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Biomedical subjects

R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 145 records · Page 8Linked to original sources
PubMed1983

The folding of ovalbumin. Renaturation in vitro versus biosynthesis in vitro.

Hen ovalbumin, the major secretory product of oviduct cells, is a 43 000-dalton glycoprotein. Many studies have led to controversy over the question of whether ovalbumin (OA) can be fully renatured after chemical denaturation. We have studied the renaturation of OA after denaturation with guanidinium chloride, urea or alkaline pH. Denatured OA displays an intrinsic viscosity consistent with nearly complete unfolding of the protein. Removal of the denaturant results in a complete reversal of the changes in intrinsic viscosity. However, closer examination of the renatured protein reveals major differences from the native form. Renatured OA (OAR) can be completely separated from the native form (OAN) by affinity chromatography on phenyl-Sepharose. OAR displays altered tryptophan fluorescence, u.v.-absorption and c.d. spectra. Only OAR binds anilinonaphthalenesulphonate (as measured by fluorescence enhancement). OAR, but not OAN, binds about 2 mol of the covalent hydrophobic affinity probe phenyl isothiocyanate/mol. Renaturation, and the production of OAR, occurs regardless of the oxidation state of the disulphide bonds, of phosphorylation of the protein, and of the presence or the absence of the single carbohydrate chain. OAR may be either monomeric or an irreversible aggregate. Which of these two states is formed depends on the protein concentration during renaturation. Monomeric and aggregated OAR can be distinguished on the basis of some spectroscopic characteristics, but they share the essential hydrophobic characteristics that distinguish them from OAN. OAN and OAR do not spontaneously interconvert. Antibodies raised to each can be made monospecific by immunoabsorption. Thus two stable forms of OA can be obtained, one of which, OAR, displays hydrophobic characteristics. OAN, but not OAR, is formed when OA is synthesized in vitro in a translation system.

Animals↗
PubMed1983

[Results and perspectives of therapy of chronic myelosis].

A survey is given on the present state of the standard therapy of chronic myeloleucosis as well as on some newer tendencies of treatment. Since in the therapy of the chronic phase of the disease and also of the blast crisis despite application of combinations of cytostatics no decisive success is to be recorded, some newer techniques--the splenectomy as well as the various kinds of the transplantation of bone marrow (autologous, syngenic, allogenic)--and their chances for success are briefly discussed.

Bone Marrow Transplantation↗
PubMed1983

Clathrin-induced pH-dependent fusion of phosphatidylcholine vesicles.

Interaction of clathrin coat protein with dioleoyl-phosphatidylcholine (DOPC) vesicles at pH 6.5 and below results in the formation of stable vesicle-clathrin complexes (Steer, C. J., Klausner, R. D., and Blumenthal, R. (1982) J. Biol. Chem. 257, 8533-8540). In this report we show by gel chromatography and sedimentation analysis that the interaction of clathrin coat protein with unilamellar dioleoyl phosphatidylcholine vesicles at pH = 6.0 results in the formation of larger structures. As shown by electron microscopy and an increase in trapped volume of both sucrose and inulin those larger structures represent fused bilayers. We examined the mixing of membrane lipid as a result of membrane fusion using resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle membrane. At a protein:lipid ratio of 1:500 there was 50% vesicle-vesicle fusion, at pH 6.0, as indicated by the change in efficiency of energy transfer between the fluorescent probes. Fusion was completed within 60 s. A number of other proteins (ovalbumin, rabbit IgG, trypsin, pronase, calmodulin, tubulin, synexin, bovine serum albumin) at 10-fold or higher concentrations, did not induce fusion of dioleoyl phosphatidylcholine vesicles, either at pH 7.4 or at pH 6.0. This system provides a model for pH-dependent and protein-mediated fusion of uncharged lipid bilayers.

Animals↗
PubMed1983

Voltage-dependent orientation of membrane proteins.

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.

Asialoglycoprotein Receptor↗
PubMed1983

Rapid changes in specific estrogen binding elicited by cGMP or cAMP in cytosol from human endometrial cells.

Addition of cGMP to cytosol of human endometrium or to cells of the endometrial cancer line HEC-1 produced severalfold increases in specific estrogen binding (EB) levels. This effect was maximal with 1 microM cGMP in the presence of 0.1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor) during incubations with [3H]estradiol. In contrast, cAMP decreased EB levels under similar conditions. The effects of cyclic nucleotides on EB levels were complete in less than 15 min in the presence of Mg2+, Mn2+, or Ca2+. The EB sites generated by the addition of cGMP during labeling of cytosol with 10 nM [3H]estradiol were found to sediment in the 8S and 4S regions of low-salt glycerol gradients. No changes in EB levels were observed when cyclic nucleotides were added to cytosol depleted of ATP by preincubation at 4 degrees C for 3 hr, but responsiveness was restored by addition of exogenous ATP. The ATP requirement and the pattern of dependence of cyclic nucleotide actions on divalent cation concentrations suggest that cGMP and cAMP effects may be mediated by kinases and may involve phosphorylations. Another possibility is that the cyclic nucleotides interact allosterically with the binder in the presence of ATP. Addition of sodium molybdate, ATP, and GTP to homogenates of endometrial tissue or HEC-1 cells produces increases in EB levels similar to those obtained by the addition of cGMP. However, these compounds are much less active when added to cytoplasm or cytosol. On the basis of these and other observations, it is hypothesized that molybdate, ATP, and GTP affect EB levels primarily by increasing cGMP concentrations through processes involving a plasma membrane-bound guanylate cyclase.

Adenosine Triphosphate↗
PubMed1983

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.

Bacterial Outer Membrane Proteins↗
PubMed1982

Aggregation and calcium-induced fusion of phosphatidylcholine vesicle-tubulin complexes.

Insertion of tubulin into the bilayer of dipalmitoyl phosphatidylcholine vesicles at the phase transition results in the formation of stable vesicle-tubulin complexes (Klausner, R. D., Kumar, N., Weinstein, J. N., Blumenthal, R., and Flavin, M. (1981) J. Biol. Chem. 256, 5879-5885). These complexes aggregated when maintained below phase transition for 10-20 min. Addition of millimolar concentrations of Ca2+, Mn2+, Zn2+, and Co2+, but not Mg2+, caused the vesicle-tubulin complexes to fuse into larger structures as shown by (a) electron microscopy, (b) increased trapped volume, and (c) changes ion resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle. There was no loss of internal aqueous contents from the vesicle-tubulin complexes during Ca2+-induced fusion. Anti-tubulin drugs had no effect on the aggregation or fusion, and vesicle-bound tubulin did not associate with microtubules when tubulin was assembled in vitro. Trypsin-treated vesicle-tubulin complexes were incapable of supporting Ca2+-induced fusion. This system provides a model for Ca2+-induced and protein-mediated nonleaky fusion of uncharged lipid bilayers.

Animals↗
PubMed1982

Interaction of liver clathrin coat protein with lipid model membranes.

Coated vesicles were prepared from rat liver homogenates by sucrose density gradient centrifugation and the clathrin coat, dissociated by mild treatment with Tris-Cl, was characterized by sodium dodecyl sulfate gel electrophoresis. At pH 6.5 and below, the soluble clathrin preparations induced a voltage-dependent increase in ion conductance across a black lipid membrane of oxidized cholesterol under conditions where the intact coated vesicles were inert. Further evidence for the interaction of clathrin with the lipid bilayer was provided by the demonstration that the fluorescent dye, carboxyfluorescein, was released from the internal aqueous space of small unilamellar dioleoyl and dipalmitoyl phosphatidylcholine vesicles upon the addition of the soluble protein. Dye release was shown to be a function of both calcium and pH. In the presence of calcium, release occurred at or above pH 8.0; in the absence of calcium, release was most prominent at pH 5.0-6.5. Formation of stable protein-phospholipid complexes was shown by KBr density gradient centrifugation. Stability of the complexes in high salt concentration suggested that electrostatic interactions were not essential to maintain the recombinants. Here, as with the black lipid membrane, incubation of the phospholipid vesicles with intact coated vesicles failed to induce release of the dye at pH 6.5 or below. Interaction of the clathrin coat protein with unilamellar vesicles at low pH resulted in a change in vesicle size and morphology as shown by electron microscopy.

Animals↗
PubMed1982

Compartmental analysis of light-induced proton movement in reconstituted bacteriorhodopsin vesicles.

Purified bacteriorhodopsin from purple membrane sheets isolated from Halobacter halobium was solubilized with a bile salt detergent, 3-[(3-cholamidopropyl) dimethyl-ammonium]-1-propanesulfonate (CHAPS). The detergent-solubilized protein was then incorporated into lecithin vesicles at either high (450:1) or low (65:1) lipid to protein ratios. Circular dichroism studies showed that the bacteriorhodopsin incorporated was in a monomeric form in the 450:1 vesicles. The 65:1 vesicles exhibited an exciton splitting characteristic of the aggregated state of bacteriorhodopsin. We then examined the light-induced movement of protons for these two preparations. Compartmental analysis was used to derive a kinetic model for the observed proton movement. The pumping was qualitatively the same for monomeric and aggregated protein. A three-compartment model provided an excellent description of proton movement in both sets of vesicles and at four different light intensities. This model demands two independent processes to account for the proton movement. The rate coefficients for both are linearly related to light intensity. However, the total flux of protons via one of these processes diminishes as a function of the hydrogen ion accumulation within the vesicles.

Bacteriorhodopsins↗
PubMed1982

Return to the family and its consequence for rehospitalization among recently discharged mental patients.

This study examined the contribution that living arrangements made to the rehospitalization rates of mental patients discharged to the community during the first 7 months they spent there. Over 20,000 mental patients discharged from psychiatric facilities in the state of New York were categorized on the basis of the living arrangements to which they were discharged. These included living alone, parental, marital, other relatives or friends, and domiciliary settings. The sample was divided in half and results of the analysis of the first sample were replicated on the second sample. Analysis of covariance and multiple regression techniques revealed that patients discharged to marital settings were rehospitalized less than those discharged to other settings, and that there were no differences in the return rates among the remaining settings. The major predictor of rehospitalization was the number of previous hospitalizations. The implications of these findings for further research on supportive as well as stressful parameters of community and family settings are discussed.

Adolescent↗
PubMed1982

Effects of cyclic nucleotides on estradiol binding in human endometrium.

The addition of molybdate to intact or homogenized cells of the endometrial adenocarcinoma line HEC-1 during incubation with [3H]estradiol ([3H]E2) at 4 C causes substantial increases in cytoplasmic E2 binding levels. A similar effect can be observed in homogenates of normal human endometrium. These effects of molybdate appear to involve activation of E2-binding sites. Fractionation of the homogenates and recombination of different fractions revealed that activation of specific E2-binding sites by by MoO4= requires cytosolic factors as well as factors associated with the cell membrane. In homogenates of neoplastic cells (HEC-1) and normal endometrium, the addition of ATP, GTP, or cGMP was also found to increase E2 binding to levels as high as those obtained by the addition of MoO4=. In contrast, the addition of cAMP was found to lower specific E2 binding levels and to counteract the effects of MoO4=, ATP, GTP, and cGMP. Levels of intracellular cAMP and cGMP can change rapidly in cells in culture. Since cGMP causes E2 binding levels to increase while cAMP causes them to decrease, changes in the levels of these two cyclic nucleotides may explain the fluctuation in concentrations of specific estrogen binders that we have previously reported to occur in cultured endometrial cells.

Adenocarcinoma↗
PubMed1982

DSM-III and structural diagnosis of borderline patients.

This study compared diagnoses arrived at through a structural interview developed by Kernberg which assesses levels of personality organization with those arrived at using DSM-III criteria of signs and symptoms among a group of hospitalized patients. Complete agreement was reached in 61 percent of the cases. Among the cases in which there was disagreement, 39 percent could nevertheless be considered to be compatible within the two systems, while the remaining cases were not. Discussion is directed to the different facets of clinical material addressed with each of these two diagnostic approaches, their discrepancies, and the underlying relationship between the two systems.

Humans↗
PubMed1982

Fluorescent membrane probes and the mechanism of maintenance of cellular asymmetry in epithelia.

Individual epithelial cells are asymmetric with respect to morphology, distribution of enzymes, lipid composition, ionic permeability, and sensitivity to drugs and hormones on the apical (mucosal) versus basolateral (serosal) membranes. It has been suggested that the tight junction, which forms the morphological boundary between apical and basolateral surfaces, helps to maintain this cellular asymmetry by forming a barrier to lateral diffusion of membrane constituents across these membranes. We have directly tested this hypothesis by selectively labeling either the apical or basolateral membrane with fluorescent probes and ascertaining 1) whether the probe can diffuse laterally in the membrane, and 2) whether it can pass through the tight junction region to the side of the cell initially not labeled. Our results show that membrane-bound lectins and some lipid probes are incapable of passing through the tight junction region. The lectins are immobile on the cell surface, but the lipid probes diffuse freely in the membrane. We propose that the ability of a lipid probe to pass through the tight junction is correlated with its ability to flip-flop to the inner monolayer of the cell membrane bilayer, and that the tight junction thus forms a barrier to lipids in the outer monolayer only. Differences in diffusion rates of different lipid probes also appear to reflect different physical characteristics of the inner versus outer membrane leaflet.

Animals↗