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Biomedical subjects

S Haas

Publications and source records attributed to S Haas.

At least 127 records · Page 7Linked to original sources
PubMed1995

Regulation of mouse myelin basic protein gene transcription by a sequence-specific single-stranded DNA-binding protein in vitro.

Cell- and stage-specific transcription of the myelin basic protein (MBP)-encoding gene (Mbp) in brain is regulated by arrays of cis-acting regulatory elements positioned at the 5'-flanking region of the gene. The proximal element between nucleotides -14 to -50, termed MB1, has previously been shown to have an important role in the cell-type-specific transcription of Mbp in cells derived from the central nervous system (CNS). Here, we utilized band-shift and in vitro transcription assays to examine the ability of MEF-2, an expression factor encoded by a cDNA isolated from mouse brain, in binding to the MB1 element and regulating transcription of the Mbp promoter. Results from the band-shift assays indicated that the bacterially produced MEF-2 recognizes specific nt in the MB1 motif, and its binding to these nt reduces the overall transcriptional activity of Mbp in a cell-free extract.

Animals↗
PubMed1995

Regulation of myelin basic protein gene transcription: identification of a distal cis-acting regulatory element.

The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial-specific transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis-acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated MB3, which is located between -93 to -130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the MB3 regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double-stranded MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G-residues within nucleotides -100 to -108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 DNA with the nuclear proteins from various cells of glial origin. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells.

Animals↗
PubMed1995

A comparative trial of a low molecular weight heparin (enoxaparin) versus standard heparin for the prophylaxis of postoperative deep vein thrombosis in general surgery.

BACKGROUND: Various studies have been performed in general surgery patients comparing low molecular weight heparin (LMWH) with standard heparin (SH) for the prevention of postoperative deep vein thrombosis (DVT), revealing contradicting results. Therefore, we have compared the efficacy and safety of a LMWH for the prevention of DVT after major general surgery. PATIENTS AND METHODS: Patients received either 20 mg LMWH (enoxaparin) once daily, or 5,000 IU SH TID, starting preoperatively in a prospective, randomized, double-blind international multicenter trial. DVT was diagnosed using fibrinogen I 125 leg scanning. Major and minor bleeding were assessed clinically. RESULTS: A total of 718 patients were randomized to LMWH, and 709 patients to SH. DVT was detected in 58 LMWH-treated patients (8.1%, 95% confidence interval [CI] 6.2% to 10.3%) and in 45 patients allocated to SH (6.3%, 95% CI 4.7% to 8.4%, P > 0.05). Major bleeding complications occurred in 11 LMWH-treated patients (1.5%, 95% CI 0.8% to 2.7%) and in 18 patients to whom standard heparin was administered (2.5%, 95% CI 1.5% to 3.9%, P > 0.05). Four LMWH-treated patients (0.6%) required reoperation for bleeding as compared to 13 patients in the SH group (1.8%, P = 0.03). CONCLUSION: This LMWH appeared as effective and safe as SH. In view of its more convenient way of administration, this LMWH might be preferred for thromboprophylaxis.

Aged↗
PubMed1995

A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells.

The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.

Animals↗
PubMed1995

c-Fos is involved in the cellular defence against the genotoxic effect of UV radiation.

The proto-oncogene c-fos encodes a nuclear protein that forms together with c-Jun or other members of the Jun family the transcription factor AP-1. The c-fos gene is inducible by UV radiation and other DNA damaging treatments which may indicate that it is required in defence against DNA damaging agents. To address this hypothesized function of c-Fos, we have compared the response of mouse fibroblasts deficient in c-Fos with the corresponding wild-type cells towards the genotoxicity of UV radiation. It is shown here that lack of c-Fos renders cells hypersensitive to the cytotoxic effect of UV light and gives rise to significant increases of UV-induced chromosomal mutations and DNA breakage. Cells lacking c-Fos were basically able to perform UV-induced repair replication, as measured by unscheduled DNA synthesis. However, with high doses of UV c-Fos deficient cells proved to be less efficient in repair synthesis than wild-type cells. Measurement of overall DNA synthesis after UV irradiation revealed that cells deficient in c-Fos are more inhibited in their recovery from the UV-induced block to replication. These data strongly suggest that c-Fos is involved in regulating the timing of DNA replication after UV irradiation by abolition of the UV-induced block to replication and thus appears to play a decisive role in the cellular defence against the genotoxic effects induced by UV radiation.

3T3 Cells↗
PubMed1995

Effect of repeated Aprosulate and Enoxaparin administration on tissue factor pathway inhibitor antigen levels.

Tissue factor pathway inhibitor (TFPI) is a naturally occurring, Kunitz-type serine protease inhibitor whose anticoagulant activity is due to an inhibition of the extrinsic coagulation pathway. Heparin injection has previously been shown to increase the plasma levels of TFPI. In this study, plasma samples were obtained from a multiple dose phase I tolerance study with a synthetic analogue of heparin, namely Aprosulate (PALLAS). Volunteers were randomized into four treatment groups: (A) 35 mg Aprosulate b.i.d. s.c.; (B) 70 mg Aprosulate b.i.d. s.c.; (C) 70 mg Aprosulate o.d. + placebo o.d. s.c.; (D) 40 mg of a low molecular weight heparin, Enoxaparin o.d. + placebo o.d. s.c. All treatments were for 7 days, with blood samples taken periodically over this time period. TFPI antigen levels were determined using Imubind TFPI ELISA kits (American Diagnostica, Greenwich, CT). TFPI antigen levels were observed to rapidly increase to levels two- to three-fold over baseline in all groups. Aprosulate caused a slightly larger increase in TFPI antigen levels than Enoxaparin, though this may be related to the doses chosen for this study. These data indicate that plasma concentrations of TFPI are increased following Aprosulate administration. TFPI may be important in mediating the antithrombotic activity of Aprosulate.

Analysis of Variance↗
PubMed1995

Comparison of proliferating cell nuclear antigen (PCNA) staining and BrdUrd-labelling index under different proliferative conditions in vitro by flow cytometry.

PC10 is a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The staining pattern in immunochemistry depends on fixation and detergent extraction treatment. The aim of this study was to validate the flow cytometric PCNA assay against Bromodeoxyuridine-labelling index (BrdUrd-LI) under different proliferative conditions in vitro. Expression of PCNA in methanol fixed cells with, and without, prior detergent extraction with EDTA/Triton was compared to BrdUrd-labelling index in NIH-3T3 fibroblasts and human Caski tumour cells in exponential phase and under confluent conditions. Serum stimulation and serum starvation conditions were studied. The results for BrdUrd-LI and PCNA-index after extraction showed good correlation for 3T3 fibroblasts and for Caski cells, with some differences for serum withdrawn Caski cells. There was no correlation between the number of cells that were positive for PCNA without extraction and BrdUrd-LI. Spheroid cells with G1-DNA-content showed an almost synchronous recruitment and progression through the cell cycle after trypsination and replating. Tightly bound PCNA paralleled this synchronicity whereas total PCNA did not change significantly. The results demonstrate that immunochemical detection of non-extractable PCNA-index gives similar results as compared with BrdUrd-labelling index under different proliferative conditions in vitro for different monolayer cell lines, whereas without extraction PCNA does not correlate with BrdUrd-LI in these fast growing cell lines due to its long half-life. PCNA expression parallels the progression through the cell cycle in V79 spheroids, a primitive model of tumour growth.

Animals↗
PubMed1995

B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein.

P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Role of cell type-specific promoters in the developmental regulation of T1, an interleukin 1 receptor homologue.

Murine T1, an orphan receptor related to interleukin 1 receptors, exhibits a bimodal expression in mouse development. The molecular analysis of cultured cell lines now reveals the contribution of alternate promoters of the T1 gene to its differential expression. In nonhemopoietic cell types, where T1 synthesis in vivo is restricted to organogenesis and neoplasia, a recently characterized AP-1-dependent promoter directs a proliferation-associated expression of the gene. In hemopoietic cells, which express the T1 receptor throughout ontogenesis in vivo, T1 gene activity is driven by a novel serum factor-independent, constitutive promoter. The tissue-specific use of constitutive versus growth factor-dependent alternate promoters thus directs the permanent activity of the T1 gene in hemopoietic tissue versus the developmentally restricted expression of the gene in nonhemopoietic tissues in vivo.

Animals↗
PubMed1995

Sulfated polysaccharide anticoagulants suppress natural killer cell activity in vitro.

In contrast to the well studied anticoagulant activity of sulfated polysaccharides (SPS), little is known about their influence on immune competent cells. Using two naturally derived SPSs (unfractionated heparin and low molecular weight heparin), one semi-synthetic SPS (pentosanpolysulfate), and one synthetic SPS (lactobionic acid) as well as the polypeptide hirudin we investigated the effect of these drugs on natural killer cell activity in vitro. We demonstrate that all SPSs tested significantly suppress the activity of natural killer cells at clinically relevant dosages. At all concentrations and at all effector:target ratios tested pentosanpolysulfate was the most potent natural killer cell inhibitor. In contrast, hirudin had no effect on natural killer cell function. Furthermore, scanning electron microscopy revealed that reduced natural killer cell activity is paralleled by decreased lymphocyte cell size and altered cell surface structures. Our results indicate that defined therapeutically applied SPSs can interfere with the tumor cell killing process.

Anticoagulants↗
PubMed1994

Expression of the myelin basic protein gene in transgenic mice expressing human neurotropic virus, JCV, early protein.

Transgenic mice containing the early region of the JC virus encoding T-antigen developed neurological disease resulting from dysmyelination in the central nervous system. In this study, we investigate expression of the myelin basic protein (MBP) gene, a major constituent of the myelin sheath, at the RNA level by Northern blot and S1 nuclease assay and at the protein level by Western blot analysis using anti-MBP antibody in two distinct transgenic lines exhibiting different degrees of dysmyelination. Results from Western blot analysis of proteins from the brains of these mice revealed great reductions in MBP levels that parallel the severity of dysmyelination in the corresponding animals. Analysis of MBP RNA by Northern and quantitative S1 assays exhibited no alterations in the transcription initiation sites of the MBP gene in these animals; however, a significant decrease in the level of MBP mRNA was detected, suggesting that T-antigen may negatively influence transcription of the MBP gene. Results from Northern and Western blot analysis of proteolipid protein revealed low-level expression of this gene. Expression of JCV T-antigen is developmentally regulated in the transgenic mice; it appears at 8 days postnatal, peaks at 15 days, and substantially decreases in 18-day-old mice. The programmed expression of JCV T-antigen, which overlaps with MBP gene transcription at the early stage of myelination, suggests the involvement of a pathway which modulates stage-specific regulation of myelin genes and viral gene expression in transgenic mice during brain development.

Animals↗