886 research outputs found

    Solution behavior, circular dichroism and 220 MHz PMR studies of the bovine myelin basic protein

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    Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties.At pH 4.8 [gamma]/2 = 0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0[middle dot]10-4 mol[middle dot]ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species.Sedimentation experiments at pH 4.8 yielded s[deg]20,w of 1.27 S at [gamma]/2 = 0.1 and 1.46 S at [gamma]/2 = 0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25[middle dot]10-7 cm2/s at [gamma]/2 = 0.1 and 0.35. The value of [function of (italic small f)]/[function of (italic small f)]0 from diffusion at pH 4.8 and [gamma]/2 = 0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, [gamma]/2 = 0.1, s[deg]20,w was 1.71 S and D[deg]20,w was estimated at 7.4[middle dot]10-7 cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1cm1% = 5.64 at 276.4 nm and 225 at 196 nm.Titration of the protein with trifluoroethanol elicited three distinct regions of conformational stability having increasing helical content as the mol fraction of trifluoroethanol increased.The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21990/1/0000400.pd

    HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8(+) CD28(-) T subset

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    BACKGROUND: T cells from HIV(+) and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8(+) CD28(-) T cells. In order to compare cytokine production from T cells from these two states, CD4(+) and CD8(+) T cells from HIV(+) aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8(+) T cell subsets CD28(+) and CD28(-) from the HIV(+) and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8(+) T cells from both HIV(+) and aged donors showed an increase of approximately 2–3 fold over controls in percentage of cells producing inflammatory cytokines IFN-γ and TNF-α. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1–2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4–5 fold) in IFN-γ and TNF-α mRNA from HIV(+) and aged CD8(+) T cells, as did ELISA for secreted IFN-γ and TNF-α (2.3–4 fold). Flow cytometric analysis showed that the CD8(+) CD28(-) T cell subset accounts for approximately 80–86% of the IFN-γ and TNF-α production from the CD8(+) subset in the aged and HIV(+) states. The CD4(+) T cell, while not significantly changed in the HIV(+) or aged states in terms of IFN-γ production, showed a small but significant increase in TNF-α production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV(+) and aged individuals, i.e. elevated serum levels and elevated CD8(+) T cell production of IFN-γ and TNF-α. Thus, the capacity for increased production of cytokines IFN-γ and TNF-α in the aged individual by the dominant CD8(+) CD28(-) subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8(+) CD28(-) T cell subsets in both the HIV(+) and the aged states may reflect a natural "endpoint" in CD8(+) T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual

    The solution behavior of the bovine myelin basic protein in the presence of anionic ligands binding behavior with the red component of trypan blue and sodium dodecyl sulfate

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    The interaction of the azo dye (2,3'-dimethyldiphenyl-7-azo-8-amino-l-napthol 3,6-disulfonic acid (TBR) and sodium dodecyl sulfate with the bovine myelin basic protein has been studied using absorbance, circular dichroism and 220 MHz PMR spectroscopy. Additional analyses of the binding reaction were carried out using light scattering, ultracentrifugal and electrophoretic techniques. A procedure for preparing pure TBR was developed. A modified structure for this synthesized TBR has been suggested. The mechanism of TBR binding to the myelin basic protein was found to be metachromatic. In addition, the interaction of TBR with the basic protein which gives rise to aggregation of the dye bound species was found to be analogous to the model proposed by Schwarz, G. and Seelig-Loffler, A. (1975) Biochim. Biophys. Acta 379,125-138) to explain the binding of acridine orange with poly([alpha]--glutamic acid). PMR spectral analyses suggested that arginine residues provide the majority of primary sites of attachment on the basic protein for TBR. The effect of sodium dodecyl sulfate binding with the bovine myelin basic protein was found to induce a minimal change in the conformation of the protein. The induction of only about 20% [alpha] helial structure could be demonstrated and the binding was reversed by raising the solution temperature to 73[deg]C. The difference in the observed behavior of basic protein arising from TBR binding as opposed to the binding of sodium dodecyl sulfate is viewed as resulting from two different binding mechanisms. The binding behavior of TBR is primarily a consequence of charge-charge interaction while the binding effects of sodium dodecyl sulfate are a consequence of hydrophobic interaction. The sodium dodecyl sulfate binding acts as a shield which limits charge-charge interaction in the basic protein molecule thus preventing aggregate formation while TBR imposes no such restraints.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21790/1/0000185.pd

    Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

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    This study was designed to assess the effect of GB virus (GBV)-C on the immune response to human immunodeficiency virus (HIV) in chronically HIV-infected and HIV- hepatitis C virus (HCV)-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ) and interleukin (IL)-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8%) presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.Universidade Federal de São Paulo (UNIFESP) Laboratório de Virologia e Imunologia Disciplina de InfectologiaFleury Medicina DiagnósticaUNIFESP, Laboratório de Virologia e Imunologia Disciplina de InfectologiaSciEL

    Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein

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    Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, pppE. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27860/1/0000273.pd

    Congruence of tissue expression profiles from Gene Expression Atlas, SAGEmap and TissueInfo databases

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    BACKGROUND: Extracting biological knowledge from large amounts of gene expression information deposited in public databases is a major challenge of the postgenomic era. Additional insights may be derived by data integration and cross-platform comparisons of expression profiles. However, database meta-analysis is complicated by differences in experimental technologies, data post-processing, database formats, and inconsistent gene and sample annotation. RESULTS: We have analysed expression profiles from three public databases: Gene Expression Atlas, SAGEmap and TissueInfo. These are repositories of oligonucleotide microarray, Serial Analysis of Gene Expression and Expressed Sequence Tag human gene expression data respectively. We devised a method, Preferential Expression Measure, to identify genes that are significantly over- or under-expressed in any given tissue. We examined intra- and inter-database consistency of Preferential Expression Measures. There was good correlation between replicate experiments of oligonucleotide microarray data, but there was less coherence in expression profiles as measured by Serial Analysis of Gene Expression and Expressed Sequence Tag counts. We investigated inter-database correlations for six tissue categories, for which data were present in the three databases. Significant positive correlations were found for brain, prostate and vascular endothelium but not for ovary, kidney, and pancreas. CONCLUSION: We show that data from Gene Expression Atlas, SAGEmap and TissueInfo can be integrated using the UniGene gene index, and that expression profiles correlate relatively well when large numbers of tags are available or when tissue cellular composition is simple. Finally, in the case of brain, we demonstrate that when PEM values show good correlation, predictions of tissue-specific expression based on integrated data are very accurate

    The advancement of blood cell research by optical tweezers

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    Demonstration of the light radiation pressure on a microscopic level by A. Ashkin led to the invention of optical tweezers (OT). Applied in the studies of living systems, OT have become a preferable instrument for the noninvasive study of microobjects, allowing manipulation and measurement of the mechanical properties of molecules, organelles, and cells. In the present paper, we overview OT applications in hemorheological research, placing emphasis on red blood cells but also discussing OT applications for the investigation of the biomechanics of leukocytes and platelets. Blood properties have always served as a primary parameter in medical diagnostics due to the interconnection with the physiological state of an organism. Despite blood testing being a well-established procedure of conventional medicine, there are still many complex processes that must be unraveled to improve our understanding and contribute to future medicine. OT are advancing single-cell research, promising new insights into individual cell characteristics compared to the traditional approaches. We review the fundamental and practical findings revealed in blood research through the optical manipulation, stretching, guiding, immobilization, and inter-/intracellular force measurements of single blood cells
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