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Biomedical subjects

A Lustig

Publications and source records attributed to A Lustig.

At least 73 records · Page 4Linked to original sources
PubMed1994

Independent folding of the domains in the hydrophilic subunit IIABman of the mannose transporter of Escherichia coli.

The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli is a homodimer of two 35-kDa subunits. Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion. Separation of tryptic fragments yields monomers of IIB and dimers of IIA, which are active and stable. To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments. Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each isolated fragment. The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and second transitions of intact IIABman. Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA. Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments. The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions. The lack of interdomain interactions suggests an unrestricted domain motion. This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain).

Calorimetry, Differential Scanning↗
PubMed1994

The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83.

The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E. coli and purified under non-denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.

Amino Acid Sequence↗
PubMed1994

Cost evaluation of a new iv device in an Israeli hospital.

A new device for IV admixtures has been developed in Israel, the Vialink minibag. It consists of a partly filled minibag, attached by rubber tubing to a sterile receptable into which various types of vials containing drugs can be firmly inserted. Time and motion studies were conducted to determine the time and cost associated with the preparation, dispensing and administration of reconstituted parenteral antibiotics via CIVAS (within minibags or Vialink bags) and preparation by nurses in wards in minibags. CIVAS with minibags was least expensive, followed by Vialink minibags. Self preparation on the ward by nurses was found to be the most expensive system. Only 55% of all IV admixtures can be prepared by the Vialink system. As only one hospital in Israel has CIVAS, the study provides a solid justification for implementing pharmacy intravenous admixture services in all Israeli hospitals. The advantages and disadvantages of each method are discussed. CIVAS (minibags and Vialink) permits the individualised preparation of sterile admixtures for each patient and provides advantages in terms of efficiency, economy and prevention of errors.

Costs and Cost Analysis↗
PubMed1994

Pharmacists as members of the healthcare team in Israel: sectoral differences.

OBJECTIVE: To examine the sectoral differences among Israeli pharmacists regarding their perception of actual versus desired contact with other members of the healthcare team, and measures necessary to achieve a closer working relationship among members. DESIGN, SETTING, AND PARTICIPANTS: Pharmacists from private, community, and hospital sectors (n = 145) completed a survey that assessed their respective views of the current and future roles of the pharmacist as a member of the healthcare team. MAIN OUTCOME MEASURES: Common to pharmacists in all three sectors is their aspiration for greater cooperation among members of the healthcare team, especially with physicians. Intersectoral differences were found in their attitude toward their role in the healthcare team: hospital pharmacists would like a better relationship with patients, private pharmacists would like closer ties with physicians, and community pharmacists would like better teamwork with the nurse. No sectoral differences were seen in how pharmacists see themselves as members of the clinical healthcare team. Only 12 percent believe they have adequate knowledge to advise on clinical pharmacotherapy. To perform this function, they indicated that more training in pharmacotherapy would be needed (98.6 percent of respondents), as well as joint courses with medical students (84.2 percent), and a modification of their internship period requiring them to work in each of the different practice sectors (73.4 percent). No sectoral differences were observed regarding the benefits of a more active role of the pharmacist in the healthcare team. CONCLUSIONS: One possible explanation for Israeli pharmacists' low professional self-image, despite the rewards of the profession, is that they lack the necessary clinical knowledge that would enable them to take a more active role in the healthcare team. Pharmacists' job satisfaction may improve if a more active advisory role in the healthcare team can be achieved.

Adult↗
PubMed1993

Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides.

The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different fractions of the same preparation. Whereas PAB II stimulated poly(A) polymerase, PAB I was an inhibitor. In analytical ultracentrifugation, the predominant form of PAB II was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a distinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The dependence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KD of 2 x 10(-9) M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retardation experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.

Animals↗
PubMed1993

[Evaluation of a new intravenous device].

Problems associated with intravenous (IV) admixture preparation include contamination by microorganisms, human error and expense. To solve some of these problems, the Vialink system was developed by Travenol, Israel. This device enables enhanced sterility in clinical settings, since in the preparation of the admixture there is only a single penetration of the rubber seal of the vial. The dilution is performed immediately prior to administration, thereby reducing the possibility of wastage due to unused admixture. In addition, Vialink offers a means of double-checking the contents of the admixtures, since the emptied vial remains attached to the infusion bag. Results of our evaluation of this new system indicate that it offers substantial savings in preparation time and expense. It was suitable for 58.2% of all IV admixtures routinely hospital-prepared. The Vialink system may be a useful strategy, as it improves the quality of the service provided by the health service team and hospital pharmacists to their patients, and results in savings in budget and manpower.

Drug Combinations↗
PubMed1992

Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.

Amino Acid Sequence↗
PubMed1992

Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.

Animals↗
PubMed1992

Professional self-image among Israeli pharmacists: sectoral differences.

OBJECTIVE: To examine sectoral differences in the professional activities and self-images of Israeli pharmacists. DESIGN, SETTING, AND PARTICIPANTS: Pharmacists from private, community, and hospital sectors (n = 145) completed a survey that assessed the rewards accrued from their professional activities as pharmacists and evaluated their professional and sectoral self-image. RESULTS: Few intersectoral differences were found among Israeli pharmacists. Most agreed that the main rewards in the public sector are secure employment, intellectual challenge, and responsibility. In the private sector, the rewards are high income, responsibility, and opportunity to counsel. The majority of pharmacists in all sectors are satisfied with their profession, and 90.7 percent would choose it again. Nevertheless, they ranked pharmacy eighth among nine professions chosen for comparison, which shows quite a low professional image. The private and hospital pharmacists attributed a higher social status to their own sectors than to the community sector. CONCLUSIONS: The inferior professional self-image held by pharmacists, despite the rewards of the profession, should be examined in greater detail.

Adult↗
PubMed1991

Density determination by analytical ultracentrifugation in a rapid dynamical gradient: application to lipid and detergent aggregates containing proteins.

A rapidly developing dynamical gradient can be formed in the analytical centrifuge when a buffer solution prepared in D2O is underlayered under the same buffer solution prepared in H2O in a specially designed double sector cell. In a short time the boundary layer spreads to form the gradient. Heavy particles (S greater than or equal to 10S) will band in the gradient corresponding to their density, which can be determined accurately. To this end, the buffer may contain density adjusting additives such as sucrose. We present results with this technique for lipid vesicles, for a Ca-antagonist bound to vesicles, as well as for a lipoprotein and reconstituted regular membrane protein-lipid arrays.

Animals↗
PubMed1991

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.

Animals↗
PubMed1991

Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

Binding, Competitive↗
PubMed1991

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Formaldehyde↗
PubMed1991

Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions.

Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.

Animals↗
PubMed1991

Weight loss programs: failing to meet ethical standards?

Ethical concerns raised by the treatment of obesity include the following: the question of whether obesity treatment is accurately described as therapy or as research given its low percentage of long-term success; the need for standardized disclosure of the possibility of harms vs the low probability of benefits; the status of voluntary and informed consent as a meaningful model of patient cooperation in treatment in light of reported findings of "treatment addiction" as a recognized syndrome; and the policy issues raised by the absence of uniform standards for and regulatory oversight of weight loss programs.

Behavior Control↗
PubMed1990

The MAS-encoded processing protease of yeast mitochondria. Overproduction and characterization of its two nonidentical subunits.

The amino-terminal presequences of proteins imported from the cytoplasm across the mitochondrial inner membrane are cleaved off by a soluble matrix-localized protease composed of two nonidentical homologous subunits. In the yeast Saccharomyces cerevisiae, these are encoded by the nuclear MAS1 and MAS2 genes. We have now constructed yeast strains in which either one or both of the genomic MAS genes are controlled by a galactose-inducible strong promoter. In these strains, the intramitochondrial concentration of each MAS-encoded subunit as well as of the holo-protease can be varied over a wide range. When overproduced, the MAS1 protein precipitates in the matrix whereas the MAS2 protein remains soluble. The MAS2 protein was obtained at a purity of 98% in milligram amounts. The purified MAS2 subunit exists largely as a soluble 52-kDa monomer. Its cleavage activity is very low and might well reflect the 2% contamination by holoprotease. Activity is restored by adding the solubilized purified MAS1 subunit. Yeast cells depleted of one or both MAS subunits continue to import precursor proteins into mitochondria, but fail to cleave them; eventually the deficient cells stop growing. This growth arrest is partly suppressed on minimal medium or under conditions in which the cells are less dependent on mitochondrial metabolism. Depletion of the MAS1 subunit causes overproduction of the MAS2 subunit.

Cell Nucleus↗
PubMed1989

Rapid isolation of OmpF porin-LPS complexes suitable for structure-function studies.

A gentle and rapid isolation procedure is described yielding fractions containing better than 95% pure OmpF porin of Escherichia coli BE with different amounts of bound lipopolysaccharide (LPS). The procedure employs continuous free-flow electrophoresis (FFE) in the presence of detergent above its critical micelle concentration. Total yields of around 45% were typically obtained when porin-enriched membrane extracts were processed. By use of analytical ultracentrifugation a molecular mass of 114,000 and a sedimentation coefficient S20,w of 5.0 S were determined for porin trimers containing approximately 1 mol of tightly bound LPS. This porin readily formed 3D crystals suitable for high-resolution X-ray diffraction analysis. Three other porin-LPS isoforms isolated by FFE revealed molecular masses of 120,000, 124,000, and 151,000, suggesting that, in addition to the tightly bound LPS, 1, 2, and 8 mol of loosely bound LPS were present per mole of porin trimer. Each of the four different isoforms was suitable for reconstitution into highly ordered protein-lipid membrane arrays. The membrane crystals obtained with the 151-kDa isoform exhibited a unit cell polymorphism similar to that previously reported.

Bacterial Outer Membrane Proteins↗