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Biomedical subjects

A Steinkasserer

Publications and source records attributed to A Steinkasserer.

At least 19 recordsLinked to original sources
PubMed1998

High level expression and structural characterisation of herpes simplex virus type I transcriptional activator VP16 (alpha-trans inducing factor).

We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>10 mg/l) of VP16. The recombinant VP16 binds to a complex of host cell transcription factors and TAATGARAT motif. Secondary structure calculations from circular dichroism measurements indicate a content of 32.0 % alpha-helix and 17.5 % beta-sheet. This is the first structural CD analysis of VP16 which will be very useful for high-throughput assay development and mechanistic studies.

Baculoviridae↗
PubMed1997

Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1 beta and TNF-alpha compared to IL-6.

A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammatory mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that closely mimics the response of the endogenous SAA2 gene to such stimuli: i.e. single treatments induce transcriptional activation by IL-1 beta and TNF-alpha to a greater extent than by IL-6 at 12-24 h. However, timecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a much greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL-6 to give a 10-fold increase in transcriptional readout over single cytokine treatments. The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore, in synergistic cytokine combinations, it blocks only the IL-1 beta driven component indicating that the effect is biological and not attributable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory signalling in a cytokine specific manner, and for the evaluation of the pro- and anti-inflammatory properties of novel natural and synthetic molecules.

Acute-Phase Proteins↗
PubMed1997

The non-immunosuppressive cyclosporin A analogue SDZ NIM 811 inhibits cyclophilin A incorporation into virions and virus replication in human immunodeficiency virus type 1-infected primary and growth-arrested T cells.

SDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin A into HIV-1 virions. SDZ NIM 811 inhibits this protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.

Anti-HIV Agents↗
PubMed1997

Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection.

Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study.

Cells, Cultured↗
PubMed1996

High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization.

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% alpha-helix and 28% beta-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.

Animals↗
PubMed1996

Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus.

Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19.

Amino Acid Sequence↗
PubMed1996

The bicyclams, a new class of potent human immunodeficiency virus inhibitors, block viral entry after binding.

The bicyclams represent a new class of highly potent and selective HIV inhibitors. Time-of-addition experiments have previously shown that these compounds interfere with an early event in the viral replicative cycle. Additional experiments have now been carried out in order to investigate in more detail the mechanism of action of these promising compounds. As described in this paper, PCR experiments revealed that no viral DNA was formed following viral infection, thus confining the target(s) of action of the bicyclams to an early stage of HIV infection. An assay, using pseudotype virions containing the envelope of HIV-1 and the genome of a plaque-forming virus (Cocal Virus), pointed to viral entry as the main target of the bicyclams. HIV-1 strains resistant to two prototype bicyclams, JM2763 and SID791 (JM3100), were raised. Results obtained with SID791 with respect to syncytium formation induced by SID791-sensitive and -resistant HIV-1 strains and the cross-resistance observed for dextran sulfate, suggest inhibition of binding and/or fusion as a plausible target of SID791. Additional experiments enabled us to exclude SID791 and JM2763 as binding inhibitors and to conclude that bicyclams block the entry of cell-bound virus. Furthermore, a monoclonal antibody recognising the V3 loop of wild-type gp120 did not bind to this region in the two bicyclam-resistant strains. Our results point to gp120 as a possible target for the HIV-inhibitory effects of the bicyclams.

Antibodies, Monoclonal↗
PubMed1996

Differential expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 in experimental glomerulonephritis.

We examined the relation between glomerular expression of chemokines from alpha-subfamily (macrophage inflammatory protein-2, MIP-2) and beta-subfamily (monocyte chemoattractant protein-1, MCP-1) and infiltration of neutrophils and monocytes in antibody mediated glomerulonephritis in rats. In the accelerated model of nephrotoxic nephritis (NTN), glomerular expression of MIP-2 and MCP-1 genes correlated with the sequential migration of neutrophil and monocyte influx, respectively. These relationships were investigated further in the heterologous phase of NTN by applying various treatments known to modulate the severity of injury. Pretreatment with bacterial lipopolysaccharide resulted in greater injury, MIP-2 expression increased 25- to 50-fold, and the glomerular neutrophil count increased two- to fourfold. Both MIP-2 mRNA levels and neutrophil infiltration were reduced by additional pretreatment with IL-6, IL-1 receptor antagonist, soluble IL-1 receptor or soluble TNF receptor (Spearman correlation coefficient r = 0.897, P < 0.005). In the heterologous phase of NTN, different pre-treatments only resulted in trivial changes in MCP-1 expression and monocyte infiltration. In conclusion, glomerular MIP-2 gene expression correlates with neutrophil infiltration both temporally during the evolution of nephritis, and when glomerular injury is modified by treatment. Glomerular MCP-1 gene expression correlates with monocyte influx. The data show chemokines of alpha- and beta-subfamilies co-operative to cause selective and sequential migration of different leukocyte subsets during development of antibody mediated glomerulonephritis.

Animals↗
PubMed1995

Characterization of binding of human beta 2-glycoprotein I to cardiolipin.

beta 2-Glycoprotein I-cardiolipin complexes are reported to be a target antigen for the binding of a subset of anti-phospholipid antibodies. The characteristics of binding of beta 2-glycoprotein I to cardiolipin are reported in this paper. Binding at neutral pH is specific, saturable, dependent on ionic strength and independent of bivalent cation. Binding at low pH is qualitatively different from that at neutral pH, and is not dependent on ionic strength. Denaturation of beta 2-glycoprotein I by heat inactivation and reduction/alkylation indicates that beta 2-glycoprotein I-cardiolipin interaction does not require the native three-dimensional structure of beta 2-glycoprotein I, implying that a linear sequence motif may be responsible. Modification of amino acid residues by KCNO treatment completely destroys binding capacity, indicating crucial involvement of lysine residues in binding of beta 2-glycoprotein I to cardiolipin. Complement factor H, which has some similar highly charged linear sequence motifs to beta 2-glycoprotein I and is composed of the same type of protein module, was found to bind to cardiolipin and inhibit the binding of beta 2-glycoprotein I to cardiolipin. Three different lysine-rich segments of the fifth domain of beta 2-glycoprotein I may be involved in binding to cardiolipin.

Amino Acid Sequence↗
PubMed1995

Cyclophilin A, the major intracellular receptor for the immunosuppressant cyclosporin A, maps to chromosome 7p11.2-p13: four pseudogenes map to chromosomes 3, 10, 14, and 18.

Cyclophilin A (CyP-A), the major intracellular receptor for the immunosuppressant cyclosporin A (CsA), is a member of the immunophilin class of proteins, which all possess peptidyl-prolyl cis-trans isomerase activity and, therefore, are believed to be involved in protein folding and/or intracellular protein transport. The CyP-A protein is encoded by a single gene; in addition, 15 pseudogenes have been identified. Recently, specific binding of CyP-A to the human immunodeficiency virus type 1 (HIV-1) gag protein has been reported. Interestingly, this interaction can be inhibited by the immunosuppressant CsA and also by nonimmunosuppressive, CyP-A-binding CsA derivatives, which were also shown to exhibit potent anti-HIV-1 activity. Results thus indicate that CyP-A may have an essential function in HIV-1 replication. Using a panel of somatic rodent-human cell hybrids and PCR technology, we localized the coding cyclophilin A gene (PPIA) on chromosome 7 and four pseudogenes (PPIP2, PPIP3, PPIP4, and PPIP6) on chromosomes 14, 10, 18, and 3, respectively. Using chromosome 7 and chromosome 10 deletion hybrid panels, we were able to localize further the coding gene to the region 7p11.2-p13, as confirmed by fluorescence in situ hybridization analysis, and one pseudogene (PPIP3) to the region 10q11.2-q23. This is the first report on the regional mapping of members of the CyP-A gene family.

Amino Acid Isomerases↗
PubMed1995

Purification and characterization of human deoxyhypusine synthase from HeLa cells.

Post-translational modification of a specific lysine residue in eukaryotic initiation factor 5A is essential for cell viability. The amino acid hypusine, which is the product of this modification, is derived in two subsequent enzyme-catalyzed reactions. We have purified and characterized the enzyme responsible for the first step in hypusine modification, deoxyhypusine synthase, from HeLa cells. The human enzyme is multimeric with a native apparent molecular weight of 150,000 consisting of subunits of 41,000. The amino acid sequences of its peptide fragments share high sequence identity with a hypothetical protein (YHRO68w) on chromosome VIII of Saccharomyces cerevisiae. This protein appears to be the deoxyhypusine synthase of yeast.

Amino Acid Sequence↗
PubMed1995

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12-p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2.

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.

Base Sequence↗