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9,177,772
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Human TNF receptor-associated factor 5 (TRAF5): cDNA cloning, expression and assignment of the TRAF5 gene to chromosome 1q32.
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Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for members of the TNF receptor superfamily. We previously identified murine TRAF5 (mTRAF5) and showed that it specifically interacts with the lymphotoxin-beta receptor (LT-beta R) and activates the transcription factor NF-kappa B. Here we have cloned the human TRAF5 homologue (hTRAF5) by cross hybridization with mTRAF5 cDNA. hTRAF5 cDNA is composed of 2894 nucleotides with a 557-amino-acid open reading frame that exhibits 77.5 and 80% identity to mTRAF5 at the nucleotide and amino acid levels, respectively. Northern blot analysis revealed that hTRAF5 mRNA is expressed in all visceral organs. Western blotting revealed that hTRAF5 protein was abundantly expressed in the human follicular dentritic cell line, FDC-1, and to a much lesser degree in several tumor cell lines. Interspecific backcross mapping revealed that Traf5 is located in the distal region of mouse chromosome 1, which shares a region of homology with human chromosome 1q. Fluorescence in situ hybridization confirmed regional localization to human chromosome 1q32.
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9,177,773
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Genomic localization of the human gene for KCNA10, a cGMP-activated K channel.
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Potassium (K) channels are important components of virtually all cells, and they play critical roles in many cellular functions. KCNA10 represents a new class of K channel specifically regulated by cGMP and postulated to mediate the effects of substances that increase intracellular cGMP. Since KCNA10 has the potential to be useful in candidate gene analysis of inherited diseases, the human gene for KCNA10 was characterized. Fluorescence in situ hybridization indicates that human KCNA10 maps to chromosome 1 at p13.1-->p22.1. Finer mapping of the gene was achieved by PCR of a set of CEPH YAC clones that spanned the region of interest. We found that YAC 818b9 contains human KCNA10. These data indicate human KCNA10 maps to 1p13.1 and resides within the genetic interval defined by microsatellite loci D1S2809 and D1S2726. That region of chromosome 1 contains another K channel gene, KCNA3.
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9,177,774
|
Elucidation of the sequence and the genomic organization of the human dentin matrix acidic phosphoprotein 1 (DMP1) gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II.
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The dentin matrix acidic phosphoprotein 1 (DMP1) gene has been mapped to human chromosome 4q21 and shown to exhibit no recombination with the autosomal dominant disorder of dentin formation, dentinogenesis imperfecta type II. In the current study, sequencing of DMP1 cDNA and genomic clones has indicated that the human gene contains an open reading frame of 1539 bp, which predicts a highly acidic, serine-rich protein of 513 amino acids. Comparison of the human DMP1-coding sequence with that of the rat, mouse, and cow indicated that the predicted protein contains a conserved hydrophobic signal peptide sequence and an Arg-Gly-Asp cell attachment sequence. The gene is encoded by six exons, the splicing phase of which is type 0, the first exon containing solely 5' untranslated sequence. Sequencing of each of the coding exons in individuals affected by dentinogenesis imperfecta type II failed to reveal any disease-specific mutations, suggesting that mutations in DMP1 are not causative of this condition at least in the two families examined in this study.
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9,177,775
|
Genomic structure, evolution, and expression of human FLII, a gelsolin and leucine-rich-repeat family member: overlap with LLGL.
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The Drosophila melanogaster flightless-I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. The homologous human FLII gene encodes a 1269-residue protein with 58% amino acid sequence identity and is deleted in Smith-Magenis syndrome. We have cloned the FLII gene and determined its nucleotide sequence (14.1 kb). FLII has 29 introns, compared with 13 in Caenorhabditis elegans and 3 in D. melanogaster. The positions of several introns are conserved in FLII-related genes and in the domains and subdomains of the gelsolin-like regions giving indications of gelsolin gene family evolution. In keeping with its function in indirect flight muscle in Drosophila, the human FLII gene was most highly expressed in muscle. The FLII gene lies adjacent to LLGL, the human homologue of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The 3' end of the FLII transcript overlaps the 3' end of the LLGL transcript, and the corresponding mouse genes Fliih and Llglh also overlap. The overlap region contains poly(A) signals for both genes and is strongly conserved between human and mouse.
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9,177,776
|
Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes.
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To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.
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9,177,777
|
Identification of a novel human kinesin-related gene (HK2) by the cDNA differential display technique.
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We have used the cDNA differential display technique to isolate genes regulated by the synthetic retinoid N-(4-hydroxyphenyl)-all-trans-retinamide (HPR), a cancer chemopreventive agent in vivo and a powerful inducer of apoptotic cell death in vitro. Here we report the identification of a novel gene, the expression of which is markedly up-regulated in tumor cells after treatment for 30-60 min with HPR. The full-length cDNA of this gene, determined by screening of a human placenta cDNA, is 3.5 kb long and contains an open reading frame of 2037 nt. The gene is > 90% homologous to the mouse KIF2, a gene belonging to the family of kinesin-related motor proteins, and we therefore named it HK2 (human kinesin 2). A shorter form of the HK2 mRNA (HK2s), containing a 57-nt deletion in the open reading frame, has also been detected. Northern analysis revealed that HK2 is widely expressed among hemopoietic and nonhemopoietic cell lines and tissues. By the use of radiation hybrids, HK2 has been localized to chromosome 5q12-q13. Kinesins constitute a superfamily of motor proteins that use energy liberated from ATP hydrolysis to move cargo along microtubules and are implicated in mechanisms of mitosis or meiosis. The role of HK2 in the growth-inhibitory and apoptotic responses elicited by HPR remains to be established.
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9,177,778
|
Physical and linkage mapping of human chromosome 17 loci to dog chromosomes 9 and 5.
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Genome mapping in the dog is in its early stages. Here we illustrate an approach to combined physical and linkage mapping of type 1 anchor (gene) loci in the dog using information on syntenic homology from human and mouse, an interbreed cross/backcross, and a strategy for isolation of dog genomic clones containing both gene-specific sequences and simple sequence repeat polymorphisms. Eleven gene loci from human chromosome 17q (HSA17q) were mapped to the centromeric two-thirds of dog chromosome 9 (CFA9), an acrocentric chromosome of medium size: P4HB, GALK1, TK1, GH1, MYL4, BRCA1, RARA, THRA1, MPO, NF1, and CRYBA1. Eight of these were also positioned on a linkage map spanning 38.6 cM. Based on combined fluorescence in situ hybridization and linkage mapping, the gene order on CFA9 is similar to that of the homologous genes on HSA17q and mouse chromosome 11 (MMU11), but in the dog the gene order is inverted with respect to the centromere. Canine loci, GALK1, TK1, GH1, MYL4, THRA1, and RARA constitute a closely linked group near the centromeric end of CFA9, spanning a genetic distance of only 4.7 cM. Canine NF1 and CRYBA1 lie distally, near the lower border of the Giemsa band adjacent to the distal one-third of CFA9. NF1 and CRYBA1 are loosely linked to the more centromeric group (31.2 cM). No HSA17 genes were found on the telomeric one-third of CFA9. Painting of dog chromosomes with a human whole chromosome 17 probe showed hybridization with only the proximal two-thirds of CFA9, consistent with the conclusion that the distal one-third corresponds to a segment or segments of other human chromosomes. Two loci, GLUT4 and PMP22, located on HSA17p, were mapped by FISH to dog chromosome 5 in a region also identified by the whole human chromosome 17 paint, indicating disruption of HSA17 syntenic homology at the centromere.
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9,177,779
|
Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.
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In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.
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9,177,782
|
An integrated transcript map of human chromosome 1p35-p36.
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The distal short arm of human chromosome 1 (1p) is rearranged in a variety of malignancies, and several genetic diseases also map to this region. We have constructed an integrated transcript map to precisely define the positions of genes and expressed sequence tags (ESTs) previously mapped to 1p35-p36, a region spanning approximately 40 Mb. To anchor the integrated map, a framework genetic map was constructed with 24 genetic markers and a marker order of 1000:1 odds, yielding an average resolution of 2.8 cM. An additional 106 genetic markers were localized relative to the framework genetic map. To place markers more precisely within 1p35-p36, a chromosome 1-specific, radiation-reduced hybrid (RH) panel was created. Individual DNA fragments of the RH panel were identified and ordered by PCR with the framework genetic map. A total of 250 markers, including 142 genes and ESTs, were mapped by PCR against the RH panel. The map has an observed resolution of 800 kb, and the results closely match and more precisely define previous mapping information for most markers. This map will help to identify candidate genes for genetic diseases mapping to distal 1p and is fully integrated with existing genetic and RH maps of the human genome.
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9,177,781
|
Plectin transcript diversity: identification and tissue distribution of variants with distinct first coding exons and rodless isoforms.
|
Plectin is a widely expressed protein that is very large in size and that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multidomain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. Moreover, hereditary gene defects in plectin cause epidermolysis bullosa simplex (EBS)-MD, a severe skin blistering disease with muscular dystrophy. Here we report the analysis of the exonintron organization of the rat plectin gene and the identification of several different isoforms on the transcriptional level. We show that of 35 coding exons identified, 4 serve as alternative first exons splicing into the same successive exon 2, which is the first of 7 exons encoding a highly conserved actin-binding domain. RNase protection mapping of transcripts containing 3 of the identified 4 alternate first exons revealed their coexpression in rat glioma C6 cells and in a series of different rat tissues that we examined. Significant variations in expression levels of first exons indicated the possibility of tissue-specific promoter usage. In addition, plectin splice variants lacking exon 31 (> 3 kb), which encodes the entire rod domain of the molecule, were identified in a variety of rat tissues. This study provides first insights into a complex plectin gene regulatory machinery with similarities to that of dystrophin.
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9,177,780
|
Construction of a 1.2-Mb contig surrounding, and molecular analysis of, the human CREB-binding protein (CBP/CREBBP) gene on chromosome 16p13.3.
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In the interest of cloning and analyzing the genes responsible for two very different diseases, the Rubinstein-Taybi syndrome (RTS) and acute myeloid leukemia (AML) associated with the somatic translocation t(8;16)(p11;p13.3), we constructed a high-resolution restriction map of contiguous cosmids (contig) covering 1.2 Mb of chromosome 16p13.3. By fluorescence in situ hybridization and Southern blot analysis, we assigned all tested RTS and t(8;16) translocation breakpoints to a 100-kb region. We have previously reported exact physical locations of these 16p breakpoints, which all disrupt one gene we mapped to this interval: the CREB-binding protein (CBP or CREBBP) gene. Intriguingly, mutations in the CBP gene are responsible for RTS as well as the t(8;16)-associated AML. CBP functions as an integrator in the assembly of various multiprotein regulatory complexes and is thus necessary for transcription in a broad range of transduction pathways. We report here the cloning, physical mapping, characterization, and full cDNA nucleotide sequence of the human CBP gene.
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9,177,783
|
Npm3: a novel, widely expressed gene encoding a protein related to the molecular chaperones nucleoplasmin and nucleophosmin.
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We report the cloning and initial characterization of the cDNAs, gene, and pseudogene of Npm3, a novel murine gene that encodes a protein related to the nuclear chaperone phosphoproteins, nucleoplasmin and nucleophosmin. Npm3 is located approximately 5 kb upstream of Fgf8 on mouse Chromosome 19 and consists of six exons spanning 2 kb. The first five exons code for an acidic protein of 19.0 kDa that contains a potential nuclear localization signal and potential phosphorylation sites for several kinases. Npm3 was expressed in all mouse tissues examined. On the basis of the similarity of Npm3 to nucleoplasmin and nucleophosmin in amino acid sequence, protein features, and exon structure, we propose that Npm3 is a new member of, and may share basic functions with, the nucleoplasmin/ nucleophosmin family of molecular chaperone proteins.
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9,177,784
|
Chromosome mapping and expression of the human interleukin-13 receptor.
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Interleukin-13 (IL-13) is a cytokine secreted by activated T cells and shares most but not all biological activities with interleukin-4 (IL-4). Both cytokines play an important role as a switch factor directing synthesis of IgE; they act on monocytes and endothelial cells, but unlike IL-4, IL-13 does not act on T cells. These cytokines have both common and distinct components in their respective receptors. Based on sequence similarity shared by cytokine receptor family members, we have identified a cDNA encoding the human IL-13 receptor (IL-13R). This cDNA was used to examine the pattern of IL-13R mRNA expression by Northern blot analyses of poly(A)+ RNA purified from different human tissues and cell lines. Among several myeloma cell lines analyzed, the U266 cell line was the only one found to express IL-13R transcripts. This cell line is also the only one described as producing IgE. The IL-13R gene was mapped to chromosome Xq24 by in situ hybridization. Interestingly, this locus is near that of the CD40 ligand gene, the product of which is also involved, like IL-13, in proliferation and IgE isotype switching of human B cells. The human IL-13R gene maps between two cytokine receptor genes located on the chromosome arm Xq region: the interleukin-2 receptor gamma chain gene (Xq13.1) and the interleukin-9 receptor gene (Xq28). The lack of nucleotide sequence similarity suggests unrelated evolutionary pathways between these receptor genes.
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9,177,785
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Structure of the human gene (COX6A2) for the heart/muscle isoform of cytochrome c oxidase subunit VIa and its chromosomal location in humans, mice, and cattle.
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We have mapped the gene for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa in three mammalian species and isolated the human COX6AH gene (HGMW-approved symbol COX6A2). The bovine gene was mapped by somatic cell hybrid mapping panels to bovine chromosome BTA 25 with 94-95% concordance. The mouse gene (Cox6ah) was mapped using an interspecific backcross panel from the cross (C57BL/6J x Mus spretus)F1 x Mus spretus probed with the mouse COX VIa-H cDNA. Cox6ah was located on distal chromosome 7, between D7Mit8 and D7Mit13. From the regions of known gene conservation among these three species, we predicted that human COX6AH would be located on chromosome 16p. We hybridized a human x rodent mapping panel of somatic cell hybrids with the human cDNA to confirm this assignment. These data taken together indicated that the human COX6AH gene is located on the short arm of chromosome 16 and facilitated the isolation of the human gene from a chromosome 16-enriched library. The human COX6AH gene spans about 1 kb and contains three exons and two small introns. The sequences of the proximal 5' flanking regions of COX6AH genes are highly conserved between human, bovine, and rodent.
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9,177,786
|
The molecular basis of the obese mutation in ob2J mice.
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The recessive ob2J mutation in mice results in an obese phenotype that is identical to that of the original ob allele. Initial studies indicated that ob2J mice fail to synthesize ob RNA in adipose tissue. Here we report the genomic organization of the mouse obese gene and establish the molecular genetic basis of the ob2J mutation. The ob2J mutation is the result of the insertion of a retroviral-like tranposon in the first intron of the ob gene. The insertion is a member of the ETn family of transposons and contains several splice acceptor and polyadenylation sites. This leads to the production of chimeric RNAs in which the ob first exon is spliced to sequences in the ETn insertion. As a consequence mature ob RNA is not synthesized, and leptin, the encoded protein, is not produced.
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9,177,787
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Structure of the human ARHG locus encoding the Rho/Rac-like RhoG GTPase.
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Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about their gene structure and regulation. RhoG, a member related to Rac and Cdc42Hs, is activated at the transcriptional level in the mid-G1 phase of stimulated fibroblasts. As a first step toward the characterization of the regulatory elements involved in serum-regulated expression, we isolated and determined the structure of the corresponding human locus (ARHG, localized in 11p15.4-p15.5). This is the first gene structure of a member of the Rho/Rac/Cdc42Hs family. At variance with Ras and Rab3A genes, ARHG contains a single intron larger than 20 kb that splits a 62-nt-long 5' noncoding first exon from the rest of the mRNA. The sequences upstream of the cap sites exhibit transcriptional activity. They are G/C-rich and devoid of TATA or CAAT boxes, as found for many housekeeping genes, including Ras genes.
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9,177,788
|
Genomic structure and expression of the human heme A:farnesyltransferase (COX10) gene.
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Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1.5-Mb tandem DNA duplication in chromosome 17p11.2-p12, while hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus. The 1.5-Mb CMT1A monomer unit duplicated in CMT1A and deleted in HNPP is flanked by two 24-kb direct repeats termed the CMT1A-REPs. Recently, sequence analysis of the CMT1A-REPs revealed that they contain an internal exon of the COX10 gene. To characterize COX10, encoding human heme A:farnesyltransferase, the genomic region was isolated and the gene structure and expression profile were determined. COX10 spans approximately 135 kb and consists of seven exons. Exons I-V are telomeric to the 1.5-Mb CMT1A monomer unit, whereas exon VII is located within this 1.5-Mb region. Exon VI is contained within the distal CMT1A-REP. All splice sites conform to the GT/AG rule. Analysis of the putative promoter region of the COX10 gene indicates that it lacks conventional TATA and CAAT boxes, but it does have several potential transcription factor-binding sites. This gene is expressed in multiple tissues with highest expression observed in the heart, skeletal muscle, and testis.
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9,177,789
|
Human synaptotagmin V (SYT5): sequence, genomic structure, and chromosomal location.
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We have determined the sequence, genomic structure, and chromosomal location of the human synaptotagmin V (SYTV) gene. The human SYTV gene encodes a 386-amino-acid product which is 91% identical to rat Syt V. The human SYTV open reading frame is interrupted by seven introns which can be alternatively spliced. Human SYTV was found to lie very close to SYTIII on chromosome 19q13.4 by PCR analysis of somatic cell hybrid DNA and by DNA hybridization to arrayed cosmids of the chromosome 19 metric physical map. This provides the first report of linked synaptotagmin genes.
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9,177,790
|
Exon-intron structure of a 2.7-kb transcript of the STM7 gene with phosphatidylinositol-4-phosphate 5-kinase activity.
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The STM7 gene encodes a novel phosphatidylinositol-4-phosphate 5-kinase (PtdInsP 5-kinase) that is subject to alternative splicing and developmental control. We have recently presented data indicating that several splice variants of STM7 incorporate elements of the X25 sequence, previously implicated in the pathogenesis of Friedreich's ataxia by the detection of an intronic GAA repeat expansion as the predominant mutation in affected individuals. We now report the exon-intron structure of STM7.I and primer sequences designed to facilitate full characterization, including details relating to a novel exon (STM7; exon 17) derived from the 3'-UTR of the PRKACG gene. The detection of a mutation(s) within these exons would provide additional support for the hypothesis that a defect in phosphoinositide metabolism gives rise to the disease phenotype.
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9,177,791
|
Mapping of the human HPC-1/syntaxin 1A gene (STX1A) to chromosome 7 band q11.2.
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We previously described the cDNA sequence of HPC-1/syntaxin 1A (HGMW-approved symbol STX1A) from rat and bovine brains. HPC-1/syntaxin 1A belongs to the syntaxin family and is apparently involved in intracellular membrane transport and the exocytosis of neurotransmitters. In this study, we isolated the cDNA and the genomic DNA clone for human HPC-1/syntaxin 1A and carried out gene mapping. Polymerase chain reaction analysis of human/rodent somatic cell hybrid panels and fluorescence in situ hybridization analysis using a genomic DNA clone provided evidence that the gene for human HPC-1/syntaxin 1A maps to chromosome region 7q11.2.
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9,177,792
|
Mapping of the gene encoding the integrin-linked kinase, ILK, to human chromosome 11p15.5-p15.4.
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We have recently reported the identification and cloning of the gene encoding p59ILK, a novel protein ser/thr kinase that is found in physiologic complexes with beta integrin subunits. ILK is a potential protoonocogene that appears to function in mediating signal transduction by beta 1 family integrins. Fluorescence in situ hybridization analysis of metaphase and decondensed free chromatin fibers localized ILK to 11p15.5-p15.4. This position was also confirmed by relational mapping using well-characterized translocations with breakpoints in chromosome band 11p15. Our results indicate that ILK maps between HBBC and CALC loci, in the 11p15.5-p15.4 band interval. This location may be important in evaluating the potential role of p59ILK in tumorigenesis since it has been shown that this region is associated with both genomic imprinting and loss of heterozygosity in certain types of tumor.
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9,177,795
|
A general approach to error estimation and optimized experiment design, applied to multislice imaging of T1 in human brain at 4.1 T.
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In this report, a procedure to optimize inversion-recovery times, in order to minimize the uncertainty in the measured T1 from 2-point multislice images of the human brain at 4.1 T, is discussed. The 2-point, 40-slice measurement employed inversion-recovery delays chosen based on the minimization of noise-based uncertainties. For comparison of the measured T1 values and uncertainties, 10-point, 3-slice measurements were also acquired. The measured T1 values using the 2-point method were 814, 1361, and 3386 ms for white matter, gray matter, and cerebral spinal fluid, respectively, in agreement with the respective T1 values of 817, 1329, and 3320 ms obtained using the 10-point measurement. The 2-point, 40-slice method was used to determine the T1 in the cortical gray matter, cerebellar gray matter, caudate nucleus, cerebral peduncle, globus pallidus, colliculus, lenticular nucleus, base of the pons, substantia nigra, thalamus, white matter, corpus callosum, and internal capsule.
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9,177,796
|
Longitudinal relaxation and diffusion measurements using magnetic resonance signals from laser-hyperpolarized 129Xe nuclei.
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Methods for T1 relaxation and diffusion measurements based on magnetic resonance signals from laser-hyperpolarized 129Xe nuclei are introduced. The methods involve optimum use of the perishable hyperpolarized magnetization of 129Xe. The necessary theoretical framework for the methods is developed, and then the methods are applied to measure the longitudinal relaxation constant, T1, and the self-diffusion constant, D, of hyperpolarized 129Xe. In a cell containing natural abundance 129Xe at 790 Torr, the T1 value was determined to be 155 +/- 5 min at 20 degrees C and at 2.0 T field. For a second cell at 896 Torr, at the same field and temperature, the T1 value was determined to be 66 +/- 2 min. At a higher field of 7.05 T, the T1 values for the two cells were found to be 185 +/- 10 and 88 +/- 5 min, respectively. The 129Xe self-diffusion constant for the first cell was measured to be 0.057 cm2/ s and for the second cell it was 0.044 cm2/s. The methods were applied to 129Xe in the gas phase, in vitro; however, they are, in principle, applicable for in vivo or ex vivo studies. The potential role of these methods in the development of newly emerging hyper-polarized 129Xe MRI applications is discussed.
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9,177,799
|
Pathological and hemodynamic study in a new model of femoral head necrosis following traumatic dislocation.
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The blood of the femoral head is thought to be supplied by vessels originating from the medial and lateral circumflex femoral arteries and via the marrow cavity of the neck. Therefore, it is difficult to induce osteonecrosis of the femoral head when the marrow cavity of the neck is preserved. In the present study, we established a new model of femoral head necrosis by dislocating the hip joint and ligating the medial and lateral circumflex femoral arteries and veins. Measurement of femoral head blood flow revealed that a marked decrease to 14.7% of the control value was achieved by both hip dislocation and ligation of blood vessels. Pathologic examination showed no necrosis with either dislocation or ligation alone, whereas at 2 and 4 weeks 80% of the animals subjected to both procedures showed widespread necrosis. These pathologic findings considered in the light of results of the blood flow measurements suggest that a decrease in femoral head blood flow below 20% of the control value is needed to cause osteonecrosis. In addition, magnetic resonance images (MRI) of the model were evaluated in the combined dislocation and ligation group at 4 weeks (n = 5). Changes on MRI were seen in 3 of 5 dogs. The necrotic changes of the femoral head are thought to be detectable on MRI within 4 weeks after ischemia without enhancement.
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9,177,798
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Vascularized pedicle bone-grafting for nontraumatic avascular necrosis of the femoral head. A 5- to 11-year follow-up.
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We investigated the results of 31 hips in 26 patients with nontraumatic (n = 20) and steroid-induced (n = 6) avascular necrosis of the femoral head (ANFH) treated with vascularized iliac pedicle bone graft (PBG). The average age at operation was 38.3 years. Three were women and 23 men. The average follow-up was 8.0 years. The Harris hip score prior to operation and at latest follow-up improved from 62 to 83; one hip collapsed and was revised with a bipolar endoprosthesis. At the final follow-up, 19 hips (63%) were clinically rated as good to excellent, 4 fair, and 7 poor. At the final follow-up, 15 of 27 hips (56%) of stage II before operation showed progressive collapse after bone grafting. In steroid-induced ANFH, in three women, 2 of 4 hips showed poor results. These results are only slightly better than those of core decompression and no better than those obtained after decompression and simple nonvascularized grafts to provide support for the subchondral bone. We concluded that vascularized PBG is sometimes indicated for ANFH in an early stage before collapse of the femoral head.
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9,177,797
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NMR of laser-polarized 129Xe in blood foam.
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Laser-polarized 129Xe dissolved in a foam preparation of fresh human blood was investigated. The NMR signal of 129Xe dissolved in blood was enhanced by creating a foam in which the dissolved 129Xe exchanged with a large reservoir of gaseous laser-polarized 129Xe. The dissolved 129Xe T1 in this system was found to be significantly shorter in oxygenated blood than in deoxygenated blood. The T1 of 129Xe dissolved in oxygenated blood foam was found to be approximately 21 (+/-5) s, and in deoxygenated blood foam to be greater than 40 s. To understand the oxygenation trend, T1 measurements were also made on plasma and hemoglobin foam preparations. The measurement technique using a foam gas-liquid exchange interface may also be useful for studying foam coarsening and other liquid physical properties.
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9,177,800
|
Results of humeral stump angulation osteotomy.
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Between 1972 and 1989 angulation osteotomy was performed on 61 patients with long humeral stumps at the Orthopaedic Hospital of Heidelberg University. Marquardt's surgical technique was used to improve function in patients who had undergone above-elbow amputation and in children with a risk of terminal osseous overgrowth. Thirty-one patients with 43 angulation osteotomies were followed up. Of the 10 adults followed up, the osteotomy had not straightened, whereas with the 33 angulation osteotomies in children, one had straightened out within 6 months, seven within 12 months and a further 12 up to 24 months after surgery. The only recognizable reason for this difference was the patient's age depending on whether humeral growth was not yet completed. Marquardt's angulation osteotomy, however, is still the only surgical technique that improves humeral stump function, providing a rotation-stable humeral prosthesis and a free-moving shoulder joint.
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9,177,801
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Mechanical testing of the tension band wire fixation in the proximal femur.
|
The mechanical stability of proximal femoral osteotomies fixed by the tension band wire technique was studied in flexion-compression and torsion tests. The fixation consisted in crossing the section with two Kirschner wires and with a wire cerclage applied to the tension surface. The study was conducted in three steps. First, cyclinders of wood were cut either transversely or at 30 degrees of inclination in relation to the long axis of the specimen, and fixed with two Kirschner wires and a wire cerclage. We concluded that the inclination of the plane of section significantly increased the stability of fixation. No significant difference was observed when oblique sections were made in the reverse orientation. Second, 30 degrees subtrochanteric varus osteotomies were performed in dog femurs, so that the section plane was transverse in one group and oblique in another, after closing the osteotomy. In both groups the fixation was achieved by two Kirschner wires that crossed the osteotomy and a wire cerclage placed on the lateral cortex (tension surface). We concluded that inclination of the osteotomy plane increased the stability of osteosynthesis in bone specimens, as already seen with the wood pieces. Third, the stability of tension band wire fixation was compared with that provided by the AO/ASIF paediatric angled plate. Varus osteotomies (30 degrees) were created at the subtrochanteric level of paired dog femurs. On one side, the femur was fixed with Kirschner wires and a wire cerclage as described previously. For the other femur, the osteotomy was fixed with the angled plate. We found that both types of fixation presented the same stability in flexion-compression tests. However, under torsion the tension band wire fixation was 30%-50% less stable than the plate fixation.
|
9,177,803
|
Tibial avulsion fracture of the posterior cruciate ligament: K-wire or screw fixation? A retrospective study of 26 patients.
|
Although the posterior cruciate ligament (PCL) is not frequently injured, a greater understanding of its role in stabilizing the knee joint, mechanism of injury and treatment has developed. Isolated avulsion injuries constitute only a subgroup of PCL injuries, but nevertheless several operative techniques have been described for the fixation of the avulsed bony fragment. In order to investigate whether K-wire or screw fixation yields better long-term results, we examined 26 patients at an average of 10.5 years after the initial operation. Clinical examination, activity level, radiographic evaluation and instrumented measurements did not reveal any significant differences. All the patients had an excellent functional result. Thus, both K-wire and screw fixation are recommended for bony PCL avulsion injuries.
|
9,177,802
|
Biomechanical analysis of the effects of single high-dose vitamin D3 on fracture healing in a healthy rabbit model.
|
In a previous ultrastructural study, the benefit of a single high dose of vitamin D3 on fracture healing in a healthy animal model was demonstrated. This study examined the biomechanical consequences of applying a single high dose of vitamin D3 in a healthy rabbit model subsequent to femoral fracture. The fracture load, the values of energy absorbed until fracture and the flexural rigidity values of the vitamin D group were significantly higher than the corresponding ones of the control group in the case of fracture. On the other hand, for intact bones, those values did not differ significantly between the two groups. It was concluded that single high-dose vitamin D3 application had positive effects on fracture healing in a healthy animal model, as far as the parameters related to mechanical strength are concerned.
|
9,177,805
|
Instrumented measurement of anterior-posterior translation in knees with chronic anterior cruciate ligament tear.
|
Anteroposterior translation of the knee joint was measured with a Knee Signature System device on 12 women and 14 men with a unilateral, chronic, isolated, anterior cruciate ligament (ACL) tear. A control group with stable knees consisted of 10 women and 10 men. Anterior translation at 178 N load of the uninjured knees was 8.0 mm (+/-2.2 mm) and in knees with an ACL tear, 14.2 mm (+/-4.2 mm). Corresponding values for anteroposterior translation were 12.1 mm (+/-2.5 mm) and 19.3 mm (+/-4.9 mm), respectively. A difference of 3 mm or more in anteroposterior translation at 178 N load between injured and uninjured knees indicated an ACL tear with 85% specificity and 88% sensitivity.
|
9,177,806
|
High stability of the Ilizarov ringfixator in a metacarpal fracture of an Arabian foal.
|
In a case of I degree open multifragmentary metacarpal fracture of a 4-week-old Arabian foal, an osteosynthesis with the Ilizarov ringfixator was performed. Immediate full weight-bearing (100 kg) was possible, demonstrating the high stability of the Ilizarov ringsystem. After 12 weeks, sufficient bony union was achieved, and the fixator could be removed. At that time, the body weight of the foal was 170 kg. In our opinion, this case proves the high stability and efficiency of the ringsystem under difficult and unusual conditions.
|
9,177,804
|
Use of preoperative vascular embolisation in spinal metastasis resection.
|
Preoperative selective embolisation was carried out on 17 patients with spinal metastases from various primary tumours. There was a significant reduction in the blood loss (2088 ml) and infusion volume requirement (3500 ml) and more favourable postoperative haemoglobin (Hb) development compared with the non-embolised but otherwise identical control group. The reduced intraoperative bleeding manifested itself in the form of greater clarity and a less complicated intraoperative course. Particularly with a dorsal approach, the reduced bleeding permitted more exact preparation and more extensive tumour resection. Preoperative embolisation is thus a valuable aid in spinal metastasis resection. Given suitable indications and exact positioning of the embolising material, no significant complications should arise. The method as a whole calls for close collaboration between interventional radiologists and spinal orthopaedists.
|
9,177,807
|
Stabilization of an inserted tricalcium phosphate spacer enhances the healing of a segmental tibial defect in sheep.
|
The effect of inserting a tricalcium phosphate (TCP) spacer stabilized by a rigid or non-rigid fixation technique on the healing of segmental tibial defects of critical size was established. The osteotomized tibiae, 11 with and 8 without TCP spacers, were fixed by an external circular device in 11 mature sheep and by plates in 8 mature sheep, respectively. Healing was evaluated roentgenographically 16 weeks after the operation. Compared with the defects without TCP spacers, enhanced stability and healing were observed in the defects with TCP spacers under an identical external fixation. Furthermore, a significantly higher incidence of healing was obtained with plate fixation than with external device fixation in the TCP-implanted defects (P < 0.04). An abundant bridging callus was roentgenograpically demonstrated in most of the healed defects, but none in the unhealed defects. The TCP spacer with its mechanical integrity enhances the stability of external fixation, and the stable immobilization provided by rigid fixation is essential for osteoconduction of an inserted TCP spacer in the healing of segmental diaphyseal defects in sheep.
|
9,177,809
|
Experimental study of the hydrodynamic effects of irrigation suction drainage.
|
The function of irrigation suction drainage, as an additional method in the treatment of bone infection, is associated with hydrodynamic problems that can only be experimentally examined. The visualization of liquid movement in the experimental model was realized by adding a colored ink to the input drain of the drainage system. When only 2000 ml of liquid was used daily (80 drops/min), the ink penetrated the experimental cavity only through the most proximal holes in the input drain. It spread slowly and irregularly through the liquid in the experimental bottle, forming pocket deposits and leaving sediment. When 6000 ml of liquid was applied daily (240 drops/min), ink quickly penetrated the cavity through all holes in the drain, leading to turbulence and dispersing equally in the bottle. Decoloration of the model bottle was faster because the output drainage was more efficient. With an increased flow, i.e., with more liquid used in the drainage, a better result was achieved. This was manifested in a swift decoloration and rinsing of the cavity.
|
9,177,808
|
Multiple chop wounds in Hong Kong. An epidemiological study of an unusual injury.
|
Knife injuries can be classified into stabbing injuries and multiple laceration or multiple chops, the latter being much more common in Chinese communities. It is the mark of criminal gang attacks with their tendency to use long knives and choppers rather than guns. The intention is often to wound rather than kill. A survey of 89 cases revealed that 90% of the victims are men, with a mean age of 27 years; 75% was admitted to the hospital at night, and in 78% of the cases the assailants were persons unknown, or so we were told by the victims. The reasons for the attacks were also not given. Most of the women victims were assaulted by their spouse. Some 74% of the patients suffered three to six lacerations; 62% of the injuries were on the extensor surfaces of the upper limbs, while the hand and the back of the trunk were also common sites. The type of management differs from that for stabbing injuries. There were no fatalities, and less than half of the patients required blood transfusion. The average hospital stay was 6.2 days. The morbidity of these injuries involves damaged tendons and nerves.
|
9,177,811
|
Chondrosarcoma secondary to synovial chondromatosis. Report of two cases and a review of the literature.
|
Malignant transformation of synovial chondromatosis into chondrosarcoma is unusual. Thirteen cases and one series have been reported; only four of them developed in the hip. The overall survival is about 50%, possibly because of the difficulty of arriving at a correct early diagnosis (radiographically and histologically) and subsequent adequate surgical therapy. We report two patients (ages 30 and 50 years) in whom synovial chondrosarcoma developed in previously excised synovial chondromatosis of the hip. The diagnosis was made with modern imaging techniques (computed tomography and magnetic resonance imaging) and verified by open biopsy. The early recognition allowed a wide limb-saving resection; both patients are disease free 3 and 2 years after surgery.
|
9,177,810
|
Rupture of flexor tendons due to pisotriquetral osteoarthritis.
|
We are reporting a case of a flexor profundus tendon rupture of the little finger due to pisotriquetral osteoarthrosis. Resection of the pisiform bone and resurfacing of the carpal tunnel with the joint capsule was performed for the osteoarthrosis and free tendon grafting for the tendon rupture. The tendon grafting and a subsequent tenolysis restored the function of the little finger. Other than radiological changes in the pisotriquetral joint, the diagnostic value of preoperative radiocarpal arthrography was emphasized in this rare complication of pisotriquetral arthrosis.
|
9,177,812
|
Fracture of ossified Achilles tendon.
|
Ruptures of Achilles tendon are quite common. We here report a case of fracture in an ossified mass of Achilles tendon in an 84-year-old male patient. The mass occurred 78 years following surgery for bilateral equinus deformities of his ankle. Clinically, he had a palpably definite gap in the ossified area of the Achilles tendon, which was confirmed by a roentgenogram and a magnetic resonance scan of his left ankle. He was treated conservatively in a plaster cast for 12 weeks. The overall functional result a year after his treatment was quite satisfactory.
|
9,177,813
|
Reasoning requirements for diagnosis of heart disease.
|
Over the past dozen years, the Heart Disease Program (HDP) has been developed to assist physicians in reasoning about cardiovascular disorders. Driven by several evaluations, the inference mechanism has progressed from a logic based model, to a Bayesian Probability Network (BPN) and finally a pseudo-Bayesian network with temporal and severity reasoning. Though aspects of cardiovascular reasoning are handled well by BPNs, temporal reasoning, homeostatic feedback mechanisms and effects of disease severities require additional inference strategies. This article discusses how these reasoning problems are handled, and deals with closely linked issues in building the user interface to collect detailed cardiovascular data and provide clear explanations of diagnoses.
|
9,177,814
|
Diagnosing congenital heart defects using the Fallot computational model.
|
This paper describes a computational model developed for the diagnosis of multiple defects. If multiple defects interact, meaning that the cues observable for multiple defects are not a sum of the cues observable for the component defects, diagnosis is particularly difficult. We developed a description and classification of the ways cues change when defects interact. A computational model (named Fallot) was implemented and a knowledge-base was constructed for the diagnosis of congenital heart defects. On each case, Fallot performs recognition-based reasoning followed by solution construction and evaluation with the cue combination methods. Fallot was tested on cases from hospital files and correctly diagnoses cases with multiple interacting defects for which conventional methods are not applicable or fail.
|
9,177,815
|
Spatio-temporal reasoning for multi-scale modeling in cardiology.
|
In this paper, we present an overview of the CARDIOLAB environment where the heart's electrical activity is modeled at distinct space and time scales. CARDIOLAB uses three models, two of which are based on cellular automata, and the third on qualitative simulation. They are combined around a blackboard for multi-scale quantitative and qualitative modeling of cardiac electrical activity. A general account on how spatio-temporal representation and reasoning methods are applied to produce heuristic associations is also presented.
|
9,177,816
|
DIAVAL, a Bayesian expert system for echocardiography.
|
DIAVAL is an expert system for the diagnosis of heart diseases, including several kinds of data, mainly from echocardiography. The first part of this paper is devoted to the causal probabilistic model which constitutes the knowledge base of the expert system in the form of a Bayesian network, emphasizing the importance of the OR gate. The second part deals with the process of diagnosis, which consists of computing the a posteriori probabilities, selecting the most probable and most relevant diagnoses, and generating a written report. It also describes the results of the evaluation of the program.
|
9,177,817
|
An expert system for diagnosis of acute myocardial infarction with ECG analysis.
|
Coronary heart disease is one of the most prevalent and costly health care problems in the world. The early and accurate diagnosis of coronary heart disease is a major problem in emergency settings. However, many primary and secondary hospitals and primary emergency units lack cardiologists on call which makes the diagnosis difficult. This paper describes an expert system for diagnosis of acute myocardial infarction developed to aid physicians without cardiology specialization. Our main goal was to develop an expert system that assists in the diagnosis and indicates the need of hospitalization in a coronary unit.
|
9,177,818
|
Allergic reactions to phenytoin in a general hospital in Singapore.
|
Phenytoin is used for the treatment and prevention of fits. We investigated all patients reported to have phenytoin allergy in our hospital and found 42 confirmed cases. Sixty-nine percent were female and 83.3% were Chinese. The mean age of the patients was 46.5 years. The reactions reported were maculopapular rash (71.4%), Stevens-Johnson syndrome (14.3%), fever (4.8%), generalized exfoliative dermatitis (2.4%), toxic epidermal necrolysis (2.4%), vasculitis (2.4%) and agranulocytosis (2.4%). In conclusion, the majority of reported allergic reactions to phenytoin were cutaneous (92.9%) and one fifth of these were potentially life-threatening.
|
9,177,819
|
Ketotifen inhibits allergen-specific T lymphocytes' responses by suppressing antigen presentation with concomitant decrease of HLA-DQ antigen on macrophages.
|
Allergen activates T lymphocytes responsive to interleukin 2 (IL-2) in allergic patients but not in normal individuals. This response was suppressed by anti-allergic agent, Ketotifen (4-(1-methyl-4-piperidylidene)-4H-benzo [4, 5] cyclohepta [1, 2-b] thiophen-10 (9H)-one hydrogen (fumarate). Prolonged culture of antigen-presenting adherent cells impaired the ability to present Dermatophagoides farinae (Df) antigen to T cells, whereas stimulation of adherent cells with recombinant interferon-gamma (IFN-gamma) restored the antigen-presenting capability. The maintained antigen presenting ability of adherent cells treated with IFN-gamma was also suppressed by Ketotifen. Fluorescence activated cell sorter (FACS) analysis disclosed that Ketotifen selectively reduced the expression of HLA-DQ antigen, crucial restriction elements in Df antigen-related responses, on macrophages but not on B cells, even in the presence of IFN-gamma. Collectively, Ketotifen prevented macrophages from inducing allergen-activated T lymphocytes' responsiveness to IL-2 at least in part by decreasing the expression of HLA-DQ antigen.
|
9,177,820
|
The abnormalities of nailfold capillaries in scleroderma as assessed by video image analysis and photomicroscopy.
|
Scleroderma is a systemic connective tissue disease in which the diagnosis in supported by morphological changes in nailfold capillary size and density. These changes are open to observer bias. In this paper we describe 2 objective methods that allow quantitative definition of capillary changes, video image analysis (VIA) and photomicroscopy. VIA was used to assess 15 healthy control subjects and 22 patients with scleroderma. Scleroderma patients had a significantly larger capillary diameter (43 microns versus 20 microns, p = 0.0001) and capillary density was reduced by a mean factor of 0.5. Image stored on computer will facilitate serial assessments of nailfold capillary changes and possibly provide information on disease progression.
|
9,177,821
|
Comparison between dual and tri-colour reagents for the analysis of lymphocyte subsets.
|
The aim of this project was to compare dual and tri-colour reagents for lymphocyte immunophenotyping. A total of 37 patient and normal specimens were immunophenotyped concurrently with the following mean values (% dual vs tri-colour): CD3 (69.4 vs 68.3) CD4 (24.0 vs 24.2) and CD19 (13.9 vs 12.6). A comparison of the results obtained using the paired t test showed that there were no significant differences for cells expressing CD3, CD4 and CD19. However, there was a significant difference in the NK (18.3 vs 16.3) cell component. A major advantage in using 3 colour immunophenotyping is the ability to analyse specimens that cannot be analysed using dual colour reagents due to debris or contamination of the gate with non-lymphocytic cells.
|
9,177,822
|
A study of cell-mediated immune response to pancreatic antigens in patients with fibrocalculous pancreatic diabetes.
|
In order to investigate whether there was any association between autoimmunity to pancreatic antigens with FCPD as well as IDDM, cell-mediated immune response to pancreatic antigens was studied by lymphoproliferation assay in 7 FCPD, 17 IDDM, 33 NIDDM patients and 102 normal controls. Optimal pancreatic antigen concentrations used were 100, 150 and 200 micrograms/ml. Positive results were considered for each concentration of antigens tested, at stimulation index (SI) > (mean +/- 2 SD) SI obtained from normal age-matched controls with the use of the corresponding concentration of antigen. The one who gave positive result with any of these optimal antigen concentrations was considered to be the responder to pancreatic antigens. With this criterion, the responders were found to be 3/7 (42.9%) FCPD, 6/17 (35.3%) IDDM and 6/33 (18.2%) NIDDM patients; while there were 11 of all 102 (10.8%) normal controls.
|
9,177,823
|
Production of mouse anti-CD4 antibodies by DNA-based immunization.
|
The intramuscular injection of plasmid DNA encoding an antigenic protein has been developed recently as a tool for immunization. DNA-based immunization was shown to generate immune responses against the encoded antigen in diverse animal species. In this report, we present the use of DNA-based immunization for the production of antibodies to CD4, a human leukocyte surface molecule. Mice were injected intramuscularly with eukaryotic expression vector containing cDNA encoding CD4 protein, termed CD4-DNA, and were subsequently assayed for anti-CD4 antibody production by indirect immunofluorescence. Sera collected from 2 of 3 inoculated mice reacted with CD4 expressing transfected COS cells and Sup-T1 cells. Anti-CD4 antibody activity was abolished by adsorption with CD4 molecule expressing cells. CD4+ cell depleted lymphocytes were also used to confirm the specificity of the anti CD4 antibodies present in immune serum. CD4-DNA immune serum reacted with approximately 1/3 of freshly isolated lymphocytes but to very few cells in the CD4+ cells-depleted preparation. CD4-DNA immunized sera was used to enumerate CD4+ cells in the peripheral blood of 6 healthy donors and 2 AIDS patients. The number of CD4+ cells estimated by DNA immunized sera was very similar to estimates using standard anti-CD4 monoclonal antibody Leu3a. DNA-based immunization is therefore capable of raising antibodies to human leukocyte surface antigens. This technology may be useful for producing antibodies to other cell surface antigens in mice or other animals.
|
9,177,824
|
Immunohistochemical characterization of a new monoclonal antibody reactive with dengue virus-infected cells in frozen tissue using immunoperoxidase technique.
|
This paper presents a novel monoclonal antibody shown to react with cytoplasmic antigens in various dengue infected human frozen organs from autopsy and necropsy specimens. Strong reactivity was found in hematopoietic cells, including immunoblasts, lymphocytes, plasma cells and macrophages of spleen, lymph node, lung, kidney and stomach. Strikingly, strong positivity was demonstrated in cerebral cortex neurones, Purkinje cells, choroid plexus and blood vessels in addition to astrocytes and microglia. Neurotropism of the virus could explain the meningitis, encephalitis, mononeuropathy and polyneuropathy observed by direct toxicity, but noted especially after an activation of mononuclear phagocytes and amplification of the immune response with subsequent vascular inflammation and formation of immune complexes.
|
9,177,825
|
AIDS--associated Kaposi's sarcoma: a rare entity at Maharaj Nakorn Chiang Mai Hospital.
|
Kaposi's sarcoma [KS] is rare in Asian countries. Since the AIDS epidemic, KS has become the most common AIDS-related cancer reported in the international literature. Up to March 1996, 4 cases of AIDS-associated KS were histologically documented at the registry at the Maharaj Nakorn Chiang Mai University Hospital, comprising 2 adult and 2 pediatric male patients. Routes of HIV exposure included intravenous injection and heterosexual contact in adult cases, and perinatal transmission and blood transfusion in the pediatric ones. KS was present as an AIDS diagnostic condition in one of the adults and in both children. In our institution, KS was second in frequency to malignant lymphoma among AIDS patients. Predomination of non-homosexual transmission of HIV infection in this region was probably a factor associated with the rarity of AIDS-associated KS.
|
9,177,826
|
V3 peptide enzyme immunoassay for serotyping HIV-1 infected pregnant Thais.
|
Previous molecular epidemiological studies show that at least 2 subtypes of HIV-1 circulate in Thailand. HIV-1 subtype B or Thai genotype B was associated with an early epidemic and was prevalent in intravenous drug users. Meanwhile, HIV-1 subtype E or Thai genotype A was becoming widespread among heterosexuals. We studied the HIV subtypes of 161 HIV-1 seropositive pregnant women. Of these, 143 pregnant patients (88.8%) tested positive for subtype E alone and 8 women (5.0%) had evidence of infection with subtype B alone. There was serologic evidence of infection with a mixture of subtypes in 7 women while the infecting subtype could not be identified in the remaining 3 women. This result agrees with previous information that subtype E predominates in Thai heterosexuals.
|
9,177,827
|
The prevalence of antinuclear, anti-dsDNA, anti-Sm and anti-RNP antibodies in a group of healthy blood donors.
|
We studied the prevalence of antinuclear (ANA), anti-double stranded DNA (dsDNA), anti-Sm and anti-RNP antibodies in a group of 93 blood donors (age range: 18-58 years). Antinuclear and anti-ds DNA antibodies were detected by immunofluorescence (IF) using HEp2 cells and Crithidia luciliae as substrates, respectively, while anti-Sm and anti-RNP antibodies were assayed by ELISA. ANA was found in 6.5% while anti-dsDNA antibodies were not detected in any of the subjects. The 98th percentile was used as cut off where values greater than 0.651 for anti-Sm and 0.601 for anti-RNP antibodies were taken to be positive. This gives a frequency of 1.1% for both antibodies. There was no significant association of antibody positivity with sex or race. We conclude that certain autoantibodies are present in low titres in the normal Malaysian Individuals, at a different frequency compared to other studies probably due to genetic, ethic or environmental factors.
|
9,177,828
|
Prevalence of anti-varicella zoster IgG antibody in undergraduate students.
|
Sera from 74 healthy Thai undergraduate students, mean age 21 + 1.7 years, were tested for the presence of IgG antibody against varicella zoster virus (anti-VZV IgG) by ELISA. Fifty-five of 74 (74.3%) individuals possessed anti-VZV IgG antibody. The presence of anti-VZV IgG was associated with a past history of varicella (p < 0.005, X2 = 33.4989). No sexual preponderance was observed. We therefore found that 1 of 4 Thai young adults was susceptible to VZV infection.
|
9,177,829
|
Syntheses of haptens containing dioxaphosphorinan methoxyacetic acid linker arms for the production of antibodies to organophosphate pesticides.
|
Four generic heterobifunctional reagents, namely 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid methyl ester, p-sulfide, 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid methyl ester, p-oxide, 2-(2-mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid bispotassium salt, p-sulfide-, and (2-methoxy-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid, methyl ester, have been synthesized and used to prepare organophosphate, thiophosphate, and dithiophosphate haptens containing a functional carboxyl group which can be used to conjugate the haptens to proteins. These hapten-protein conjugates have been used as antigens for preparing polyclonal sera against all classes of organophosphate pesticides. The eight examples used protein-hapten conjugates of chlorpyrifos, parathion, diazinon, paraoxon, azinphos, dimethoate, demeton, and dichlorvos. These were all immunogenic and resulted in sera containing antibodies that recognized the corresponding parent pesticide with high specificity.
|
9,177,830
|
DNA binding and cleavage by a cationic manganese porphyrin-peptide nucleic acid conjugate.
|
A cationic manganese porphyrin-peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage experiments were performed with a 247-base pair restriction DNA fragment containing a 10-base pair homopurine binding target for the PNA. Oxidative activation by this Mn porphyrin-PNA conjugate leads to sequence specific, 3'-staggered cleavage of both DNA strands near the strand displacement junction. Furthermore, the Mn porphyrin-PNA porphyrin conjugates bind over 100-fold better to double-stranded DNA compared to the native PNA.
|
9,177,833
|
Radiolabeling of epidermal growth factor with 99mTc and in vivo localization following intracerebral injection into normal and glioma-bearing rats.
|
High grade gliomas may have amplified expression of the epidermal growth factor receptor (EGFR) gene c-erb-B, which often is associated with increased expression of transmembrane EGFR. The purpose of the present study was to develop a method for labeling EGF with 99mTc and to determine whether the resulting radioligand would localize, following intracerebral injection, in rats bearing EGFR-positive gliomas. EGF has a relatively low molecular mass (approximately 6 kDa) compared to monoclonal antibodies, and this has allowed smaller bioconjugates, which should diffuse more rapidly within the brain and more effectively target disseminated glioma cells, to be constructed. In the present study, EGF has been labeled with either 131I or 99mTc, and in vitro uptake of the resulting radioligand has been investigated using C6EGFR rat glioma cells, which had been transfected with the EGFR gene. Cellular uptake of 131I radioactivity peaked after approximately 30 min of incubation with [131I]EGF, following which time it declined, while 99mTc radioactivity continued to increase over a 6 h incubation with [99mTc]-EGF. To determine if radiolabeled EGF had in vivo tumor-localizing properties, C6EGFR glioma cells were implanted stereotactically into the brains of Fischer rats. Four weeks later, either 99mTc- or 131I-labeled EGF was injected intracerebrally into normal or glioma-bearing animals using the same stereotactic coordinates. External gamma scintigraphy revealed that 131I radioactivity disappeared rapidly from the brain regions of tumor-bearing animals compared to 99mTc, approximately 50% of which remained in the tumor for up to 12 h. In contrast, only approximately 20% remained in the brains of non-tumor-bearing animals after 6 h. These studies are the first to describe a method for radiolabeling EGF with 99mTc and to detect it by external scintigraphy in the brains of tumor-bearing animals.
|
9,177,831
|
Nucleosides and nucleotides. 160. Synthesis of oligodeoxyribonucleotides containing 5-(N-aminoalkyl)carbamoyl-2'-deoxyuridines by a new postsynthetic modification method and their thermal stability and nuclease-resistance properties.
|
Heptadecadeoxynucleotides containing 5-(N-aminoethyl- or N-aminohexyl)carbamoyl-2'-deoxyuridines (E or H) were synthesized using a newly developed postsynthetic modification method. As a convertible nucleoside unit, 5-methoxycarbonyl-2'-deoxyuridine (1) was initially incorporated into oligodeoxynucleotides (ODNs) according to the phosphoramidite method at various positions using a DNA synthesizer. Fully protected ODNs attached to a solid support were treated with alkyldiamines such as ethylenediamine and 1,6-hexanediamine to give the above modified ODNs. The thermal stability, resistance toward nuclease digestion, and stability in fetal calf serum of the modified ODNs were studied. An increase in the number of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (H) in the ODNs was found to effectively stabilize duplex formation with both the corresponding complementary DNA and RNA and protect against nucleolytic hydrolysis by snake venom phosphodiesterase. In particular, the half-life of ODN 19, which contained four H residues, was about 162 h in the presence of the nuclease. Furthermore, 19 was also stable in medium containing 10% fetal calf serum with a t1/2 of about 48 h, while t1/2 for the corresponding unmodified ODN was 13 min.
|
9,177,832
|
Synthesis of conjugates for a barbiturate screening assay.
|
Novel derivatives of barbiturates functionalized with free carboxylic acids were designed and synthesized. Coupling of 5-cyclopentyl-5-carboxycrotylbarbituric acid via its active ester to an aminofluorescein derivative produced a fluorescent tracer. Conjugation of the 5-cyclopentenyl-5-carboxyethylbarbituric acid via its mixed anhydride to thyroglobulin allowed for subsequent development of a polyclonal antibody which was evaluated for binding in a fluorescence polarization immunoassay format with various barbiturates. The binding studies showed good cross-reactivity of a variety of barbiturates containing both aromatic and aliphatic 5-substituents with the tested antisera. The relationship between the immunogen architecture, the chemical structure of the binding analytes, and the characteristics of the antisera is also presented.
|
9,177,834
|
Synthesis of a new photoreactive derivative of dipyridamole and its use in the manufacture of artificial surfaces with low thrombogenicity.
|
Photoimmobilization of dipyridamole (Persantin) was accomplished through the use of a new synthetic conjugate molecule, 1. Persantin is a powerful inhibitor of platelet activation and aggregation and is widely used as a vasodilator. Conjugate 1 consists of triply protected dipyridamole [three of the four hydroxyl groups carry a tert-butyldimethylsilyl (TBDMS) protective group) and the photoreactive 4-azidobenzoyl group. A short hydrophilic spacer chain, derived from triethylene glycol, separates the protected dipyridamole system and the photoreactive group. Compound 1 was immobilized on polyurethane sheets (Pellethane D-55) through irradiation with ultraviolet (UV) light, and the protective groups were removed afterward. The resulting modified polyurethane surfaces were characterized by different physicochemical techniques: UV extinction, contact angle measurements (captive bubble technique), and X-ray photoelectron spectroscopy (XPS). The UV extinction measurements showed the presence of 13 +/- 1 nmol of immobilized dipyridamole/cm2. The contact angle measurements revealed that the modified surface was markedly more hydrophilic than the control (i.e. unmodified polyurethane). XPS measurements clearly established the presence of immobilized dipyridamole in the outermost layers of the modified surface. This was especially clear from the XPS spectra recorded at a low take-off angle (approximately 6 degrees). Furthermore, the XPS spectra showed that the TBDMS protective groups had been quantitatively removed during the deprotection/washing treatment. The in vitro blood compatibility of the modified surface was studied with the thrombin generation assay as developed in our group, as well as with scanning electron microscopy. The thrombin generation test produced a lag time of 1275 s for the modified surface, as opposed to 569 s for the control. Scanning electron microscopy showed that far fewer platelets adhere to the modified surface (approximately 7 x 10(3)/mm2) as compared to the control (approximately 6 x 10(2)/mm2). Taken together, the experimental data reveal that the modified surface has excellent blood compatibility in vitro. It is discussed that the use of conjugate 1 leads to simultaneous exposure of dipyridamole at the modified surface and to a marked increase of the surface hydrophilicity, which is likely to hamper adsorption of plasma proteins. The combination of these effects is uniquely related to the molecular buildup of 1. Conjugate 1 will be used in future work that is aimed at preparing small-caliber polyurethane vascular grafts with a blood compatible lumenal surface.
|
9,177,836
|
Critical parameters for adduct formation of the carcinogen (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide with oligonucleotides.
|
Various parameters relevant for the formation of dG adducts produced in the reaction of individual benzo[a]pyrene diol epoxide (BPDE) stereoisomers with oligonucleotides have been studied. Reaction time, temperature, pH, molar ratio of diol epoxide and oligonucleotide, base sequence, and buffer system were shown to affect the amount of (+)-anti-BPDE dG adducts formed. Optimum experimental conditions for dG adduct formation were different depending on the base sequence context of the oligonucleotide employed [5'-d(CCTATAGATATCC) or 5'-d(CCTATTGCTATCC)]. In general, low temperature to allow a longer reaction time, slightly alkaline Tris-HCl (pH 7.5-8.0) or alkaline phosphate buffer (pH 11), low concentration of organic solvent, and a molar excess of (+)-anti-BPDE promote dG adduct formation with an oligonucleotide. Low incubation temperature and Tris-HCl buffer also favor dG adduct formation of (-)-anti-BPDE and both enantiomers of syn-BPDE to both 5'-d(CCTATAGATATCC) and 5'-d(CCTATTGCTATCC).
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9,177,840
|
Synthesis and characterization of rhenium-complexed alpha-melanotropin analogs.
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Receptor binding peptides labeled with medically important radionuclides such as technetium and rhenium are an important tool for the imaging and treatment of many forms of cancer. This paper describes a method of labeling peptides with rhenium using a natural amino acid chelating moiety. The structural characteristics of this chelate moiety, N-acetyl-cysteine-glycine-cysteine-glycine (NAc-CGCG) complexed with nonradioactive rhenium, have been investigated. The stability of this peptide-metal complex has been evaluated on the tracer level using radioactive rhenium-186. The rhenium-bound peptide has been appended to the N termini of receptor binding alpha-melanocyte stimulating hormone (alpha-MSH, NAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) fragments via solid phase peptide synthesis. Bioassays and receptor binding studies of the resulting complexes demonstrate that the fragments retained biological activity and exhibited receptor binding constants ranging from 0.3 to 1.1 nM. This method could provide a general means of labeling bioactive peptide fragments that would simplify product purification and characterization.
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9,177,837
|
A 15-base acridine-conjugated oligodeoxynucleotide forms triplex DNA with its IL-2R alpha promoter target with greatly improved avidity.
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Attachment of 6,9-diamino-2-methoxyacridine to the 5' end of a purine-rich oligodeoxynucleotide targeting a 15 bp oligopurine oligopyrimidine stretch in the promoter region of the interleukin-2 receptor alpha chain (IL-2R alpha) gene results in an approximately 500-fold increase in its triplex forming avidity as determined by both band shift assay and DMS footprinting (Kd lowered from 2.5 microM to 5 nM). This oligonucleotide participates in Mg(2+)-dependent three-stranded DNA formation in which it is oriented antiparallel relative to the purine strand of the target duplex as determined by acridine moiety sensitized photoreactivity with the target duplex DNA. The oligonucleotides used in these studies were synthesized with a 3-amino-2-hydroxypropyl group at the 3' end to protect against exonucleolytic degradation for future in vivo applications. The 3'-amino group underwent partial removal, probably during the NaOH deprotection step. Both the 3'-amino and the 3'-free forms of the oligo have the same binding avidity and specificity. The interaction of the third strand with its target is sequence specific and can be essentially abolished by a point G-->T transversion 4 bases away from the 3' end of the target oligopurine block or severely reduced by other mutations within the target duplex. Thus, the attachment of the acridine moiety to the 5' end of the oligonucleotide does not seem to substantially compromise the sequence specificity of binding. Additionally, the oligonucleotide composed of G and A nucleotides was found to be superior to the oligonucleotide containing G and T residues since the difference in avidity of binding to the same target site was 17-fold.
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9,177,839
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Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells.
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Bispecific antibodies that bind to a tumor antigen and the T cell receptor (TCR) redirect cytotoxic T lymphocytes (CTL) to lyse tumor cells which have escaped normal immune recognition mechanisms. One well-characterized tumor antigen, the folate receptor (FR), is expressed on most ovarian carcinomas and some types of brain cancer. Recently, it was shown that conjugates of folate and anti-TCR antibodies are extremely potent bispecific agents that target tumor cells expressing the high-affinity folate receptor, but not normal cells expressing only the reduced folate carrier protein. In this paper, it is shown that the size of these conjugates can be reduced to the smallest bispecific agent yet described (30 kDa) by attaching folate to a single-chain antibody, scFv, of the anti-TCR antibody KJ16. The scFv/folate conjugates are as effective as IgG/folate conjugates in mediating lysis of FR4 tumor cells by CTL. The optimal folate density was in the range of 5-15 folate molecules per scFv or IgG molecule, which yielded half-maximal lysis values (EC50) of approximately 40 pM (1.2 ng/mL for scFv). Finally, the scFv/folate conjugates could efficiently target tumor cells even in the presence of free folic acid at concentrations that are normally found in serum. Compared to conventional bispecific antibodies, the small size of scFv/folate conjugates may prove advantageous in the ability to penetrate tumors and in reduced immunogenicity.
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9,177,843
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Regiospecific solid-phase synthesis of branched oligonucleotides. Effect of vicinal 2',5'- (or 2',3'-) and 3',5'-phosphodiester linkages on the formation of hairpin DNA.
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A general procedure for the solid-phase regiospecific synthesis of branched oligonucleotides (bNA) analogues using readily available phosphoramidite reagents has been developed. The key feature of this method is use of the solid-phase phosphoramidite procedure to assemble linear oligonucleotide sequences and sequential removal of the phosphate (beta-cyanoethyl or methyl) and silyl protecting groups without detaching the nascent oligonucleotide from the solid support. Conversion of the phosphate backbone into the more stable phosphodiester linkages allows for removal of the 2'-O-tert-butyldimethylsilyl protecting group without cleavage or isomerization at the branch point. This method allows for the formation of branched oligonucleotides with sequences of arbitrary base composition, length, and orientation around the branch point junction, including a "Y"-shaped octadecamer d(TACTA)-rA[2',5'd(GTATGT)]3',5'd(CAAGTT). Studies to explore structural effects in the use of a branched adenosine as replacement for nucleotide loops in duplex and triplex DNA are also described. Branched oligonucleotides of the type rA[2',5'dCndA10-5']3',5'dCndT10-3' and rA[2',5'dCn3',3'dA10-5']3',5'dCnT10-3' form hairpin duplexes with thermal stability comparable to or better than that of one with a natural deoxynucleotide loop.
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9,177,841
|
Kinetic analysis of sequence-specific alkylation of DNA by pyrimidine oligodeoxyribonucleotide-directed triple-helix formation.
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Attachment of a nondiffusible bromoacetyl electrophile to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence-specific alkylation of a guanine base in duplex DNA two base pairs to the 5'-side of a local triple-helical complex. Products resulting from reaction of 5'-ETTTTMeCTTTTMeCMeCTTTMeCTTTT-3' at 37 degrees C with a 29 base pair target duplex are determined by a gel mobility analysis to be oligonucleotides terminating in 5'- and 3' -phosphate functional groups, consistent with a mechanism involving alkylation, glycosidic bond cleavage, and base-promoted strand cleavage. The guanine-(linker)-oligonucleotide conjugate formed upon triple-helix-mediated alkylation at the N7 position of a guanine base in a 60 base pair duplex was identified by enzymatic phosphodiester hydrolysis of the alkylation products followed by reversed phase HPLC analysis. To determine the rate enhancement achieved by oligonucleotide-directed alkylation of duplex DNA, a comparison of rates of alkylation at N7 of guanine in double-stranded DNA by the N-bromoacetyloligonucleotide and 2-bromoacetamide was performed by a polyacrylamide gel assay. The reaction within the triple-helical complex on a restriction fragment was determined at 200 nM N-bromoacetyloligonucleotide to have a first-order rate constant k1 of (2.7 +/- 0.5) x 10(-5) S(-1) (t1/2 = 7.2 h). The reaction of 2-bromoacetamide with a 39 base pair duplex of sequence corresponding to the restriction fragment targeted by triple-helix formation was determined to have a second-order rate constant k2 of (3.6 +/- 0.3) x 10(-5) M(-1) S(-1). A comparison of the first-order and second-order rate constants for the unimolecular and bimolecular alkylation reactions provides an effective molarity of 0.8 M for bromoacetyl within the triple-helical complex.
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9,177,845
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Synthesis of new d-propoxyphene derivatives and the development of a microparticle-based immunoassay for the detection of propoxyphene and norpropoxyphene.
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The synthesis of [S-(R,S)]-4-[[methyl[2-methyl-3-(1-oxopropoxy)-3, 4-diphenylbutyl]amino]-1-oxobutoxy]-2,5-pyrrolidinedione+ ++ (propoxyphene active ester, 2) is described. This was used as an intermediate to prepare a propoxyphene immunogen, [S-(R,S)]-4-[methyl][2-methyl-3-(1-oxopropoxy)-3,4-diphenylbuty l]-amino]- 1-oxobutyl-Bovine Thyroglobulin (3). This immunogen was then used to generate antibodies which demonstrate good cross-reactivity to d-propoxyphene, d-norpropoxyphene, and other propoxyphene metabolites. In addition, these antibodies were shown to have very low cross-reactivity to methadone, a structurally related compound. The introduction of an aminomethyl benzoate spacer into the propoxyphene active ester (2), followed by the activation of the carboxylic acid, provided for a more stable active ester (5). This stable active ester, together with the antibodies generated from the propoxyphene immunogen, has led to the development of an immunoassay based on the Kinetic Interaction of Microparticles in Solution (KIMS).
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9,177,835
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99mTc-labeled sigma-receptor-binding complex: synthesis, characterization, and specific binding to human ductal breast carcinoma (T47D) cells.
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sigma-Receptors have recently been shown to be expressed in a variety of human tumor cells. In an attempt to prepare 99mTc chelates that would bind to sigma-receptors and be useful for imaging sigma-receptor-positive tumors, we have synthesized and characterized a bisaminothiol (BAT) chelate appended with a sigma-receptor pharmacophore. The synthesis of target ligand VII was accomplished in three steps starting from bicyclic imidazolidino[1,2-d]dithiazapine. The labeling of the BAT ligand with 99mTc was carried out in high yields (> 80%) using stannous tartarate as a reducing agent, resulting in the target sigma-receptor-binding chelate [99mTc]BAT-EN6, III. Similarly, 99gTc chelate with ligand VII was prepared from ammonium pertechnetate by reduction with stannous tartarate. 99nTc-radiolabeled chelate was purified by reversed phase HPLC, and cell binding with human breast ductal carcinoma (T47D) was performed. A high degree of specific binding (90-97%) was obtained when sigma-receptor ligands such as halogenated phenylethylenediamines were used to determine nonspecific binding. A modest affinity dose-dependent inhibition of binding was found with BD1008, I, and 4-IPEMP, II (IC50 = 47 +/- 2 and 59 +/- 5 nM, respectively), known sigma-ligands. No specific binding was found with [99mTc]BAT, VIII [without appended sigma-pharmacophore (N-alkyl-substituted ethylenediamine)], showing that biological activity resulted from the pendent pharmacophore. 99gTc complex was found to be a potent inhibitor (Ki = 42.7 +/- 8.5 nM) of [3H]DTG binding in guinea pig brain membranes. Scatchard analysis of [99mTc]BAT-EN6 (spiked with [99gTc]BAT-EN6) binding in T47D breast cancer cells showed a saturable binding, with a Kd of 43.5 +/- 14.7 nM and a Bmax of 3121 +/- 130 fmol/(mg of protein). A biodistribution study of [99mTc]BAT-EN6 chelates in Sprague Dawley rats showed hepatic clearance, as expected. A blocking study at 4 h postinjection using 2 mumol of BD1008 with [99mTc]BAT-EN6 showed a significant decrease of radiopharmaceutical in liver (15.32 vs 22.31% ID/organ) and kidney (1.01 vs 2.21% ID/ organ), organs known to possess high concentrations of sigma-receptors. These results imply that [99mTc]BAT-EN6 binds with high affinity to sigma-receptors expressed in human breast tumor cells, and it may be useful for imaging breast cancer.
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9,177,842
|
Biodistribution and catabolism of Ga-67-labeled anti-Tac dsFv fragment.
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The disulfide-linked fragment (dsFv) of the antibody to the alpha subunit of the IL2 receptor has been radiolabeled with a [Ga-67] Ga-2-(p-SCN-Bz)-NOTA derivative linked through an isothiocyanato group to either the epsilon-amino group of lysine or the alpha-amino group of the N-terminal amino acids. This low molecular weight protein (LMWP) has been proposed as a tumor diagnostic agent. However, > 60% of the injected dose localized in the mouse kidney. The major catabolites (> 95%) in the kidney were identified as the Ga-2-(p-SCN-Bz)-NOTA conjugate with either lysine or methionine, with no evidence of transchelation of Ga-67. Since different amino acids in the dsFv were radiolabeled according to this procedure, it was possible to study the relative residence times of the various catabolites. The methionine conjugate had a significantly shorter residence time than the lysine conjugate in the same kidney. Labeling the appropriate amino acid in a LMWP may lead to reduced residence times and increased diagnostic or therapeutic ratios.
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9,177,838
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New coupling reagents for the preparation of disulfide cross-linked conjugates with increased stability.
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To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.
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9,177,844
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Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.
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Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.
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9,177,848
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Preparation, characterization, and biological evaluation of technetium(V) and rhenium(V) complexes of novel heterocyclic tetradentate N3S ligands.
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Various tetradentate N3S ligands which contain pyridyl, morpholino, or imidazolyl moieties were prepared and labeled with technetium and rhenium. Metal complexation of the ligands occurred efficiently over the pH range from 2 to 11. Ligands possessing the S-THP (tetrahydropyranyl)-protected mercapto group labeled efficiently even under alkaline conditions, and among the three types of heterocyclic metal complexes, a marked difference in stability was observed; rhenium complexes decomposed to ReO4 whereas technetium complexes decomposed to TcO2/TcO4. In general, imidazolyl complexes of both technetium and rhenium were very stable in saline; less than 10% decomposition after 24 h. The technetium histidyl complex and technetium pyridyl complex were quite stable even under cysteine challenge; less than 10% decomposition after 24 h. The rhenium and technetium morpholino complexes were very unstable; greater than 10% decomposition after only 1 h in saline and greater than 25% decomposition in 1 h under cysteine challenge. Profound pharmacokinetic differences among these metal complexes were also observed in rat biodistribution studies. The neutral pyridyl complexes exhibited high blood and liver uptake and slow clearance from these tissues. The replacement of a hydroxyl group by a carboxyl group, which resulted in an anionic complex at physiological pH, resulted in a dramatic decrease in blood and liver uptake. The neutral imidazolyl complex exhibited marked reduction in blood uptake and much faster clearance from blood and liver compared to the neutral pyridyl complex. Finally, the anionic histidyl complex, which contains both the imidazolyl and carboxyl groups, had the most favorable pharmacokinetic properties in that it exhibited very low blood, liver, and kidney uptakes and a rapid clearance from the body via the renal system. The combination of the high stability and favorable pharmacokinetic properties of the imidazolyl complexes should render them useful for targeted delivery of the medically important isotopes.
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9,177,846
|
Structural determination of the conjugate of human serum albumin with a mitomycin C derivative, KW-2149, by matrix-assisted laser desorption/ionization mass spectrometry.
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A new mitomycin C derivative, KW-2149, is known to form a covalent conjugate with human serum albumin (HSA). This conjugate exhibits 1/20 of the anticellular activity of unconjugated KW-2149. Structural studies of this conjugate were carried out using a combination of enzymatic digestion, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The tryptic peptide T5 (residues 21-41) was the only peptide found to be modified by KW-2149 moieties, the [(gamma-L-glutamylamino)ethyl]thio group or the (2-aminoethyl)thio group, through a disulfide bond. Although the latter peptide lost its mitomycin C moiety in the course of tryptic digestion, these data strongly suggest that KW-2149 was bound to Cys-34, the only free cysteine on HSA.
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9,177,854
|
Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes.
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This study directly compared the behaviour of chondrocytes in porous matrices comprising different collagen types and different pore diameters. There was a dramatic difference in the morphology of the cells in the type I and type II collagen matrices. The cells in the type II collagen matrix retained their chondrocytic morphology and synthesized glycosaminoglycans, while in the type I matrix the chondrocytes displayed a fibroblastic morphology with less biosynthetic activity than those in the type II. Small pore diameter affected morphology initially in the type I matrices and showed a higher increase of DNA content, but with time the cells lost the chondrocytic morphology. Our results demonstrate the marked influence of collagen type and pore characteristics on the phenotypic expression of seeded chondrocytes.
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9,177,851
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Peptide targeting and delivery across the blood-brain barrier utilizing synthetic triglyceride esters: design, synthesis, and bioactivity.
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As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carrier-targeting system. We have developed a prodrug design strategy that is designed to utilize membrane-bound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptide-carrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood-brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.
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9,177,852
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Conventional and high-yield synthesis of DTPA-conjugated peptides: application of a monoreactive DTPA to DTPA-D-Phe1-octreotide synthesis.
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Successful imaging of somatostatin receptor-positive tumors with 111In-DTPA-D-Phe1-octreotide has stimulated development of peptide radiopharmaceuticals using DTPA as the chelating agent. However, use of cyclic DTPA dianhydride (cDTPA) resulted in low synthetic yields of DTPA-peptide by either solution or solid-phase syntheses. This paper reports a novel high-yield synthetic procedure for DTPA-D-Phe1-octreotide that is applicable to other peptides of interest using a monoreactive DTPA derivative. A monoreactive DTPA that possesses one free terminal carboxylic acid along with four carboxylates protected with tert-butyl ester (mDTPA) was synthesized. Fmoc-Thr(tBu)-ol, prepared from Fmoc-Thr(tBu)-OH, was loaded onto 2-chlorotrityl chloride resin. After construction of the peptide chains by Fmoc chemistry, mDTPA was coupled to the alpha amine group of the peptide on the resin in the presence of 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Treatment of the mDTPA-peptide-resin with trifluoroacetic acid-thioanisole removed the protecting groups and liberated [Cys(Acm)2,7]-octreotide-D-Phe1-DTPA from the resin. Iodine oxidation of the DTPA-peptide, followed by the reversed-phase HPLC purification, produced DTPA-D-Phe1-octreotide in overall 31.8% yield based on the starting Fmoc-Thr(tBu)-ol-resin. The final product gave a single peak on analytical HPLC, and amino acid analysis and mass spectrometry confirmed the integrity of the product. 111In radiolabeling of the product provided 111In-DTPA-D-Phe1-octreotide with > 95% radiochemical yield, as confirmed by analytical reversed-phase HPLC, TLC, and CAE. These finding indicated that use of mDTPA during solid-phase peptide synthesis greatly increased the synthetic yield of DTPA-D-Phe1-octreotide, due to the absence of nonselective reactions that are unavoidable when cDTPA is used. These results also suggested that mDTPA would be a versatile reagent to introduce DTPA with high yield into peptides of interest.
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9,177,847
|
Electrospray mass spectrometry of alpha and beta chains of selected hemoglobins and their TNBA and TNB conjugates.
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The molecular weights of alpha and beta hemoglobin chains from 15 different vertebrate animal sources and 2 common human variants were determined by electrospray mass spectrometry and compared to the calculated masses based on published amino acid sequences. Conjugates were prepared for 14 of the globins using 2 traditional colorimetric derivatizing reagents, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and trinitrobenzenesulfonic acid (TNBS), and the mass of each conjugate was determined by mass spectrometry.
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9,177,855
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Calcium phosphate ceramic coatings as carriers of vancomycin.
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Infection in the setting of total joint arthroplasty remains a challenging problem. Attention has turned to developing methods of local delivery of antibiotics for prophylaxis. Vancomycin loaded into calcium phosphate ceramic coatings on titanium alloy substrates is a clinically relevant concept in the setting of total joint arthroplasty. Drug loading was accomplished by immersion of ceramic-coated discs in vancomycin-containing simulated physiological solution; in some experiments drug loading by immersion was followed by lipid coating in egg phosphatidylcholine solutions. The kinetics of vancomycin release and the efficacy of drug inhibition of Staphylococcus aureus were determined in vitro in comparison to the release from currently used antibiotic-laden poly(methyl methacrylate) (PMMA). The loading by immersion provided effective release and inhibition at early time points (up to 24 h); however, the lipid-coated samples demonstrated significant release and effective bacterial inhibition up to 72 h. The two-step procedure, i.e. drug loading followed by lipid coating in order to slow antibiotic elution, is more effective than the conventional one-step loading. The study indicated that the osteoconductive calcium phosphate coatings have the potential to serve as drug carriers to prevent infection in the setting of total joint arthroplasty.
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9,177,856
|
Effect of different surface finishing and of hydroxyapatite coatings on passive and corrosion current of Ti6Al4V alloy in simulated physiological solution.
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Direct and alternating current electrochemical tests were carried out on Ti6Al4V with different surface finishing and with hydroxyapatite (HA) coatings. Sand-blasting and rough titanium deposits obtained by vacuum plasma spraying (VPS) bring about an increase of passive and corrosion current density (c.d.) with respect to smooth Ti6Al4V, as a consequence of the augmentation of the real surface. The presence of HA deposits obtained by VPS causes an increase of passive and corrosion c.d. of the metallic substrate of about one order of magnitude and this should be taken into account in view of human body applications.
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9,177,857
|
Structural and electrochemical examinations of PACVD TiO2 films in Ringer solution.
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The conditions for obtaining titanium dioxide from the substrates titanium tetrachloride and oxygen and applying this to a surgical stainless steel of the type 316L by the plasma assisted chemical vapour deposition method have been determined. It was established that, during the process, titanium dioxide anatase is created, Crystallizing in a tetragonal lattice. During exposure of the 316L steel with the titanium dioxide coating, in Ringer's solution, protective properties of this covering improve. After 120 h the coating adopts superior barrier characteristics. Titanium dioxide covering increases the resistivity of steel of the type 316L to pitting corrosion and general corrosion. Any damage or partial removal of the coating does not cause an increased galvanic corrosion of the substrate.
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9,177,858
|
Rietveld analysis and Fourier maps of hydroxyapatite.
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Rietveld analysis was applied to the X-ray powder diffraction data of well-crystallized hydroxyapatites synthesized at 80 degrees C and pH 7.4. The least-squares refinement program adopted for Rietveld analysis showed good pattern fitting with the calculated profile. The a- and c-axis dimensions, a-0.943046 + 0.000126 nm and c-0.687485 +/- 0.000104 nm, were obtained and compared with the data estimated from a single peak. The lattice dimensions obtained by Rietveld analysis were much closer to the values of a single crystal, a = 0.9432 nm and c-0.6881nm. Fourier analysis was also performed. The maps of electron densities expressed the existing possibility of each atom composing hydroxyapatite in the crystal structure. In particular, columnar calcium and screw-axis calcium were clearly revealed. These data agreed well with the computer graphics model of hydroxyapatite reported previously.
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9,177,849
|
Use of designed peptide linkers and recombinant hemoglobin mutants for drug delivery: in vitro release of an angiotensin II analog and kinetic modeling of delivery.
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We have designed and tested specific peptide linkers for the glutathione-mediated reductive release of the angiotensin II analog N-acetyl-CGDKVYIHPF attached to recombinant human hemoglobin mutants by a disulfide bond. Inclusion of negatively charged residues decreased the rate of release by as much as 5-fold for the N-terminal linker DCD when compared to that of the control linker CG. Two different surface cysteine mutants in the second domain of the alpha,alpha-chain of recombinant human hemoglobin, D75C and K16C, were examined for their effect on the release of peptide by reduced glutathione. The reaction of the D75C-peptide conjugate with glutathione for release of the peptide is slow, with a second-order rate constant of 2.1 M-1 S-1, allowing the possibility of long term delivery. The rate of peptide release from the K16C vs the D75C mutant was decreased 15-fold. Thus, different peptide release rates can be obtained by changing both peptide linker residues and the surface location of peptide attachment. Kinetic modeling of this release using either measured or literature values for different parameters suggests boundary conditions for application to the in vivo release of peptidomimetics, small molecules, or other drugs bound to the hemoglobin surface.
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9,177,850
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Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1MN gp120.
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Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent alpha-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropy])-triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the epsilon-amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53000 +/- 42000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.
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9,177,853
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Heterobifunctional cross-linkers containing 4,9-dioxa-1,12-dodecanediamine spacers.
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A series of heterobifunctional linker arms has been prepared by functionalization of (tert-butoxycarbonyl)-4,9-dioxa-1,12-dodecanediamine [tBOC-HN(CH2)3 O(CH2)4 O(CH2)3 NH2] with anhydrides or acid chloride.
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9,177,859
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Characterization of protein release through glucose-sensitive hydrogel membranes.
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Glucose-sensitive phase-reversible hydrogels have been prepared based on the specific interaction between polymer-bound glucose and concanavalin A (Con-A). The main goal of this study was to characterize the release of model proteins (insulin and lysozyme) through the hydrogel membrane as the free glucose concentration in the environment was changed. The diffusion of the model proteins through the hydrogel membrane was examined using a diffusion cell. Porous poly(hydroxyethyl methacrylate) (PHEMA) membranes were used to sandwich the mixture of glucose-containing polymers and Con-A in between the donor and receptor chambers. The porous PHEMA membranes allowed diffusion of glucose, insulin and lysozyme, while preventing loss of glucose-containing polymers and Con-A in the sol state. The release rate of model proteins through the glucose-sensitive hydrogel membrane was dependent on the concentration of free glucose. The release rate of the proteins did not remain constant, however, due to the change in free glucose concentration resulting from diffusion of glucose from the receptor chamber to the donor chamber. This study demonstrated the possibility that the glucose-sensitive phase-reversible hydrogels can be used to regulate the insulin release as a function of the free glucose concentration in the environment.
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9,177,860
|
Denatured thiolated collagen. I. Synthesis and characterization.
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A new thiolating reagent is used to introduce sulphur groups into denatured atelocollagen. The procedure is easy to control and applicable on a large scale. The reagent is a reactive dicarboxylic acid compound containing sulphur in the form of a disulphide functionality. It is prepared by reacting N,N'-disuccinoylcystamine with 1,1'-carbonyldiimidazole. When this reagent is added to a solution of denatured atelocollagen in dimethylsulphoxide, amide bonds are formed between the carbonyl functions of the reagent and epsilon-NH2 of lysine and hydroxylysine residues from the protein. The disulphide groups introduced can then be reduced by reaction with 1,4-dithiothreitol to give the-SH form of the modified protein. Control of the stoichiometry between the reagent and the protein can lead to varying modification levels. A maximum level of 0.33 mmol SH per gram of protein can be attained, which corresponds to complete thiolation of the lysine and hydroxylysine residues. Thiolated denatured atelocollagen exhibits gelatin-like behaviour, by being highly soluble in water at all pH values and by forming heat-reversible gels.
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9,177,861
|
Denatured thiolated collagen. II. Cross-linking by oxidation.
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We have recently described a new method for the thiolation of denatured collagen, which allows precise amounts of SH groups to be attached onto the protein backbone. The oxidation of denatured thiolated collagen produces disulphide cross-linking. The cross-linking of these products has been studied, optimized and compared to the cross-linking of native and denatured collagen with 0.5% aqueous glutaraldehyde. Films have been prepared and their tensile mechanical properties and biodegradation rates with trypsin and collagenase have been evaluated. Our results indicate that the cross-linking in oxidized thiolated collagen depends on the number of the disulphide bridges formed and on their intermolecular versus intramolecular repartition. Since the number of disulphide bridges can be controlled by the level of thiol in the denatured collagen and by the oxidation procedure, it is possible to control the mechanical properties and the biodegradation rates of these new materials. Under optimized conditions, oxidized denatured thiolated collagen films are more resistant and rigid than glutaraldehyde-cross-linked collagen films Cross-linked thiolated collagen materials are also more resistant to collagenase degradation. However, because of the loss of the triple-helical structure, they are more susceptible to trypsin degradation relative to glutaraldehyde-cross-linked triple-helical collagen. Denatured collagen cross-linked by physiological bridges such as disulphide bridges, with controllable mechanical properties and biodegradation rates, is of considerable interest in biomedical applications.
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9,177,862
|
Adsorption of alpha-1-microglobulin from biological fluids onto polymer surfaces.
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A recent study in our laboratory has identified the potential role of urine-derived alpha-1-microglobulin (alpha-1-m) in mediating Pseudomonas aeruginosa adhesion to polystyrene, while other workers have suggested a possible role of the protein in the immunological response. Due to the ubiquitous presence of alpha-1-m in body fluids, the adsorption of the protein from serum, cerebrospinal fluid, urine and used continuous ambulatory peritoneal dialysis fluid onto polystyrene was investigated. The treated surfaces were sequentially immersed in water and increasingly concentrated isopropanol-water solutions in order to selectively desorb bound proteins on the basis of their binding strength. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the wash supernatants showed different protein desorption profiles for each biological fluid, despite the qualitative similarity between the protein composition of the fluids, and highlighted the uptake of alpha-1-m from each fluid to the surface. In the case of urine, the analysis was extended to commercial polyurethane and silicone stents. The ease of desorption of urine-derived alpha-1-m could be correlated with surface hydrophobicity of the stent biomaterial.
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9,177,863
|
Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays.
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A basic structural motif of lamellar bone is the arrays of parallel collagen fibrils, with successive arrays having different orientations to form a plywood-like structure. Measurements of the angles between adjacent arrays from cryomicrotomed and vitrified thin sections of demineralized rat bone, cut approximately parallel to the lamellar boundary plane, show that most angles are around 30 degrees, although a subset are around 70 degrees. A structural model for collagen organization based on these measurements is proposed in which an individual lamellar unit (thick and thin lamellae together with transition zones) is composed of five arrays of parallel collagen fibrils, each offset by 30 degrees.
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9,177,864
|
A murine model of human myeloma bone disease.
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Myeloma causes a devastating and unique form of osteolytic bone disease. Although osteoclast activation is responsible for bone destruction, the precise mechanisms by which myeloma cells increase osteoclast activity have not been defined. An animal model of human myeloma bone disease would help in clarification of these mechanisms. Multiple myeloma occurs spontaneously in aging C57 BL/KaLwRij mice and has all of the features of the disease in humans, including the characteristic bone lesions. The disease can be induced in normal C57 BL/KaLwRij mice by inoculation of fresh marrow-derived cells from mice with myeloma, but this model is difficult to study because of variability in the number of myeloma cells in marrow-derived preparations. To develop a better animal model of human myeloma bone disease, we have established and subcloned a cell line from this murine myeloma and found that it causes osteolytic bone lesions in mice characteristic of human myeloma bone disease. The cell line produces interleukin-6, but grows independent of exogenous interleukin-6. Mice inoculated intravenously with the cultured cells predictably develop an identical disease to the mice injected intravenously with fresh bone-marrow-derived myeloma cells, including monoclonal gammopathy and radiologic bone lesions. We found that some of the mice became hypercalcemic, and the bone lesions are characterized by increased osteoclast activity. We found identical results when we inoculated Nu/Bg/XID mice with cultured murine myeloma cells. Because we can inoculate mice with precise numbers of cells and predict accurately when the mice will develop bone lesions, become hypercalcemic, and die, this should be a convenient model for determining the mechanisms by which the myeloma cells cause osteoclast activation in this model of human myeloma bone disease.
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9,177,865
|
Systemic administration of an anabolic dose of PGE2 in young rats increases the osteogenic capacity of bone marrow.
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Prostaglandin E2 (PGE2) possesses significant anabolic properties when administered systemically (i.e., it increases bone formation and, consequently, bone mass). We recently characterized the effects of a 3 week administration of 6 mg/kg PGE2 into young rats and showed it increases cortical and cancellous bone mass and mechanical strength in long bones and bone density in the calvaria. We also found that a single dose of PGE2 induces the expression of early-response genes (c-fos, c-jun, and egr-1) in bone marrow cells within these two types of bone. These observations, together with findings by others of new cancellous bone formation in PGE2-treated animals, suggested that recruitment of osteoblasts from their precursors is a major mechanism of the anabolic effect of PGE2. To test this hypothesis directly, we injected PGE2 (6 mg/kg) or vehicle into 4-week-old rats for 2 weeks and then assessed the osteogenic potential of bone marrow in an ex vivo culture system. Primary and first-passage bone marrow cultures were established in the presence of beta-glycerophosphate, ascorbate, and dexamethasone, and osteogenic differentiation was measured by bone nodule formation and alkaline phosphatase activity. This regimen increased bone mass expressed as femoral ash weight by 4.7% and tibial cancellous bone area by 38.3%. Nodule formation at 21 days was increased in both primary and first-passage cultures from PGE2-treated rats despite seeding of the same number of marrow cells. Alkaline phosphatase activity was elevated in both primary and first-passage cultures from PGE2-treated rats beginning 6-10 days after culture initiation. Cell proliferation was only slightly elevated in cultures from PGE2-treated rats. These data strongly suggest that in vivo administration of PGE2 induces the proliferation or differentiation of osteoprogenitor cells in bone marrow, and this effect takes a major part in its anabolic effect in vivo.
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9,177,866
|
Osteocytes and bone lining cells: which are the best candidates for mechano-sensors in cancellous bone?
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Previously, we have investigated the possible role of osteocytes as mechano-sensors, and mediators of bone turnover. It was found that the proposed regulatory mechanism produced morphologies of trabecular bone, under particular loading conditions, which were consistent with morphogenesis and adaptation as seen in reality. The main objective of this study was to discern whether lining cells or osteoblasts could possibly play a similar role as effectively with regard to their capacity for self-optimization of the trabecular architecture, in terms of a low apparent mass to stiffness ratio. For that purpose the earlier analyses with osteocytes as mechano-sensors, distributed throughout the bone, were repeated for mechano-sensors located at bone surfaces only. Compared to the osteocyte model, the surface cell remodeling algorithm was reluctant to change its architecture, which implies that it is less sensitive to changes in the loading pattern. This resulted in less efficient bone adaptation, which was reflected by a considerably higher relative mass for a similar apparent stiffness in the loading direction. In other words, more mass is needed to obtain an equally stiff structure, at the apparent level, with respect to the externally applied loads. Furthermore, stresses and strains at the tissue level vary across a much wider range, relative to the osteocyte model, where the higher incidence of elevated strains indicates an increased failure risk. Therefore, we conclude that mechanical information at the bone surface may not be sufficient to adequately regulate functional bone adaptation.
|
9,177,867
|
Expression of gelatinases within the trabecular bone compartment of ovariectomized and parathyroidectomized adult female rats.
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Ovariectomized (ovx) and parathyroidectomized (ptx) rat models of disturbed bone metabolism have been widely used in evaluating bone changes resulting from hormonal depletion, and are characterized by elevated and depressed bone turnover, respectively. We report here the expression of gelatinases extracted from native trabecular bone in these models. Nine-month-old female Sprague-Dawley rats were sacrificed after 3 weeks following ovx or 10 days post ptx to determine the influence of these procedures on the levels of proximal tibial bone tissue gelatinases. Identification and quantitation of these enzymes were performed via gelatin gel zymography of native tissue extracts and laser densitometry of developed gels, respectively. In the ptx model, a reduction in tissue levels of pro- and active-MMP-2 and a 45 kDa activated fragment was seen, whereas ovx exhibited significant increases in these enzymes. The MMPs are therefore clearly under the influence of factors known to modulate bone remodeling in vivo. The study of MMP levels directly extracted from bone using these experimental models may assist in developing management regimes for metabolic bone diseases through the use of drugs aimed at controlling turnover.
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9,177,868
|
Long-bone biomechanics in mice selected for body conformation.
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Two lines of mice divergently selected from the control strain (CBi) against the positive phenotypic correlation between body weight (b.w.) and tail (skeletal) length were obtained (CBi/C: high weight, short tail; CBi/L: low weight, long tail). The selected animals showed a different relationship between body and skeletal masses. To compare the adequacy between biomass and load-bearing ability of the skeleton, and to describe the eventual role of bone mechanostat in the production of these changes, cross-sectional and bending properties of both femur diaphyses were determined in CBi, CBi/C, and CBi/L adult mice of both genders. Cortical bone material quality (elastic modulus) was reduced in the selected lines (p < 0.001), significantly less in CBi/C than in CBi/L. In contrast, cross-sectional design (b.w.-adjusted values of moment of inertia, CSMI) was largely improved (p < 0.001), significantly more in CBi/C than in CBi/L. These effects determined a greater stiffness and strength in CBi/C than in CBi/L or CBi weight-paired mice. The elevations of the negative regression lines between elastic modulus and CSMI ("distribution/quality" curves) decreased in the order CBi/C > CBi/L > CBi. Data show that selection improved diaphyseal stiffness and strength in CBi/C animals because of an architectural overcompensation for the reduced bone material quality. Therefore, an inadequate control of long-bone architectural design as a function of the mechanical quality of cortical bone and b.w. bearing could have been induced in that line. Assuming bone mechanostatic regulation to be genetically programmed, some of the corresponding biological determinants should be transmitted independently, because artificial selection separately affected material quality and architectural design. The possibility of transmission of an inadequate mechanostatic function (inability to adapt bone modeling to bone material quality as a function of the biomass to be supported) was also shown, as some genotypes could express architectural modifications that largely exceed bone material quality deterioration.
|
9,177,869
|
Effects of 1- and 6-month spaceflight on bone mass and biochemistry in two humans.
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The bone mineral density and the biochemical parameters exploring bone cell activities were analyzed in two cosmonauts who spent 1 and 6 months, respectively, in the Russian MIR station. Measurements were performed before the flight, after the flight, and after a recovery period. At the end of the first month, peripheral QCT measurements indicated a slight decrease of trabecular bone mass in the distal tibial metaphysis. However, after 6 months of spaceflight, a more marked loss of trabecular and cortical bones was observed in the tibia, and was still significant after 6 month recovery in the trabecular compartment, whereas a decrease was no longer observed in the cortical envelope. No change was observed in either compartment of the distal radius at any time. Ultrasound BUA of the calcaneus was greatly reduced by the first month, followed by a more dramatic decrease after month 6. Ultrasound SOS detected no change. Parameters reflecting bone formation activity appeared to be depressed after both missions. In contrast, no dramatic change in resorption parameters was observed, except for a trend toward an increase in pyridinoline. In conclusion, the lower weight-bearing bones appeared more sensitive than the upper ones in terms of spaceflight-induced bone loss. This probably explained the absence of marked systemic biochemical data changes. This study further suggests that recovery in the tibial trabecular compartment 6 months after landing was not completed after a 6 month mission.
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9,177,870
|
Evaluation of vertebral volumetric vs. areal bone mineral density during growth.
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Bone mineral "density" (BMD) measured by dual-energy X-ray absorptiometry (DEXA) does not represent the volumetric density (grams per cubic centimeter), but rather the areal density (grams per square centimeter). This distinction is important during growth. The purpose of this study was to measure vertebral dimensions in cadavers of young pigtail macaques (Macaca nemestrina), and to derive equations to predict the volumetric bone density from noninvasive measurements. We measured the areal bone density by DEXA, vertebral volume by underwater weighing, mineral content by ashing, dimensions of lumbar vertebrae by calipers, and dimensions of vertebrae by radiography. Somatometric measurements of the female lumbar vertebral bodies showed that the shape changed during growth. The bone mineral content from the densitometer correlated significantly with the ash weight (r = 0.99, error 8.7%). The correlation coefficient between the volumetric bone mineral density and areal BMD measurement was significant (r = 0.68, p < 0.0001) with a 9.5% error; this improved significantly to 0.82 (7.2% error) when the BMD was divided by the vertebral depth from the radiograph. A real BMD showed a strong correlation with age (r = 0.82, p < 0.0001), with an average increase of 7.4%/year. In contrast, volumetric mineral density showed a weak relationship with age (r = 0.43, p < 0.01), for an average increase of 1.5%/year. When studying bone mineral density during growth, the differences between volumetric and areal bone mineral density should be taken into consideration.
|
9,177,871
|
1-alpha-Hydroxyvitamin D3 treatment decreases bone turnover and modulates calcium-regulating hormones in early postmenopausal women.
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50 Japanese women within 10 years after menopause (mean age 52.5 years) were studied to determine the effects of 0.75 microgram of 1-alpha-hydroxyvitamin D3 [1-alpha-(OH)D3] with calcium (150 mg/day) (treated group: N = 25) and calcium only (control group: N = 25) for 12 months on bone mass and metabolism. Their L2-4 BMD measurements were 1.5 SD below the mean value of Japanese young, normal women. L2-4 BMDs increased significantly in the treated group (+2.1%; p < 0.01), but decreased significantly in controls (-2.1%; p < 0.01). Although serum calcium and creatinine remained unchanged in both groups, phosphorus levels increased significantly in the treated group (p < 0.01). Urinary calcium/creatinine (Cr) increased in both groups. Urinary pyridinoline/Cr and deoxypyridinoline/Cr decreased significantly in the treated group (p < 0.05), but not in the control group. Serum osteocalcin levels remained unchanged in both groups. Intact parathyroid hormone levels decreased significantly (p < 0.05) and calcitonin levels significantly increased in the treated group (p < 0.05), but these changes were not observed in the control group. These data clearly demonstrate that 0.75 microgram of 1-alpha-(OH)D3 maintained bone mass by reducing bone resorption by modulation of calcium-regulating hormones. Temporarily increased urinary calcium excretion was observed in control group, but did not appear to be effective in modulating bone turnover.
|
9,177,872
|
Bone turnover in neonates: changes of urinary excretion rate of collagen type I cross-linked peptides during the first days of life and influence of gestational age.
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New markers have been used to monitor the changes of bone turnover occurring during growth. Data on bone turnover rate during the perinatal period are, however, very scarce. In the present study we evaluated bone turnover rate, assessed by the measurement of urinary N-terminal telopeptide of type I collagen (NTx) concentrations, at different gestational ages, and we documented the trend of bone turnover rate occurring in the first days after birth. Urine samples were obtained from 83 healthy full term newborn infants, 16 preterm, and 17 infants of diabetic mothers (IDMs). The first miction after birth was collected. Urine samples were also collected 24 and 48 h after birth. NTx was measured by an enzyme-linked immunosorbent assay (Osteomark, Ostex International, Inc. Seattle, WA). The relationship between NTx at birth and all the other variables has been evaluated using multiple regression analysis. The changes of NTx excretion over time and the effect of the groups were studied by multivariate analysis of variance (MANOVA) for repeated measures. We found a remarkable association between gestational age and NTx concentrations at birth (R = 0.56; p < 0.00001). NTx concentrations showed a progressive decrement, reaching a nadir between the 38th and the 42nd week of gestation. The NTx concentrations changed significantly during the first 48 h of life in the three groups. Moreover, preterm infants had NTx excretion values at birth significantly higher than full term infants (p < 0.001), whereas NTx excretion rates of IDMs were not different from those of the other two groups of subjects. In conclusion, gestational age seems to be the major determinant of bone turnover in neonates; NTx excretion rate is higher before term, it slows in proximity of delivery, and it increases significantly during the first 48 h of life. Preterm infants have higher bone turnover rate than full term infants. NTx excretion rate of IDMs was comparable with those of the control subjects.
|
9,177,874
|
Fluid resuscitation following a burn injury: implications of a mathematical model of microvascular exchange.
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A validated mathematical model of microvascular exchange in thermally injured humans has been used to predict the consequences of different forms of resuscitation and potential modes of action of pharmaceuticals on the distribution and transport of fluid and macromolecules in the body. Specially, for 10 and/or 50 per cent burn surface area injuries, predictions are presented for no resuscitation, resuscitation with the Parkland formula (a high fluid and low protein formulation) and resuscitation with the Evans formula (a low fluid and high protein formulation). As expected, Parkland formula resuscitation leads to interstitial accumulation of excess fluid, while use of the Evans formula leads to interstitial accumulation of excessive amounts of proteins. The hypothetical effects of pharmaceuticals on the transport barrier properties of the microvascular barrier and on the highly negative tissue pressure generated postburn in the injured tissue were also investigated. Simulations predict a relatively greater amelioration of the acute postburn edema through modulation of the postburn tissue pressure effects.
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