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Biomedical subjects

M Rubinstein

Publications and source records attributed to M Rubinstein.

At least 19 recordsLinked to original sources
PubMed1992

Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.

Animals↗
PubMed1992

An inducer of NKCF (NK cytotoxic factor) release: localization on target-cell membrane and initial characterization.

Natural killer (NK) cells are probably involved in the elimination of virus-infected cells and of certain tumor cells. NK cell-mediated cytotoxicity (NK-CMC) was extensively studied and was found to consist of several steps. Following recognition and conjugation between the effector and the target cell, the latter one induces release of NK cytotoxic factor (NKCF) from the effector cells. The NKCF binds to the target cell which is subsequently killed. None of the molecules involved in these steps was completely characterized. In the present study it is demonstrated that isolated membranes of target cells can effectively induce the release of NKCF. Furthermore, the activity of such isolated membranes was found to be modulated by interferon (IFN) treatment of the cells prior to membrane isolation. It was therefore concluded that an NKCF-inducing structure (NKIS) is present on plasma membranes and is distinct from the NK-recognition structure. Similarly, the sensitivity to NK-CMC could be transferred from sensitive cells to IFN-gamma-treated (NK-resistant) cells by membrane fusion with the aid of Sendai virus envelope glycoproteins. It is proposed that transfer of NKIS is responsible for the acquired sensitivity to NK-CMC. In addition, it is shown that NKIS activity was recovered following membrane solubilization and reconstitution. Its level on cell surface was modulated by treatment of cells with tunicamycin, thus indicating that NKIS was probably a cell surface glycoprotein.

Cell Membrane↗
PubMed1992

Expression of active human interferon-beta in transgenic plants.

Tobacco plants were transformed with the human gene for interferon-beta (IFN-beta). Transformation was determined by the polymerase chain reaction (PCR), and expression was determined by Western blot analysis, by purifying the IFN from the transgenic plants, and by bioassays indicating its activity in human cells. Plants expressing IFN-beta were self-pollinated. IFN-beta-expressing progeny plants were selected and produced active IFN-beta, indicating stable transformation.

Base Sequence↗
PubMed1992

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Animals↗
PubMed1992

Interleukin-6 protects ductal breast carcinoma cells from MHC-unrestricted cell-mediated cytotoxicity.

Interleukin-6 (IL-6) has various biological activities including growth stimulation and maturation of B cells into antibody-producing cells, growth stimulation of murine hybridoma and plasmacytoma cells, induction of acute phase proteins, activation of T cell functions, triggering differentiation of various hematopoietic cells, and inhibition of growth of the human ductal breast carcinoma cell line T-47. Recently it was found that IL-6 also has the ability to enhance natural killer (NK) cell activity. In the present study the possible role of IL-6 as a regulator of NK cell-mediated cytotoxicity (NK-CMC) was evaluated. It was found that IL-6 reduced the sensitivity of T-47 cells (a ductal breast carcinoma cell line) to NK-CMC. The mechanism of IL-6-induced protection was studied. IL-6 had no effect on the level of conjugate formation between T-47 cells and NK cells. However, IL-6 reduced the number of dead conjugated T-47 cells. IL-6-treated T-47 cells were also found to be as sensitive as the nontreated cells to the lytic effect of NK cytotoxic factor (NKCF). However, IL-6 appeared to reduce the ability of T-47 cells to induce release of NKCF from NK cells following conjugate formation. Therefore, it is suggested that IL-6 protects T-47 cells from natural killing by the same mechanism as interferon.

Breast Neoplasms↗
PubMed1991

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.

Base Sequence↗
PubMed1991

Pharmacokinetics of recombinant interferon alpha-C.

Recombinant interferon alpha-C (rIFN alpha-C, Interpharm), is a new type of alpha-interferon that has a specific activity of 1-2 x 10(9) units/mg protein. The pharmacokinetics of rIFN alpha-C were studied in 11 patients with metastatic renal-cell carcinoma. A total of 10 million units IFN alpha-C were injected intramuscularly and the serum level of IFN was evaluated up to 72 h post-administration. Measurable IFN concentrations appeared in the serum as early as 0.5 h, and levels peaked at 4-6 h (Cmax = 53.2 +/- 4.6 units/ml). Relatively high levels persisted for 24 h and declined thereafter with an apparent half-life of 3-4 h. The mean area under the serum-concentration curve (AUC) was 1,259 +/- 145 units h ml-1, indicating good bioavailability of the preparation from the intramuscular injection.

Aged↗
PubMed1991

Cyclosporin A regulates the expression of HLA-DR on human monocytes by two different mechanisms.

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-gamma in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-gamma, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-gamma receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-alpha also inhibited the IFN-gamma-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-alpha is proposed.

Cycloheximide↗
PubMed1991

The effect of bilateral sympathectomy on noise induced temporary threshold shift.

The cochlea is innervated by sympathetic nerves originating or passing the superior cervical ganglion. The termination of one type (the vascular independent) is in the habenular region close to the auditory nerve fibers, and the other, the perivascular type, is associated with blood vessels, particularly in the spiral vessel of the tympanic lip. Suggested functions have so far received partial evidence in the literature. Borg (1982) suggested the protective value of sympathectomy of the ear in noise. Our experiments further elaborate this protective value, as it was seen that bilateral cervical sympathectomy diminished the temporary threshold shift in awake, sound exposed GP.

Acoustic Stimulation↗
PubMed1991

Sedation effect on temporary threshold shift induced by acoustic overstimulation.

The mechanism by which noise damages the inner ear has not as yet been fully elucidated. Experiments were done to study the influence of the sedation in temporary threshold shift (TS) induced by acoustic overstimulation, as barbiturates were found to improve the brain's tolerance to ischemia. Four groups of guinea pigs (GP) were used. The temporary TS was decreased with the reduction of the temperature in awake, as well as sedated, sound-exposed GP. However, the temporary TS in the sedated, but normothermic GP was as great as in the awake, normothermic group. The high temperature counteracts the protection effect of the sedation in nose-induced hearing loss.

Acoustic Stimulation↗
PubMed1991

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Cell Death↗
PubMed1991

Monoclonal antibodies to the soluble human IL-6 receptor: affinity purification, ELISA, and inhibition of ligand binding.

Soluble IL-6 receptor (IL-6-R) purified to homogeneity from normal human urine was used for immunization of mice and rabbits. Spleen cells derived from a mouse showing a high binding titer to IL-6-R in an inverted solid phase radioimmunoassay (IsRIA) and in a Western blotting analysis were fused to mouse myeloma cells. The hybridomas were screened by the IsRIA, and 30 positive clones were isolated and characterized. They were suitable for affinity purification of the IL-6-R and for its detection by Western blot analysis, by ELISA and by sandwich type sRIA. Most of them inhibited the binding of labeled IL-6-R to IL-6 in a solid phase RIA.

Animals↗
PubMed1991

The antiproliferative effect of cyclosporine on hematopoietic and lymphoblastoid cell lines--common mechanistic elements with interferon-alpha.

Cyclosporine, but not its nonimmunosuppressive analog cyclosporine H (CsH), caused in a variety of hematopoietic cell types a growth arrest in the G0/G1 phase of the cell cycle. This arrest was associated with a significant reduction in the c-myc mRNA levels, which could be observed already 1 hr following CsA treatment. Similarity between the antiproliferative effects of CsA and IFN-alpha was observed. Thus, the IFN-alpha sensitive human B-lymphoblastoid cell line Daudi was also sensitive to CsA while an IFN-alpha resistant variant of Daudi cells was found to be resistant to CsA as well. Inhibition of protein synthesis with cycloheximide during IFN-alpha or CsA treatment blocked their ability to reduce the expression of c-myc. Depletion of protein kinase C (PKC) activity from cells by pretreatment of Daudi cells with phorbol.12-myristate 13-acetate (PMA) abolished the G0/G1 arrest induced by both CsA and IFN-alpha. Combinations of low concentrations of CsA and IFN-alpha had synergistic effects on cell-cycle distribution and on c-myc mRNA level, suggesting that CsA and IFN-alpha differ in some features of their antiproliferative action. This conclusion was supported by the observation that a CsA-resistant variant of Daudi cells was found to retain its sensitivity to IFN-alpha. In addition, reduction of ornithine decarboxylase mRNA expression was obtained with IFN-alpha but not with CsA. Taken together, our results suggest that CsA and IFN-alpha share some common element(s) in the pathways of their antiproliferative activity. The possible mechanisms of their antigrowth effects and the clinical significance of our findings are discussed.

Blotting, Northern↗
PubMed1991

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

Amino Acid Sequence↗
PubMed1991

The prevalence of tardive dyskinesia.

A total of 2250 subjects from psychiatric and geriatric settings was examined for abnormal involuntary movements by the same team of trained raters employing a standard examination technique and rating scale. "Spontaneous" dyskinesia rates were 1.3% among 400 healthy elderly people surveyed at senior citizens centers, 4.8% among medical geriatric inpatients and ranged from 0 to 2% among psychiatric patients never exposed to neuroleptics. For samples of neuroleptic-treated patients, prevalence rates ranged from 13.3% among patients at a voluntary psychiatric hospital to 36.1% among state hospital patients. Logistic regression analyses revealed a large effect of age on tardive dyskinesia prevalence and an interaction of age with sex. Among younger subjects, men had higher rates; among subjects over age 40, rates were higher for women. Edentulousness and presence of other neurological disorders were possible contributors to high rates for the elderly. Even with control for age, sex and duration of neuroleptic exposure, prevalence differed markedly across study site.

Adult↗
PubMed1991

A clinical and polysomnographic comparison of neuroleptic-induced akathisia and the idiopathic restless legs syndrome.

Neuroleptic-induced akathisia (NIA) is motor restlessness caused by dopamine receptor blocking antipsychotic agents. Nine patients with NIA and 11 patients with idiopathic restless legs syndrome (RLS) were studied polysomnographically. The sleep disturbances were milder in NIA than idiopathic RLS but increased numbers of awakenings and decreased sleep efficiencies were common to both groups. In addition, RLS patients demonstrated prolonged sleep latencies. Periodic movements in sleep (PMS) were present in only 5 of 9 patients with NIA but in all 11 patients with idiopathic RLS. In no NIA patient did we see the multiple, large amplitude, violent, resting myoclonic jerks of the legs that we saw during wakefulness in some of our more severe cases of idiopathic RLS. NIA patients tended to experience inner restlessness and idiopathic RLS patients tended to experience leg paresthesias as an antecedent to motor restlessness. Idiopathic RLS patients had symptoms that were worse at night and in repose far more frequently than patients with NIA. NIA and idiopathic RLS have similarities and differences. Because both NIA and idiopathic RLS are characterized by motor restlessness and sleep disturbances, the pharmacodynamics of antipsychotic medications may give clues as to both the cause and treatment of idiopathic RLS.

Adult↗
PubMed1990

A method for preparing biologically active aqueous cyclosporin A solutions avoiding the use of detergents or organic solvents.

The immunosuppressive agent cyclosporin A (CsA) is highly hydrophobic and is, therefore, being taken for in vitro use directly from ethanolic stock solutions. In the present study it is demonstrated that ethanol itself affects several immunological activities in vitro, thus interfering with the immunosuppressive effects of CsA. As an alternative, it is demonstrated that CsA can be solubilized and retain its activity in aqueous solutions of polyvinyl pyrrolidone (PVP). Unlike ethanol, PVP has no effect on the various immunological activities in vitro and therefore the immunosuppressive effects of CsA can be evaluated without interference.

2',5'-Oligoadenylate Synthetase↗