Influence of Agonist, Shear Rate, and Perfusion Time on Nitric Oxide Inhibition of Platelet Deposition

Nitric oxide (NO) is a physiological species involved in inhibition of platelet adhesion and aggregation. A novel NO delivery device was utilized to quantitatively assess the effects of gaseous NO on platelet deposition to agonist-coated biomaterials in the presence of a platelet suspension. Platelet deposition was evaluated as a function of agonist (collagen, fibrinogen, or IgG), shear rate (250, 500, and 750 s^–1), and perfusion time (5, 7.5, and 15 min). The minimal aqueous surface NO concentrations and fluxes necessary for significant inhibition of platelet deposition were quantified. Platelet deposition was completely inhibited at a gaseous NO exposure of 0.1 ppm, irrespective of the platelet agonist, shear rate, and perfusion time. The corresponding aqueous surface NO concentration was 0.09 nM at 250 s^–1 as predicted by a validated model. Surface fluxes ranged between 0.3 and 0.6 femtomoles cm^–2 s^–1. The results of this study are useful for establishing generalized guidelines (i.e., NO flux requirements in the presence of agonists, shear rate, and perfusion time) for the design and development of suitable biomaterials incorporating NO to reduce platelet deposition. Further studies incorporating blood, rather than platelet suspensions, are required to provide a more complete assessment of the required NO flux necessary to inhibit platelet deposition. © 2000 Biomedical Engineering Society. PAC00: 8717-d, 8719Tt     

Occurrence of lymphocytotoxins in multi-case rheumatoid arthritis families: relation to HLA.

The presence of lymphocytotoxic antibodies (LCA) and their association with HLA haplotypes has been studied in 27 multi-case rheumatoid arthritis (RA) families (13 multiplex and 14 simplex) in Northern India. Of the total 59 RA patients, 69.4% had cytotoxins in their sera as compared with 2.5% of healthy controls. No differences were observed in the frequency of LCA in relation to sex and rheumatoid factor. LCA against B cells were significantly more predominant than those against T cells. Twenty families studied for correlation of HLA with LCA showed greater intensity of reaction with DR4+ haplotypes, particularly in simplex families. Similarly, the frequency of LCA among patients and unaffected parents was greater in simplex compared with multiplex families. Haplotype sharing with the patient was increased in the relatives positive for cytotoxins in these families. An immunogenetic contribution made by the affected parent and a common environmental stimulus may be responsible for the increased production of LCA in multi-case families with RA.     

Multiple cis-regulatory elements and the yeast sulphur regulatory network are required for the regulation of the yeast glutathione transporter, Hgt1p

HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4 background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4 strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the –356 to –364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at –333 to –340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at –300 to –285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells.     

DFNA54, a third locus for low-frequency hearing loss

Nonsyndromic hereditary hearing impairment (NSHHI) is a highly heterogeneous disorder with more than 90 loci mapped, of which nearly one-half of the responsible genes are identified. In dominant NSSHI hearing loss is typically biased towards the high frequencies while low-frequency hearing loss is unusual. Only two NSHHI loci, DFNA1 and DFNA6/14/38, are associated with predominantly low- frequency loss. We mapped the loci harboring the gene responsible for autosomal dominant low-frequency hearing loss in a multigenerational family. The pedigree of a Swiss family with low-frequency hearing loss was established. Using genomic DNA, DFNA1 and DFNA6/14/38 were excluded by linkage analysis or by direct sequencing of the responsible gene. Genome-wide linkage analysis was performed using commercially available microsatellite markers. Two-point linkage analysis demonstrated linkage to chromosome 5q31, the locus for DFNA15, with a lod score of 6.32 at recombination fraction =0 for marker D5S436. Critical recombinations were seen at markers D5S1972 and D5S410. Sequencing of the corresponding gene POU4F3 yielded no pathogenic mutation segregating with the affected members. In addition to Wolfram syndrome gene 1 (DFNA6/14/38) and diaphanous (DFNA1) there is evidence for a third gene involved in low-frequency hearing loss located at DFNA15. Because of the differences in auditory phenotype and the absence of pathogenic mutation in the coding region of POU4F3 it is likely that there is a second gene in 5q31, designated DFNA54, associated with NSHHI.     

Identification of amino acid residues of anthrax protective antigen involved in binding with lethal factor

Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for the translocation of LF to the cytosol. The binding of LF to cell surface receptor-bound PA is a prerequisite for the formation of lethal toxin. It has been hypothesized that hydrophobic residues P184, L187, F202, L203, P205, I207, I210, W226, and F236 of domain 1b of PA play an important role in the binding of PA to LF. These residues are normally buried in the 83-kDA version of PA, PA83, as determined by the crystal structure of PA. However, they become exposed due to the conformational change brought about by the cleavage of PA83 to PA63 by a cell surface protease. Mutation of the above-mentioned residues to alanine resulted in mutant proteins that were able to bind to the cell surface receptors and also to be specifically cleaved by the cellular proteases. All the mutant proteins except the F202A, L203A, P205A, and I207A mutants were able to bind to LF and were also toxic to macrophage cells in combination with LF. It was concluded that residues 202, 203, 205, and 207 of PA are essential for the binding of LF to PA.     

Ultraviolet light-induced modification of crosslinked hyaluronan gels

Hyaluronan (HA) gels (hylans) crosslinked with divinyl sulfone (DVS) are highly biocompatible and can be structurally modified to obtain desired mechanical properties that are attractive for their use as tissue-engineering scaffolds. However, unmodified hylan gels are not good substrates for cell attachment or infiltration, likely as a result of their smooth surface and the highly anionic nature of HA. This study investigated whether the cell-adhering characteristics of hylan gels could be enhanced by irradiation with ultraviolet (UV) light, with or without prior dehydration. The attachment and proliferation of neonatal rat smooth muscle cells atop these gels was compared with that on unmodified (control; C) or dehydrated (D) gels. UV-induced changes to gel structure and chemistry were characterized by confocal and electron microscopy, and fluorphore-assisted carbohydrate electrophoresis (FACE). Cell attachment was sparse on both unmodified (C) and dehydrated (D) gels. Significantly higher levels of cell attachment were observed on the surface of irradiated (UV) and dehydrated-irradiated (DUV) gels, likely because of texturing of the gel surface by UV light. In addition, dehydration of gels before UV irradiation created irregular pore-like structures through which cells appeared to migrate into the interior. FACE assays demonstrated that UV-irradiation alters the chemistry of HA, causing limited breakdown of HA chains and DVS crosslinks within gel and possibly creating new crosslinks that have not yet been identified. Because the hylan gels are altered structurally and chemically, binding of cells to the material is likely to be more permanent than possible by other approaches, such as coating of cell-adhesive matrix factors on the gel surface, described previously. The significance of this work is that we have developed a technique for the modification of DVS-crosslinked HA (hylans) to enhance their performance as a cellular scaffold for tissue-engineering applications.     

Risk factors for HIV infection in injection drug users and evidence for onward transmission of HIV to their sexual partners in Chennai, India

OBJECTIVES: Determining HIV prevalence in injection drug users (IDUs) and their regular sex partners in Chennai, India. METHODS: A total of 226 IDUs and their regular sex partners were enrolled during April-July 2003. After informed consent was obtained, a semistructured questionnaire was administered and serum was tested for HIV antibody. RESULTS: The HIV seroprevalence was 30% (68/226) in IDUs and 5% in their regular sex partners (11/226). While in 25% of couples only the male partner was HIV positive, 5% of the couples were concordant for HIV infection and 70% were HIV negative. Fifty-seven percent of the HIV-positive IDUs and 45% of the HIV-infected women thought that they had "no chance" or "very little chance" of getting HIV, reflecting low HIV risk perception. More than 20% IDUs reported borrowing or lending of injection equipment. In univariate analyses "sex" and "condom use" with sex workers had no bearing but "more than twice a day injecting frequency," "history of incarceration," "tattoos," "recruitment from northern part of the city," and ever-injecting drugs in drug-selling places had significant association with HIV infection in IDUs. In an adjusted model, the odds of HIV infection were 2 times higher among IDUs who had ever injected drugs in drug-selling places and 6 times higher in those who were recruited from the northern part of central Chennai. CONCLUSION: Reducing sharing of injection equipment and unsafe tattooing through targeted and environmental interventions, increasing HIV risk perception, and promoting safer sex practices among IDUs and their sex partners are urgent program needs.