule' approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane alpha-helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.
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9,178,512
|
A new type of hydrophilic carbon paste electrodes for biosensor manufacturing: binder paste electrodes.
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The effect of two types of carbon pastes and two osmium-based redox mediators on the response of amperometric enzyme electrodes for glucose was examined. A hydrophobic mediator and a hydrophilic cationic mediator were prepared and mixed in a paste that contained either mineral oil as the pasting liquid, or a polycationic electrolyte without oil. It was found that the current densities were increased by a factor of 25 when the oil-based paste was replaced by the hydrophilic one (binder paste, BP) and five- to six-fold when the hydrophilic mediator was used in place of the hydrophobic. The linear range for the glucose oxidase electrodes was extended to concentrations higher than 60 mM. The glucose electrodes were preliminary optimized and their half-life time reached more than 12 h when operated continuously under vigorous stirring when the 'pasting' polyelectrolyte was crosslinked. At a working potential of 400 mV versus the Ag/AgCl saturated electrode, the saturating current densities per geometric surface area were 1.2 mA/cm2 +/- 0.2 (n = 7). These 'binder paste electrodes' are the first reported bulk modified electrodes without hydrophobic pasting liquid or cover membranes, and present an interesting research and application tool.
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9,178,513
|
Electrochemiluminescence flow injection immunoassay for atrazine.
|
Antibodies to atrazine were labelled with glucose oxidase and used in colorimetric enzyme linked immunosorbent assays. Transparent aminosilanized indium tin oxide coated glass electrodes were derivatized with aminodextran covalently modified with atrazine caproic acid. The labelled antibodies were used to investigate the derivatized electrodes colorimetrically and the electrodes were use in an electrochemiluminescence flow injection analyser. Electrochemiluminescence immunoassay for atrazine in the range 0-10 ppb showed that it was possible to detect less than 0.1 ppb, the precautionary limit for pesticides in drinking water recommended by the European Commission.
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9,178,511
|
Diel activity of Ixodes ricinus Acari:ixodidae at two locations near Stockholm, Sweden.
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The diel 'activity', i.e. availability, of Ixodes ricinus larvae, nymphs and adults was investigated in a meadow and a forest habitat near Stockholm during 1991-1993. Generally, the immature ticks were more prevalent in the forest than in the meadow. In the meadow, the mean larval and adult numbers varied significantly between 4 h time intervals with the peak activity from 2300 to 0300 h. In the forest, the tick numbers did not differ significantly between the time intervals. The association of the tick activity with certain meteorological variables was strongest in the meadow, where the mean numbers of all tick stages were negatively correlated with the temperature. The relative humidity was positively correlated only with the mean numbers of larvae. In contrast, the larval activity in the forest was positively and negatively correlated with the temperature and relative humidity, respectively, while the nymphal and adult activity showed no association with these climatic variables. The impact of the host activity on the tick diel activity is also discussed.
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9,178,514
|
A cell-based immunobiosensor with engineered molecular recognition--Part I: Design feasibility.
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A novel bioelectronic sensor is described in which living immune cells are transformed into unique biotransducer couples by engineering their molecular recognition for preselected antigens of clinical interest. This 'hybrid' biosensor, constructed with mast cells interfaced to a microfabricated thermoelectric device with the use of biomolecular linkages, is capable of detecting antigens in real time by transducing minute heat changes arising from antigen-induced mast cell activation processes. The thermoelectric approach was selected based upon preliminary bioenergetic calculations which indicated that metabolic changes arising from mast cell antigen recognition result in a significant increase in exothermic heat relative to basal metabolic conditions. Experimental studies confirmed that mast cell activation and degranulation can be discriminated theramally from basal metabolic activity. Results obtained from microcalorimetry experiments using cultured mast cells (MC/9) mucosal-like mast cell line), and harvested mast cells (rat peritoneal mast cells) indicated that detectable increases in heat output (-3 +/- 0.5 pW/cell, mean peak output) immediately followed cell activation. The construction of a miniature hybrid immunobiosensor device was made possible by bioelectronic coupling achieved with the use of cellular adhesive proteins that immobilized non-adherent (MC/9) cells as well as adherent (RBL-2H3 rat basophilic leukemia) cells to the thermopile. Results from preliminary tests conducted on a hybrid biosensor prototype validated the design feasibility of a miniature, living cell immunodiagnostic biosensor. Such cell-based hybrid biosensor approaches may greatly extend the capability for selective, rapid, on-site, antigen detection for a wide range of clinically relevant antigens and offer new approaches to in vitro diagnostics.
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9,178,515
|
Potential of microsensor-based feedback bioactuators for biophysical cancer treatment.
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Solid tumors usually exhibit a poorly organized vascularization and a deviant energy metabolism which result in an acidic pH and large hypoxic areas in the tumor microenvironment. A lot of cell biological data support the hypothesis that such physico-chemical conditions are promoters of the microevolution of malignant cells, inhibitors of the immune response, and co-factors for tumor cell invasion. Our experimental in vitro analyses and computer simulations indicate that the efficiency of immunotherapies and classical methods for cancer treatment might be improved if a physico-chemical microenvironment could be restored which reflects that found in normal tissue. In order to monitor and manipulate the tumor microenvironment, we suggest utilizing silicon-based feedback bioactuators which are controlled by on-line microsensors. These miniaturized bioactuators play the role of 'pH clamps' and can be implanted directly at the sites of inoperable tumors and metastases where they can reconstitute normal physico-chemical conditions. The drug application scheme can be precisely controlled by an integrated microprocessor. The paper summarizes the current state of development of such microsensor-based feedback bioactuators and outlines their potential for biophysical cancer treatment.
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9,178,516
|
Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling.
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A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.
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9,178,517
|
Voltammetric enzyme sensor for urea using mercaptohydroquinone-modified gold electrode as the base transducer.
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A voltammetric urea-sensing electrode was prepared by combining a lipid-attached urease layer with a 2,5-dihydroxythiophenol-modified gold electrode. A self-assembled monolayer of dihydroxythiophenol was prepared on the gold surface by soaking the electrode into an ethanolic solution containing the modifier. A layer of the lipid-attached enzyme and that of acetyl cellulose overcoat were successively made on the dihydroxythiophenol-modified electrode by applying a dip-coating procedure. The addition of urea in a test solution (10 mM phosphate buffer, pH 7.0) brought about an increase of pH near the urease layer. The pH shift accompanied a negative shift of the anodic peak, which corresponded to the electro-oxidation of dihydroxyphenol moiety to form quinone, on the linear sweep voltammograms for the urease/dihydroxythiophenol electrode. The concentration of urea (0.2-5 mM) could be determined by measuring the electrode current at -0.05 V versus Ag/AgCl from the voltammogram. The electrode was applied to the determination of urea in human urine; the measurement of electrode current at such a low potential provided the urea determination without any electrochemical interference from L-ascorbic acid and uric acid.
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9,178,518
|
Effectiveness of protein A for antibody immobilization for a fiber optic biosensor.
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The fiber optic biosensor performs fluoroimmunoassays at the surface of multimode optical fibers. The effectiveness of protein A, an immunoglobulin binding protein, for antibody immobilization on the surface of these fiber probes has been investigated. No difference was observed in the binding of fluorescently-labeled goat-IgG by rabbit anti-goat IgG regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal as probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes since antibody-antigen complexes have been successfully eluted from protein A under low pH conditions. Protein A probes coated with rabbit anti-goat IgG obtained nearly identical signal levels at 500 and 5000 ng/ml of Cy5.5 goat IgG five consecutive times following regeneration with glycine-HCl, 2% acetic acid, pH 2.5.
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9,178,519
|
Indonesian medicinal plants. XIX. 1) Chemical structures of four additional resin-glycosides, mammosides A, B, H1, and H2, from the tuber of Merremia mammosa (Convolvulaceae).
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Four new resin-glycosides, named mammosides A (10), B (11), H1 (12), and H2 (13), were isolated from the tuber of Merremia mammosa (Convolvulaceae), a Jamu raw material. Their chemical structures have been elucidated on the bases of chemical and physicochemical evidence, including synthesis of a glycosidic acid designated as mammoside I.
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9,178,520
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Simple approach to optically active drimane sesquiterpenes based on enzymatic resolution.
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When the beta-acyloxy esters (+/-)-10 and (+/-)-11 were exposed to the lipase OF-360 from Candida rugosa or immobilized lipase OF-360 in a water-saturated organic solvent, the hydrolyzed product (8aS)-6 was obtained in high chemical (40%) and optical (> 99% ee) yields. The absolute structure of (8aS)-6 was confirmed by the fact that (8aS)-6 was converted into an authentic sample gamma-keto nitrile (8aS)-17. Treatment of the diol (+/-)-12 with isopropenyl acetate in the presence of the lipase Godo E-4 from Pseudomonas sp. provided the unchanged (8aR)-12 (89% ee) in 42% yield.
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9,178,522
|
Synthesis and dual antagonistic activity against thromboxane A2 and leukotriene D4 of [4-[1-(benzenesulfonamido)alkyl]phenyl]alkanoic acid derivatives.
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In order to find new antiasthmatic agents with dual antagonistic activity against thromboxane A2 (TXA2) and leukotriene D4 (LTD4) receptors, synthesis and pharmacological evaluation of various [4-[1-(benzenesulfonamido)-alkyl]phenyl]alkanoic acid derivatives were undertaken. TXA2 and LTD4 antagonistic activities in vitro were evaluated by measuring the inhibitory effects on L-46619-induced contraction of guinea-pig trachea and LTD4-induced contraction of guinea-pig ileum and trachea. Several compounds showed satisfactory dual antagonistic activities, and their effect (after oral administration) on LTD4-induced bronchoconstriction in guinea-pig in vivo was examined. The results demonstrated that both 4-[4-[1-(4-chlorobenzenesulfonamido)hexyl]phenyl]butyric acid (12e) and 4-[4-[1-(4-chlorobenzenesulfonamido)-5-methylhexyl]phenyl]bu tyric acid (12m) possessed good anti-LTD4 activities. Compounds 12e and 12m were then evaluated for other related pharmacological effects involving the arachidonic acid cascade. These compounds appear to be hybrid eicosanoids antagonists having antagonistic activity against contraction of guinea-pig trachea induced by prostaglandin D2 (PGD2) and PGF2 sigma, as well as TXA2 and LTD4 antagonistic activities.
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9,178,521
|
Synthesis, affinity at 5-HT2A, 5-HT2B and 5-HT2C serotonin receptors and structure-activity relationships of a series of cyproheptadine analogues.
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Cyproheptadine is a drug that shows high affinity for type 2 (5-HT2) receptors. We studied a series of compounds obtained by modification of the tricyclic system of Cyp (dibenzocycloheptadiene): 2f (thioxanthene), 2g (xanthene), 2h (dihydrodibenzocycloheptadiene), 2j (diphenyl), 2i (fluorene), and 3b (phenylmethyl). Their activities at the rat cerebral cortex 5-HT2A receptor were (pKi +/- S.E.M.): 8.80 +/- 0.11 (Cyp), 8.60 +/- 0.07 (2f), 8.40 +/- 0.02 (2g), 8.05 +/- 0.03 (2h), 7.87 +/- 0.12 (2j), 6.70 +/- 0.02 (2i) and 6.45 +/- 0.02 (3b); those at the rat stomach fundus 5-HT2B receptor (pA2 +/- S.E.M.) were: 9.14 +/- 0.25 (Cyp), 8.49 +/- 0.07 (2f), 7.58 +/- 0.58 (2g), 7.02 +/- 0.14 (2h), 6.07 +/- 0.20 (2j), and undetectable (2i, 3b): and those at the pig choroidal plexus 5-HT2C receptor (pKi +/- S.E.M.) were: 8.71 +/- 0.08 (Cyp), 8.68 +/- 0.01 (2f), 8.58 +/- 0.20 (2g), 7.95 +/- 0.05 (2h), 7.57 +/- 0.04 (2j), 6.98 +/- 0.04 (2i) and 6.63 +/- 0.20 (3b). The slopes did not differ significantly from unity. The compounds exhibited the same order of activities at every type of receptor, and the most active molecules presented certain steric (butterfly conformation of the tricyclic system) and electrostatic (proton affinity on the top of the central rings) patterns. It is concluded that the activity of cyproheptadine derivatives at 5-HT2 receptors is related to these molecular features, which make feasible a common disposition to interact with all three 5-HT2 subtypes.
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9,178,523
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Medicinal foodstuffs. VI. 1) Histamine release inhibitors from kidney bean, the seeds of Phaseolus vulgaris L.: chemical structures of sandosaponins A and B.
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Two new olean-12-ene-type triterpene oligoglycosides, named sandosaponins A and B, were isolated from kidney bean, the seed of Phaseolus vulgaris L., together with three known saponins, soyasaponins I and V and dehydrosoyasaponin 1. The structures of sandosaponins A and B were determined on the basis of chemical and physicochemical evidence, which included the chemical derivation of sandosapogenol from a known sapogenol, soyasapogenol B. Five saponins obtained from kidney bean were found to inhibit histamine release from rat exudate cells induced by an antigen-antibody reaction and, among them, sandosaponins A and B showed the most potent inhibitory activity.
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9,178,524
|
Biotransformation of (-)-epicatechin 3-O-gallate by human intestinal bacteria.
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The biotransformation of (-)-epicatechin 3-O-gallate (1) and related compounds was undertaken using a human fecal suspension. Of fifteen metabolites isolated, four compounds were new, namely, two epimers of 1-(3'-hydroxyphenyl)-3-(2",4".6"-trihydroxyphenyl)propan-2-ols (6, 19); 2",3"-dihydroxyphenoxyl 3-(3',4'-dihydroxyphenyl)propionate (14) and 1-(3',4'-dihydroxyphenyl)-3-(2",4",6"-trihydroxyphenyl)propan-2-ol (18). (-)-Epicatechin (2), (-)-epigallocatechin (16) and their 3-O-gallates (1, 17) were extensively metabolized by a human fecal suspension after incubation for 24 h, whereas the gallates (1, 17) resisted any degradation by a rat fecal suspension, even after a prolonged incubation time (48 h), suggesting a difference in metabolic ability between two intestinal bacterial mixtures from different species.
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9,178,526
|
Synthesis of spin labels for ESR imaging of living rat head.
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Spin labels (7, 10, 13, 16, 22, 27) were synthesized from piperidinyloxyl (1), pyrrolidinyloxyl (2), and oxazolidinyloxyl (3). These compounds were injected into the carotid artery of anesthetized rats, and the ESR spectra of the rat brain were immediately recorded by the use of an L-band ESR spectrometer. Based on the spectra obtained, we considered whether or not these spin labels can pass the blood brain barrier and bind to brain tissue components.
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9,178,525
|
The development of a cascade impactor simulator based on adhesion force measurements to aid the development of dry powder inhalations.
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Adhesion and friction forces are the main physical factors determining the re-suspension of a micronized drug from carrier particles during inhalation. Hence, it appears useful to link adhesion and friction force measurements to the in vitro testing of dry powder inhalations, namely the assessment of the mass median aerodynamic diameter (MMAD) using an eight-stage Andersen cascade impactor. Interactive mixtures of micronized Salmeterol Xinafoate adhered to irrespirable lactose monohydrate carrier particles were used as model dosage forms. The adhesion force between the drug and carrier particles was assessed using a centrifuge technique, and the MMAD was determined under standardized working conditions using the Andersen-Cascade impactor (Mark II). A cascade impactor simulator (CIS), which is a computer program containing a re-suspension model to assess the amount of drug detached from the carrier particles during inhalation, was developed and validated using the experimental data. It could be shown, that the CIS provided a good estimate of the loss of drug due to adhesion to the carrier particles and the loss of drug on the cascade impactor walls. Small deviations between the theoretical and experimental mass median aerodynamic particle diameters however were found. These deviations were shown to be mainly due to the experimental error introduced by the cascade impactor, and that the error due to the experimental adhesion measurements is negligibly small. Hence, the CIS developed could be a useful tool in early development stages of dry powder inhalations to predict the in vitro aerodynamic performance of drug particles.
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9,178,527
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Inclusion of trithia[5]heterohelicene by various serum albumins. An effective probe for chiral discrimination.
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Racemic 2-hydroxymethyltrithia[5]heterohelicene (2-HT), which has a labile helical structure was incorporated into serum albumins of nine species in 1% ethanolic aqueous solution, giving 1:1 complexes which exhibited induced circular dichroism spectra with species specificity. The application of 2-HT to bovine serum as a probe for chiral recognition revealed that the serum itself manifested an apparent chiral discrimination between enantiomers of 2-HT.
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9,178,528
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Synthesis and biological evaluation of 7-hydroxy-3,4-diphenyl-1,2-dihydroisoquinolines as new 4-hydroxytamoxifen analogues.
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A phenolic 3,4-diphenyl-1,2-dihydroisoquinoline derivative (4a) as a new 4-hydroxytamoxifen analogue and a related compound (4c) were synthesized from 3,4-diphenyl-1,2,3,4-tetrahydroisoquinolin-4-ols (5a, c), which were prepared by intramolecular Barbier reaction of N-(2-iodobenzyl)phenacylamines. Anti-proliferative activities of 4a,c and 5a,c, as well as 4b and 5b prepared previously, against human mammary carcinoma MCF-7 cell line and human nasopharyngeal carcinoma KB cell line were evaluated. The 3,4-diphenyl-1,2-dihydroisoquinoline derivatives (4a,c) and isoquinolin-4-ols (5a,b) were active against MCF-7 cells and were nearly equipotent to the corresponding nonphenolic compound (1a). The mechanism of the anti-proliferative activity of 4a-c against MCF-7 cells is discussed.
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9,178,529
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Garsubellin A, a novel polyprenylated phloroglucin derivative, increasing choline acetyltransferase (ChAT) activity in postnatal rat septal neuron cultures.
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Garsubellin A (1), a novel polyprenylated phloroglucin derivative, has been isolated from the wood of Garcinia subelliptica and its structure has been elucidated by spectroscopic analyses. Compound 1 could increase the ChAT activity at 10 microM in P10 rat septal neuron cultures.
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9,178,530
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Ultrastructural localization of acid phosphatase in some bacteria, after treatment with Lubrol W1.
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The ultracytochemical localization of acid phosphatase from some bacteria (Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa) was dependent on the changes in the lipoprotein content of the membranes as a result of the action of the Lubrol W1.
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9,178,531
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Drug resistance in Detroit River gram-negative bacilli.
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Detroit River Gram-negative bacilli were examined for resistance to agents of interest to public health. The total recoverable population and the lactose-fermenting organisms existed at approximately 10(5) and 10(2) colony forming units per litre, respectively. Lactose-nonfermenting and lactose-fermenting isolates demonstrated resistance to six and four of nine antimicrobial agents, respectively, when tested by a paper disc procedure. Multiple resistance in lactose-nonfermenting organisms included up to five agents. Lactose-fermenting isolates produced multiple resistance to two antibiotics. Only 7% of antibiotic resistance strains were proven to contain plasmids. Biochemical testing indicated that the most common group of resistant bacteria was Pseudomonas fluorescens. Comparison of protein profiles produced by polyacrylamide gel electrophoresis indicated that there was variation between P. fluorescens strains demonstrating the same multiple resistance.
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9,178,532
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Acinetobacter radioresistens metabolizing aromatic compounds. 1. Optimization of the operative conditions for phenol degradation.
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A strain of Acinetobacter radioresistens was able to utilize phenol as the only carbon and energy source, after an acclimatization period of 3 days in which increasing phenol concentrations from 50 to 200 mg/l were supplied. At 30 degrees C, the complete phenol utilization in batch degradation tests occurred in 2.5-3 h at pH 7 and 8, but it increased strongly at pH 6 (over 40 h). No microbial growth was detected at 40 degrees C, while at 20 degrees C (pH 7-8) the time necessary for complete phenol degradation was about twofold longer than that at 30 degrees C (pH 7-8) revealing a good capability of the strain as a seed-micro-organism for enhancing phenol degradation. The bacterial growth in acclimatized cultures, evaluated with the viable cell count, always displayed a trend consistent with the use of phenol as a substrate with an eventual lag phase and then an exponential phase, while in the non-acclimatized cultures an initial stage of cellular death was observed.
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9,178,533
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A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies.
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Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.
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9,178,534
|
Pre-existence and emergence of drug resistance in HIV-1 infection.
|
Antiviral treatment of HIV-1 infection often fails because of the rapid emergence of resistant virus within weeks of the start of therapy. This raises the question of whether resistant viruses pre-exist in drug-naive patients or whether it is produced after the start of therapy. Here we compare the likelihood of pre-existence with the likelihood of production of resistant virus during therapy. We show that provided resistant virus pre-exists, then a stronger therapy may lead to a greater initial reduction of virus load, but will also cause a faster rise of resistant virus. In this case the total benefit of treatment is independent of the degree of inhibition of sensitive virus. If, on the other hand, resistant mutants do not pre-exist, then the emergence of resistance during treatment depends on the efficacy of the drug. If the drug is sufficiently potent to eradicate sensitive virus, then the probability that resistant mutants first appear during therapy is smaller than the probability that they existed before therapy. If the drug cannot eradicate the sensitive virus, then after sufficiently long time resistant mutants will appear. However, mutants that are unlikely to pre-exist may taken long time to appear.
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9,178,535
|
Mirror agnosia.
|
Normal people rarely confuse the mirror image of an object with a real object so long as they realize they are looking into a mirror. We report a new neurological sign, 'mirror agnosia', following right parietal lesions in which this ability is severely compromised. We studied four right hemisphere stroke patients who had left visual field 'neglect'. i.e. they were indifferent to objects in their left visual field even though they were not blind. We then placed a vertical parasagittal mirror on each patients' right so that they could clearly see the reflection of objects placed in the (neglected) visual field. When shown a candy or pen on their left, the patients kept banging their hand into the mirror or groped behind it attempting to grab the reflection; they did not reach for the real object on the left, even though they were mentally quite lucid and knew they were looking into a mirror. Remarkably, all four patients kept complaining that the object was 'in the mirror', 'outside my reach' or 'behind the mirror'. Thus, even the patients' ability to make simple logical inferences about mirrors has been selectively warped to accommodate the strange new sensory world that they now inhabit. The finding may have implications for understanding how the brain creates representations of mirror reflections.
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9,178,536
|
Influences on the global structure of cortical maps.
|
Cortical maps often contain global spatial structure: however, theoretical accounts for their development have generally concentrated on reproducing only local structure. We show that the elastic net model of cortical map formation can closely approximate the global structure of the ocular dominance column map observed in macaque primary visual cortex. A key component is the assumption of spatially non-uniform and anisotropic correlations in the retina. This work shows how genetic and epigenetic effects could combine to establish characteristic global structure in cortical maps.
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9,178,537
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Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels.
|
We describe the application of three-dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type I collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site: and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.
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9,178,538
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Implications of recent geological investigations of the Mozambique Channel for the mammalian colonization of Madagascar.
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Madagascar separated from continental Africa during the break-up of Gondwanaland early in the Cretaceous. The presence of several terrestrial mammalian groups on Madagascar is paradoxical as (i) these groups postdate the departure of Madagascar from Africa: and ii) terrestrial mammals are poor dispersers across wide water barriers. Recent geological studies focusing on the Davie Fracture Zone of the Mozambique Channel offer a resolution to this situation, by suggesting the presence of a land-bridge from the mid-Eocene to the early Miocene, an interval that matches the ages of Madagascar's mammalian groups.
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9,178,539
|
Localization of S1- and S2-like immunoreactivity in the nervous system of the brittle star Amphipholis squamata (Delle Chiaje 1828).
|
The recent isolation and characterization of the SALMFanide neuropeptides S1 GFNSALMFamide; and S2 (SGPYSFNSGLTFamide) from the sea stars. Asterias rubens and Asterias forbesi have initiated numerous studies on their morphological localization and distribution within the phylum Echinodermata. It has been shown by immunocytochemistry and radioimmunoassay that these peptides are widely distributed in the nervous system of some asteroids, echinoids and ophiuroids. A physiological approach has also shown that S1 and S2 potentiate the luminescence of the small ophiuroid Amphipholis squamata. In the present study. S1- and S2-like immunoreactivity have been localized in A. squamata by immunocytochemistry on both wholemount preparation and histological sections. The results reveal a widespread neuronal distribution of S1-like immunoreactivity in the circumoral ring, radial nerve cord, and tube feet. S1-like immunoreactivity was found to be associated with axons and cell bodies in both the ectoneural and hyponeural components of the nervous. S2-like immunoreactivity was detected only in the ectoneural plenus of the circumoral ring and radial nerve cord.
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9,178,540
|
Genetic and behavioural evidence of monogamy in a mammal, Kirk's dik-dik (Madoqua kirkii).
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Little is known about the mating behaviour of monogamous mammals. Here, we present behavioural and genetic evidence of fidelity in a socially monogamous dwarf antelope, Kirk's dik-dik. DNA microsatellite analysis revealed no evidence of extra-pair paternity (EPP) in dik-diks: mothers' partners matched the paternal genotype in all 12 juveniles tested. One likely reason for the absence of EPP is that males guard their mates closely during oestrus and over-mark all female scent, thereby reducing the likelihood of other males attempting to mate. In addition, males may be limited in their ability to search for extra-pair populations (EPCs) by activities associated with pair-bond maintenance. Year-round, males maintained proximity within pairs, followed their females' activity patterns, and spent approximately 64% of their time with their partners. However, males did attempt to obtain EPCs when the opportunity arose, and genetic monogamy in dik-diks is probably best explained by the behaviour of females: in contrast to many monogamous female birds, female dik-diks do not appear to seek EPC partners. We propose that females avoid extra-pair males because they are unable to mate with them without instigating a potentially dangerous conflict.
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9,178,541
|
Selection and fitness in bacteriocin-producing bacteria.
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Bacteriocins are proteinaceous anticompetitor molecules produced by bacteria against closely related species. A number of theoretical models have been used to explain experimental data that indicate high polymorphisms among bacteriocins and a frequency-dependent nature of selection for bacteriocin-producing strains. The majority of these experimental data were, however, obtained from investigations into the colicin group of bacteriocins produced by Gram-negative bacteria. The conclusions drawn from these models have been extrapolated to other bacteriocins and allelopathic compounds in general. Examination of more recent experimental investigations into the bacteriocins of Gram-positive bacteria indicate a lower degree of polymorphism and a less frequency dependent mode of selection among these strains them among the colicin-producing strains. Here we examine these contradictions in the light of the assumptions and conclusions of the theoretical models and reported data. We show that fitness costs as indicated by decreased relative maximum growth rate associated with bacteriocin production may be much lower in many cases than is assumed in the present models. A lower fitness cost associated with bacteriocin production adequately explains the newer data from Gram-positive bacteria cited here, and indicates that extrapolation of existing models to all bacteriocins and other allelopathic compounds is not appropriate.
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9,178,542
|
Historical rainforest contractions, localized extinctions and patterns of vertebrate endemism in the rainforests of Australia's wet tropics.
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The spatial patterns in the distributions of vertebrates in the rainforests of the wet tropics biogeographic region of north-eastern Australia were examined to form hypotheses on the processes that have shaped vertebrate assemblages and patterns of species richness and regional endemism. These rainforests occur in a relatively narrow and discontinuous strip along the coast of north-eastern Australia. We found that the number of regionally endemic species and the proportion of regional endemics present in each subregion are both strongly related to the geographic shape of subregional patches of rainforest, independent of rainforest area, within Australian tropical rainforests. Shape has a more significant influence on regional endemism than area, and area has a stronger influence on species richness. These patterns were congruent for all terrestrial vertebrate classes manuals, birds, reptiles and frogst, and for the four groups combined. Our results suggest that the combination of current rainforest area and shape are an index of the relative susceptibility of each area of rainforest to historical contractions, with the implication that historical habitat fluctuations, coupled with subsequent localized extinctions species sifting; have been extremely important processes in determining current patterns of endemism in Australia's wet tropical rainforests. This hypothesis is supported by the highly nested structure of the subregional distribution patterns.
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9,178,543
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Genotypic variation among different phenotypes within aphid clones.
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Most aphid species Hemiptera: Aphididae are parthenogenetic between periods of sexual reproduction. They are also highly polyphenic, with different adult morphs occurring in the life cycle, piz. winged, wingless, asexual and sexual. It is assumed that aphids born in a parthenogenetic clonal lineage are genetically identical regardless of the final adult form with the exception of sexual forms). Using the randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) we have found that different asexual adult phenotypes winged and wingless of some clones of two cereal aphid species (the grain aphid, Sitobion avenae (F.) and the bird-cherry aphid. Rhopalosiphum padi (L.) may be distinguished by the presence or absence of one or more RAPD-PCR bands. In three of nine clones examined, such differences were found, and Southern blotting and hybridization of the discriminating bands confirmed these to be of aphid origin, rather than due to endosymbiotic bacteria or contaminating fungi. The main 248 and 296 bp bands, in the two species respectively, were sequenced and found to be A/T rich. The smaller band showed 57% homology with white striated muscle over a stretch of 90 bp. Genomic DNA treated with dimethyl sulphoxide to remove secondary structures still showed differences in RAPD-PCR profiles between winged and wingless morphs within the unusual clones. This discovery may be widespread and therefore it is important to understand the phenomenon in relation to clonal organisms.
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9,178,544
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CSlo encodes calcium-activated potassium channels in the chick's cochlea.
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Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila 'slowpoke' gene. By homology screening with mouse Sla cDNA, we have isolated a full-length clone (cSlo1) from a chick's cochlear cDNA library, rSlol had greater than 90% identity with mouse Slo at the amino acid level, and was even better matched to a human brain Slo at the amino and carboxy termini. cSlol had none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection of HIEK 293 cells demonstrated that cSlol encoded a potassium channel whose conductance averaged 224 pS at +60 mV in symmetrical 140 mM K. Macroscopic currents through cSlol channels were blocked by scorpion toxin or tetraethyl ammonium, and were voltage and calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells.
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9,178,545
|
Molecular evolution of imprinted genes: no evidence for antagonistic coevolution.
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Genomically imprinted genes are those for which expression is dependent on the sex of the parent from which they are derived. Numerous theories have been proposed for the evolution of genomic imprinting: one theory is that it is an intra-individual manifestation of classical parent -offspring conflict. This theory is unique in predicting that an arms race may develop between maternally and paternally derived genes for the control of foetal growth demands. Such antagonistic coevolution may be mediated through changes in the structure of the proteins concerned. Comparable coevolution is the most likely explanation for the rapid changes seen in antigenic components of parasites and antigen recognition components of immune systems. We have examined the evolution of insulin-like growth factor Igf2, and its antagonistic receptor Igf2r) and find that in contrast to immune genes, at the sites of mutual binding they are highly conserved. In addition, we have analysed the rate of molecular evolution of seven imprinted genes including Igf2 and Igf2r), sequenced in both mouse and rat, and had that this is the same as that of nonimprinted receptors and significantly lower than that of immune genes controlling for differences in mutation rates. Contrary to the expectations of the conflict hypothesis, we hence find no evidence for antagonistic coevolution of imprinted genes mediated by changes in sequence.
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9,178,547
|
An adaptationist view of apoptosis.
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A cell's decision whether to undergo apoptosis (cell suicide) is examined here from an adaptationist perspective, rather than a mechanistic one. External and internal inputs to the cell's protein-based information processing network are used in making this decision, with the cell factoring in its replaceability. A system in which each cell takes primary responsibility for deciding its own fate has great adaptive value because it harnesses each cell's self-knowledge rather than waiting for external cues to be recognized by other cells. Cell self-destruction can be an important selective mechanism, potentially leading to better performance of tissues over time. However, reliance on cells to monitor themselves has a flaw, since cells may incur selfish mutations that impair their apoptotic responsibility. The tight control exerted over somatic cells serves to check selfish genes involved in neoplasia and viral infections. Germ cells appear to be similarly monitored, both by other germ cells and by supporting follicular or Sertoli cells, thus maintaining the advantages offered by an apoptotic system. The adaptationist approach views the limited replacement of neurons and cardiac myocytes as likely to have net survival value. The linkage of these cells into a network with their neighbors throughout a lifetime allows for a precisely functioning team of cells expected to compensate for gradual declines in individual cell functionality. Replacement of apoptotic cells with naive cells might decrease brain functionality and might risk upsetting the conduction of cardiac impulses. The evolutionary viewpoint lends itself to new hypotheses, but only the boldest speculator would have predicted a system in which cells are given primary responsibility for deciding whether to kill themselves when they deem it beneficial to the organism.
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9,178,546
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Neural responses to salient visual stimuli.
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The neural mechanisms involved in the selective processing of salient or behaviourally important stimuli are uncertain. We used an aversive conditioning paradigm in human volunteer subjects to manipulate the salience of visual stimuli (emotionally expressive faces) presented during positron emission tomography (PET) neuroimaging. Increases in salience, and conflicts between the innate and acquired value of the stimuli, produced augmented activation of the pulvinar nucleus of the right thalamus. Furthermore, this pulvinar activity correlated positively with responses in structures hypothesized to mediate value in the brain right amygdala and basal forebrain (including the cholinergic nucleus basalis of Meynert). The results provide evidence that the pulvinar nucleus of the thalamus plays a crucial modulatory role in selective visual processing, and that changes in perceptual salience are mediated by value-dependent plasticity in pulvinar responses.
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9,178,548
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Studies on biosynthesis of brassinosteroids.
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Biosynthesis of steroidal plant hormones, brassinosteroids, was studied using the cell culture system of Catharanthus roseus. Feeding labeled compounds of possible intermediates to the cultured cells, followed by analyzing the metabolites by gas chromatography-mass spectrometry disclosed the pathways from a plant sterol, campesterol, to brassinolide. There are two pathways, named the early C6-oxidation pathway and late C6-oxidation pathway, both of which would be operating in a wide variety of plants. Recent findings of brassinosteroid-deficient mutants of Arabidopsis and the garden pea by several groups, and the possible blocked steps of the mutants in the biosynthetic pathways are also introduced.
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9,178,549
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Effect of dietary n-3/n-6 fatty acid ratio on the total count, fatty acid composition, and histamine and leukotriene concentrations of mast cells in tunica mucosa bronchiorum of type I allergic guinea pig.
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To search for the most effective dietary n-3/n-6 ratio to suppress the type I allergic response, we performed basic experiments that applied parameters, associated with the type I allergy. Guinea pigs fed on diets containing lipids with the n-3/n-6 ratio at different levels and the polyunsaturated fatty acid/saturated fatty acid ratio of a fixed level were sensitized with ovalbumin and reared for two weeks. The lowest or critical level of the n-3/n-6 ratio which produced a significant difference in the parameters was as follows: about 2.0 for the response of mast cells and eosinophils; 0.5 and 1.0, respectively, for the uptake of n-3 and n-6 polyunsaturated fatty acids and decreased histamine production; and 0.2 for decreased leukotriene B4 and total leukotrienes 4, and increased leukotrienes 5/leukotrienes 4. The critical level of the n-3/n-6 ratio thus differed widely according to the parameter. Overall, the upper limit for the dietary n-3/n-6 ratio to suppress antigen-induced type I allergic responses is suggested to be around 1.0.
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9,178,550
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Oxygen sensitivity of NifA protein of Azospirillum lipoferum FS as suggested by gene cloning and expression in Escherichia coli.
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We cloned and sequenced a 2.8-kb SalI fragment of Azospirillum lipoferum FS as a homologue of the Klebsiella oxytoca nifA gene. The amino acid sequence deduced from an open reading frame of 1872 bases showed 91% identity to that of the A. brasilense NifA, and the putative central sigma54 interaction domain was conserved as well as the C-terminal DNA-binding domain. The NifA function on the nifH promoter was examined in Escherichia coli using a combination of a nifA driver plasmid and a nifH-lacZ reporter plasmid, in which the transcriptional activation of the nifH promoter by the NifA was evaluated with the beta-galactosidase activity. The A. lipoferum NifA activated the nifH promoter solely under microaerobic conditions, while the K. oxytoca NifA activated it irrespective of the oxygen condition. These observations suggest that oxygen sensitivity is an intrinsic property of the A. lipoferum NifA.
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9,178,551
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In vitro and in vivo anti-platelet effects of enzymatic hydrolysates of collagen and collagen-related peptides.
|
Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined for their inhibitory effects on platelet aggregation induced by the addition of ADP. Human platelet aggregation was suppressed by more than 50% with each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at a concentration of 0.3 mM. The inhibitory effects of these peptides were about 10 times higher in human PRP than in rat PRP. Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg, and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1 to 0.8 mM even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg and enzymatic hydrolysates of collagen suppressed the decrease in platelet count for endotoxin-induced DIC in rats. Collagen itself has been regarded as a potent inducer of platelet aggregation, but these findings suggest that collagen-related peptides and enzymatic hydrolysates of collagen prevent platelet aggregation.
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9,178,553
|
Characterization of the protein and glycan moieties in different forms of bovine lactoferrin.
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The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52, and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments (residues 64-471, 130-471, and 472-689) of LF-a coincide with those of the 54, 47, and 30 kDa fragments of LF-b. respectively. All these fragments, which were positive by PAS staining, were not stained after being treated with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses were the same as those of the treated fragments of LF-b. The 58 and 52 kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin, while the 54 and 47 kDa fragments of LF-b hardly bound to it.
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9,178,552
|
Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.
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Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40%. In contrast with normal cells, the cells cultured in the presence of PI-PLC showed needle-like appendages which seemed to have been formed due to portions of the cell remaining adhered to the culture dish as the cell shrank. When LLC-PK1 cells were treated with PI-PLC, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells. Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation. These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation. Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis is effective for investigating the function of GPI-anchor proteins.
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9,178,554
|
Thermal gelation profile changes in reconstituted actomyosin due to storage under a high salt concentration and low temperature.
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Changes in the heat-induced gelation properties of reconstituted rabbit skeletal actomyosin stored under a high salt concentration at pH 6.0 and 0 degree C were investigated at different weight ratios of actin to myosin by using dynamic rheological and biochemical measurements. The addition of actin resulted in a pronounced peak maximum at about 50 degrees C and an accompanying temporary reduction in the range at about 50 degrees C to 60 degrees C. The more the initial actin concentration was increased, the greater was the area of the peak/shoulder. However, this area was markedly diminished with increasing storage time. As a result, the dynamic rheological pattern was transformed from an actomyosin type into a myosin type. The relationship between the G' value at 80 degrees C and the actin/myosin weight ratio was curvilinear, with a peak at the ratio of 0.05, immediately after storage was started. This profile changed during storage, depending on the extent to denaturation of actin and myosin in the reconstituted actomyosin (RAM). The G' value of actomyosin in 0.5 M KCl with a small actin/myosin ratio of 0.05 decreased to one-half of its initial value after 7 days of storage, whereas the G' value with a large actin/myosin ratio of 0.225 increased by about 1.6 times. In 1.5 M KCl, all the G' values declined to the level with myosin alone after 7 days of storage. The time-course plots of the remaining actin concentration in RAM at different weight ratios of actin to myosin after being treated with 0.5 M or 1.5 M KCl showed a decrease in the actin content with increasing storage time, and an increase in the KCl concentration to 1.5 M KCl promoted the denaturation of actin in RAM faster than with 0.5 M KCl. The surface hydrophobicity of each RAM sample progressively increased with increasing storage time, while little significant increase in the sulfhydryl (SH) content during storage was observed. It is concluded that changes in the heat-induced gelation properties of actomyosin during storage are largely attributable to the denaturation of actin rather than to the denaturation of myosin or to quantitative changes in the SH content and hydrophobicity.
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9,178,555
|
Characterization of 30-kDa fragments derived from beta-conglycinin degradation process during germination and seedling growth of soybean.
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The degradation process of beta-conglycinin, a vicilin-type glycosylated storage protein of soybean seeds, during germination and seedling growth was examined by concanavalin A blotting combined with polyacrylamide gel electrophoresis. We detected and analyzed the structures of key intermediary fragments of 30 kDa derived from beta-conglycinin degradation, they were proved to be single-domain type subunits of beta-conglycinin. We show here a degradation process of beta-conglycinin: beta-conglycinin is subjected to limited proteolysis at exposed regions on the molecular surface, like domain junctions, generating 30-kDa single-domain fragments before non-specific proteolysis.
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9,178,557
|
Purification of amylases and other enzymes by a forced-affinity chromatography method.
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An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.
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9,178,556
|
Efficient expression of mono- and diacylglycerol lipase gene from Penicillium camembertii U-150 in Aspergillus oryzae under the control of its own promoter.
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The gene, mdlA, coding for mono- and diacylglycerol lipase from Penicillium camembertii U-150 was expressed efficiently in Aspergillus oryzae under the control of its own promoter. The gene product was secreted into the culture medium with a highest productivity of 1 g/liter and correctly processed at both N- and C-termini. KEX2-like processing was suggested to occur at the C-terminus in both A. oryzae and P. camembertii. Specific activity and substrate specificity of the purified recombinant protein were also almost the same to that of native protein but the extent of N-glycosylation in the recombinant protein was about half of that of the native protein. The presence of introns did not seem to affect the gene expression. The mdlA expression was induced by lipids and regulated transcriptionally in A. oryzae as well as P. camembertii. Promoter deletion analysis showed that the region between the positions at -382 and -554 bp from the translation initiation point was important to the higher expression of mdlA. The promoter sequence of mdlA was compared to that of the Geotrichum candidum lipase gene, which is also reported to be inducible by lipids, with three commonly observed oligonucleotide sequences.
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9,178,558
|
Lipid peroxidation in linoleic acid micelles caused by H2O2 in the presence of myoglobin.
|
We investigated the lipid peroxidation in linoleic acid micells caused by H2O2 in the presence of metmyoglobin by monitoring the oxygen consumption. O2 consumption usually consisted of two phases. In the first phase, it occurred slowly and linearly until the concentration of linoleic acid hydroperoxide reached a certain value, rapid consumption, presumably by a chain reaction, then followed in the second phase. No effects of diethylenetriaminepentaacetic acid (DTPA) on the induction period (the period during the first phase) and the maximum oxygen consumption rate (MOCR) in the second phase indicate that free ferric ions liberated from myoglobin had no role in any phases during the lipid peroxidation. The differing dose effects of ascorbic acid, alpha-tocopherol, and sodium nitrite on the induction period and MOCR reflect their respective antioxidative mechanisms during lipid peroxidation.
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9,178,559
|
High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli.
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We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase.
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9,178,560
|
Effects of dietary sesaminol and sesamin on eicosanoid production and immunoglobulin level in rats given ethanol.
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The effects of sesaminol and sesamin on the ethanol-induced modulation of immune indices related to food allergy were examined in rats given a low (10%)-casein diet. Chronic ethanol drinking, at the dietary level of 23% (w/w), significantly increased the plasma IgA and IgM concentrations, irrespective of the presence of 0.1% and 0.2% sesaminol, but the effects disappeared with 0.2% sesamin. A significant IgG-elevating effect of these lignans was also found. In contrast, the concentration of plasma IgE was not influenced by the dietary manipulation. Although ethanol drinking did not influence splenic leukotriene B4 production, sesaminol tended to decrease it dose dependently, while sesamin increased the plasma prostaglandin E2 concentration. These results suggest that sesaminol and sesamin seems to have a diverse effect on the plasma levels of immunoglobulins and eicosanoids.
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9,178,561
|
High level expression of XMP aminase in Escherichia coli and its application for the industrial production of 5'-guanylic acid.
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To improve the efficiency of the enzymatic conversion of 5'-xanthylic acid (XMP) to 5'-guanylic acid (GMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA gene in Escherichia coli. By connecting the PL promoter of lambda phage, the SD sequence of trpL of E. coli, and ATG, at a suitable position upstream of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides of the upstream region of the guaA gene on pPLA66 showed that the C-terminal region of the guaB gene, which encodes IMP dehydrogenase, was conserved and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66 showed an increase in the activity of approximately 370 times when compared with that of the strain MM294, and the amount of the enzyme protein represented approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated in a 5-liter jar fermentor using a medium which contained mainly corn steep liquor. The culture broth had high XMP aminase activity. In the conversion reaction using mixed broths consisted of 600 ml of XMP-fermentation broth of Corynebacterium ummoniagenes KY13203 and 30 ml of cultured broth of E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added to the reaction mixture to make the cell membrane permeable to nucleotides. After 23 h of the reaction, 70 mg/ml (131 mM) of GMP.Na2.7H2O was accumulated from 83 mg/ml (155 mM) of XMP.Na3.7H2O, without addition of ATP. The molar conversion yield was approx. 85%. The facts that the cell membrane was treated to allow nucleotides to permeate and that the conversion reaction proceeded well enough in spite of a small amount of E. coli cells indicate ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E. coli cells. Therefore, it was considered that the coupling reaction between these two kind of strains was established.
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9,178,562
|
Sequential binding of Staphylococcal gamma-hemolysin to human erythrocytes and complex formation of the hemolysin on the cell surface.
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Staphylococcal gamma-hemolysin consists of two protein components, F (or H gamma I) and H gamma II. To elucidate the mode of action of gamma-hemolysin, we studied the binding order of F and H gamma II to human erythrocytes and the cell-bound state of the two components. The binding of F to human erythrocytes preceded the binding of H gamma II to the cells, and thereafter hemolysis occurred. Western immunoblot analysis of the cell-bound gamma-hemolysin indicated that F and H gamma II components form high-molecular-mass (150-250 kDa) complexes on the erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins. Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K. Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of H gamma II to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes.
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9,178,563
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Purification and characterization of a dipeptidyl carboxypeptidase from Pseudomonas sp. WO24.
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A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. WO24. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular mass of 74,000 Da by SDS-PAGE and 72,000 Da by gel filtration, indicating that it is monomeric. The isoelectric point was 5.2 and optimum pH was 6.5-7.0. It showed a specific activity of 780 mumol/min/mg, which is the highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly bonds, or tripeptides. The enzymatic activity was completely inhibited by 0.001 mM EDTA and 0.1 mM O-phenanthroline, but it was not affected by general serine and cysteine protease inhibitors. Addition of Zn2+ completely restored the original activity of the inactivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease. The characteristics of the purified enzyme are slightly different from those of the DCPs from Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and considerably from those of the DCP from Bacillus pumilus.
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9,178,564
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Acyl amino acid derivatives as novel inhibitors of influenza neuraminidase.
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We searched our chemical collection to identify non-N-acetylneuraminate (NeuAC) inhibitors of influenza neuraminidase (NA). Of the 62 acyl derivatives tested, several acyl amino acids, but not acyl alkanolamine derivatives, were effective and inhibited the NA activity in a dose-dependent manner. N-3-Hydroxymyristoyl D-cysteine and N-myristoyl-O-caproyl-D-serine were the more potent compounds and inhibited the enzyme in a noncompetitive manner (Ki = 102 and 125 microM, respectively) without respect to the substrate. An important consideration for the choice of inhibitor is the selectivity of the inhibition. These compounds were selective inhibitors of viral NA and effective for any variant enzyme, but the enzymes from V. cholerae and human placenta were insensitive. Accordingly, the acyl amino acid derivatives may be expected to be inhibitors without cellular toxicity and may serve as lead compounds for anti-influenza agents.
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9,178,565
|
Cloning and sequencing of a cDNA encoding alpha-glucosidase from sugar beet.
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A cDNA encoding sugar beet alpha-glucosidase was cloned from a library constructed from mRNA of suspension-cultured cells. The cDNA, 3056 bp in length, had an open reading frame encoding a polypeptide of 913 amino acid residues with a molecular mass of 102,078 Da, included only one of four regions which were conserved in the alpha-amylase family of enzymes. The deduced amino acid sequence from the analysis of the cDNA contained the sequences of the proteolysis peptides and the active site region peptide of sugar beet alpha-glucosidase. The primary structure indicated relatively high homology in the range of 28.2 to 54.3% to those for other alpha-glucosidases. The highest homology was found in barley alpha-glucosidase.
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9,178,566
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Isolation and partial amino acid sequence of bacteriocins produced by Lactobacillus acidophilus.
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Bacteriocins produced by Lactobacillus acidophilus JCM 1023, JCM 1028, JCM 1021, JCM 1229, and JCM 5342 were active against closely related lactobacilli. These bacteriocins were purified and partial sequenced. Bacteriocin activities of L. acidophilus JCM 1023 and JCM 1028 were associated with two components. On the basis of N-terminal amino acid sequencing and the molecular masses, it is interpreted that these two-component bacteriocins are identical to acidocin J1132, a bacteriocin from L. acidophilus JCM 1132 [Tahara et al., Appl. Environ. Microbiol., 62, 892-897 (1996)]. Other bacteriocins were single-peptide bacteriocins.
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9,178,567
|
Stimulating effect of ileal pancreaticobiliary secretion on ileal apolipoprotein A-IV mRNA expression in fasted rats.
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The effect of pancreaticobiliary secretion on the intestinal expression of the apo A-IV gene was examined in fasted rats. Pancreaticobiliary diversion, but not biliary diversion alone, into the ileum increased the ileal apo A-IV mRNA expression by 24 h post-operation. Jejunal apo A-IV mRNA was reduced by biliary exclusion. The data suggest that the biliary constituent plays an important role in the apo A-IV gene expression in the entire length of the small intestine, and that up-regulation of the apo A-IV gene requires exocrine pancreatic in addition to biliary secretion.
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9,178,568
|
Biological activity of purpurogallin.
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Purpurogallin showed antibacterial activity toward gram-positive bacteria. Strong activity against methicillin-resistant Staphylococcus aureus [minimal inhibitory concentration (MIC) against methicillin of 1600 micrograms/ml] was found, with MIC of 11.0 micrograms/ml. Purpurogallin inhibited the growth of all tested plants and decreased the chlorophyll content in the cotyledons of Brassica campestris subsp. rapa. It showed potent inhibitory activity against prolyl endopeptidase (the 50% inhibitory concentration was 1.6 x 10(-5) M), unlike its analogues, hinokitiol and tropolone.
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9,178,569
|
Selective incorporation of polyunsaturated fatty acids into organelle phospholipids of animal cells.
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The selective incorporation of linoleic (18:2(n-6)) and docosahexaenoic (22:6(n-3)) acids into phospholipids of mitochondria, endoplasmic reticulum, and plasma membrane was investigated by changing the ratio of 22:6(n-3) against 18:2(n-6) in a medium, in which Chinese hamster V79-R cells were grown. In those organelles, 18:2(n-6) and its elongation product (eicosadienoic acid) (20:2 (n-6)) were predominantly incorporated into phosphatidylcholine. However, 22:6(n-3) was incorporated more selectively into phosphatidylethanolamine than 18:2(n-6) and 20:2(n-6).
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9,178,570
|
Zinc finger-like motif conserved in a family of RNA binding proteins.
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NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys2-His2 zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.
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9,178,571
|
A gene homologous to the Streptomyces chymotrypsin-like protease (SAM-p20) gene is tandemly located.
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A gene encoding a homolog of the Streptomyces chymotrypsin-like serine protease, SAM-P20, was identified downstream of the sam-p20 gene and designated SAM-P20D. This gene has two tandem Shine Dalgarno sequences and two initiation codons. We have established vector systems with the function of tyrosinase gene-bone melanin pigmentation as a reporter for sam-p20D gene expression in Streptomyces coelicolor in order to identify the promoter and terminator activities. Using this system, the sam-p20D gene was suggested to be transcribed monocistronically.
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9,178,573
|
Structure and activity of a new form of the glutamate transporter of the nematode Caenorhabditis elegans.
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A Caenorhabditis elegans cDNA for a glutamate transporter was cloned and examined in this study. The predicted protein is 11 residues shorter at the N-terminus than Ceglut-1, which we previously reported. The protein, when expressed in Xenopus laevis oocytes, showed much higher glutamate transport activity than Ceglut-1, suggesting that the N-terminal sequence is critical in glutamate transport.
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9,178,572
|
Peculiar archaea found in Japanese paddy soils.
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Archaeal 16S rDNA clones retrieved from paddy soil DNA were sequenced. Among 100 clones, 88 clones were assigned to methanogens and nine clones were assigned to crenarchaeota. However, three of the nine clones were phylogenetically far from the cultured crenarchaeota and closely related to marine planktonic archaea. The other three clones showed extremely novel 16S rDNA sequences and were phylogenetically far from both Crenarchaeola and Euryarchaeota. This paper reports the ubiquitous presence of crenarchaeotal and extremely novel clones in paddy soils.
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9,178,574
|
Comparison of hemisphere size in wild and domestic geese.
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The hemisphere size has been studied in wild and domestic geese (Anser fabalis, n = 77: A. albifrons, n = 48; A. anser, n = 8; A. anser f. domestica, n = 10), which were classified according to particular age category (adult or immature). Material used in studies was fixed in 4% solution of formaldehyde. The brain weight as well as hemispheres' length, width and height was measured with standard method. Morphometric characteristic of geese's brains has been presented in absolute and relative way (by parallel use of index and allometric approach). The appropriate ontogenic, taxonomic and domestication comparisons were carried out. The greatest differences referred to absolute and relative width and height of hemispheres.
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9,178,576
|
Presence of cerium-cytochemical reactions of glomerular phosphatases of normal gerbil Meriones crassus: an ultrastructural localization study.
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Phosphatase cytochemical activity in the normal glomerulus of the desert gerbil Meriones crassus was demonstrated using cerium ions as capturing agents. Three major enzymes have been recognized: sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), alkaline phosphatase (ALPase) and acid phosphatase (ACPase). However, cytochemical staining for these markers to map their localizations and distributions reveal a high positivity of Na(+)-K(+)-ATPase. This appeared as uniform dense precipitates surrounding the glomerular basement membrane (GBM) and the plasma membranes of the epithelial and endothelial cells of the glomerular layers. Negligible ALKase reaction product being over the glomerular epithelia including the GBM. In contrast, the cytochemical profiles of ACPase was unusual, with dense reaction products extensively covering the endoplasmic reticulum at the region of Golgi apparatus products lysosomes (GERL) complex, including its cisternal and tubular elements and the lysosomal-vacuolar apparatus of the glomerular epithelial cells. All other subcellular organelles showed no activity. For Na(+)-K(+)-ATPase, the reaction product was successive when acetate buffer (as decalcifying agent, pH 5.0) was used. This reaction was still seen when a medium containing levamisole was used. Cytochemical controls for all enzymes were incubated in substrate-free media including those using levamisole as an inhibitor of ALPase. The data presented, which is reported for the first time, is not an attempt to determine the contribution of the selected phosphatases in the glomerular physiology and pathology. Such findings may, nevertheless, have functional implications in the fact that these markers may be involved in the ultrafiltration and other metabolic activities of the glomerulus at the molecular and/or cellular level. In addition to earlier morphological and recent histochemical work, the present study updates and recognizes information to be used as a baseline to which the gerbil model can now be employed to investigate the behavioural adaptations of the desert rodents.
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9,178,575
|
Subpopulations of stromal cells from long-term human bone marrow cultures: ontogeny of progenitor cells and expression of growth hormone receptors.
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Long-term culture of bone marrow derived stromal colony forming cells (S-CFC) in matrix and nutrient defined agar medium resulted in stromal cell colonies that pass sequentially through three distinct morphological stages: firstly, aggregated loose syncytium of round to avoid cells (stage I), a second developmental stage of large branching colonies in which the cells become enlarged, elongated with cytoplasmic projections forming a loosely anastomized network with adjacent cells (stage II), and finally cells become dissociated, loosing their long, thin cytoplasmic filaments and breaking their contacts with one another, but remain large and retain a bi-polar nature (stage III). Cells were also grown in liquid medium in a culture microenvironment closely resembling conditions of haemopoiesis in vitro. Using a panel of well defined monoclonal antibodies reactive against the rat, rabbit and human growth hormone receptors, this study found immunochemical evidence of the presence and localization of binding sites of growth hormone (GH) in the cell membrane and extra-nuclear Golgi area of long-term bone marrow derived human stromal cells in liquid and semi-solid nutrient agar mediums. GH-receptor immunoreactivity was present in small proliferating progenitor cells, myofibroblast-like cells, large reticular fibroblast cells, adipocytes and endothelial cells. Only MAb known to be reactive against human tissue resulted in strong immunoreactivity. The expression of GH-receptors not only on small proliferating, but also on the well differentiated cells, indicates a role for growth hormone on non-progenitor cells. GH-receptor immunoreactivity on differentiating and/or differentiated cells suggests that GH is also necessary for, or has a trophic function in differentiation. We propose that direct GH action is necessary not only for differentiation of progenitor cells as implied by the dual effector hypothesis, but also their subsequent clonal expansion, differentiation and maintenance.
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