Biomedical subjects

R Margalit

Publications and source records attributed to R Margalit.

At least 37 records · Page 2Linked to original sources

PubMed1996

Comparison of the properties of native and pentaammineruthenium(III)-modified xylanase.

Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were purified and then modified by the attachment of pentaammineruthenium, thereby resulting in the generation of a xylanase with veratryl alcohol oxidase activity. Hydrolytic activity of T. reesei xyn II on soluble xylans was unchanged by modification with pentaammineruthenium; however, modification of B. pumilus xynA greatly reduced xylan hydrolysis unless the active site of the xylanase was protected with xylose during the modification. The presence of histidine, cysteine, or reduced glutathione during xylan hydrolysis greatly increased the xylanase activity of the pentaammineruthenium-modified B. pumilus xylanase. Glycine, glutamic acid, methionine, or oxidized glutathione had no effect on xylanase activity.

Alcohol Oxidoreductases↗

PubMed1995

Liposome-encapsulated silver sulfadiazine (SSD) for the topical treatment of infected burns: thermodynamics of drug encapsulation and kinetics of drug release.

Liposomes encapsulating silver sulfadiazine (SSD), the drug of choice for topical treatment of infected burns, are investigated as an improved delivery system that could act as a locally targeted sustained-release drug depot. This communication reports the first stage of the investigation and is focused on (a) the development of spectrophotometric assays for liposome-encapsulated and for free (aqueous soluble) and SSD, (b) on evaluation of the efficiency of encapsulation and kinetics of drug release. DMSO containing 140 mM NH3 was found to be the best solvent for dissolution of the liposomes and for determination of their SSD content. Peak absorption of liposome-originating SSD in this solvent is at 263 nm with em values of 23 x 10(3)-26 x 10(3). Peak absorption of SSD in aqueous solutions is at 254 nm with em magnitudes varying from 2 x 10(3) to 23 x 10(3), depending on the electrolytic composition of the system. Kinetic studies of drug release and separations by centrifugation and by gel-exclusion chromatography all indicate that the SSD in the liposomal system is distributed among three states: encapsulated, soluble unencapsulated, and stable (unencapsulated) aggregates that reside in the aqueous phase in which the liposomes are suspended. The liposomal SSD systems were found to meet the essential requirements of high-efficiency encapsulation and sustained drug release. Encapsulation efficiencies of > 80% at 10 mM lipid, reaching up to 95% at 100 mM lipid, were obtained. The release of encapsulated SSD follows first-order kinetics, with half-life up to 24 hr and with sensitivity to the electrolytes in the system. It is concluded that SSD-liposomal systems are feasible, have potential benefits over treatment with free SSD, and merit further pursuit into providing local targeting.

Burns↗

PubMed1995

A disaccharide that inhibits tumor necrosis factor alpha is formed from the extracellular matrix by the enzyme heparanase.

The activation of T cells by antigens or mitogens leads to the secretion of cytokines and enzymes that shape the inflammatory response. Among these molecular mediators of inflammation is a heparanase enzyme that degrades the heparan sulfate scaffold of the extracellular matrix (ECM). Activated T cells use heparanase to penetrate the ECM and gain access to the tissues. We now report that among the breakdown products of the ECM generated by heparanase is a trisulfated disaccharide that can inhibit delayed-type hypersensitivity (DTH) in mice. This inhibition of T-cell mediated inflammation in vivo was associated with an inhibitory effect of the disaccharide on the production of biologically active tumor necrosis factor alpha (TNF-alpha) by activated T cells in vitro; the trisulfated disaccharide did not affect T-cell viability or responsiveness generally. Both the in vivo and in vitro effects of the disaccharide manifested a bell-shaped dose-response curve. The inhibitory effects of the trisulfated disaccharide were lost if the sulfate groups were removed. Thus, the disaccharide, which may be a natural product of inflammation, can regulate the functional nature of the response by the T cell to activation. Such a feedback control mechanism could enable the T cell to assess the extent of tissue degradation and adjust its behavior accordingly.

Animals↗

PubMed1995

Liposome-mediated drug targeting in topical and regional therapies.

Liposome-mediated drug targeting is reviewed in four major categories of topical and regional therapies: wounds and burns, ocular, intraperitoneal, and pulmonary. A survey of the data in the field is preceded by definitions of carrier-mediated drug targeting, in particular for topical and regional treatments. The ability of liposomes to meet essential requirements for task performance and liposome surface-modification as the major approach to endow liposomes with targeting abilities are reviewed. Analysis of current findings in the field shows that (1) most studies explored regular liposomes that were unable to meet the essential requirements for targeting and (2) in vivo drug targeting in topical and regional therapies has been achieved rarely and seldom attempted, yet there are encouraging indications from a few studies that using surface-modified liposomes such targeting is feasible. Both established and novel liposomal systems attest to this feasibility and point out future directions. The former can be found by revisiting immunoliposomes that were initially designed for systemic administration but might well fit topical and regional cases. The latter is exemplified by bioadhesive liposomes, designed specifically for topical/regional therapies. It is concluded that careful implementation of such approaches could be successful for the achievement of liposome-mediated drug targeting in topical and regional therapies.

Administration, Topical↗

PubMed1994

Veratryl alcohol oxidase activity of a chemically modified cellulase protein.

A cellulase, cellobiohydrolase I (CBH I) from Trichoderma reesei was chemically modified by covalent attachment of pentaammine ruthenium (III) without loss in hydrolytic activity. Data suggest that such a modification endowed CBH I with oxidoreductase activity. The modified enzyme was able to carry out hydrogen peroxide-dependent oxidation of veratryl alcohol, a substrate for lignin peroxidase, at a rate of 0.148 mumol substrate oxidized min-1 mumol-1 enzyme. The effects of pH, temperature, and substrate concentration on the oxidation reaction were examined. The optimal temperature was determined to be 45 degrees C, and the optimal pH was 4.3. The Km and Vmax for veratryl alcohol were determined to be 3.519 mM and 52.27 microM min-1, respectively. Tartrate at concentrations as low as 0.10 mM was found to inhibit the reaction.

Alcohol Oxidoreductases↗

PubMed1994

Bioadhesive, collagen-modified liposomes: molecular and cellular level studies on the kinetics of drug release and on binding to cell monolayers.

Liposomes, modified by covalently-anchoring collagen to their surface, were investigated for their abilities to be bioadhesive and to act as sustained-release drug carriers. These bioadhesive liposomes have the potential to induce significant improvements in topical and regional therapies. The major findings for uni-(ULV) and multilamellar (MLV) bioadhesive liposomes are: (a) Both ULV and MLV release small molecular weight drugs over prolonged periods. For example, rate constants of (6 +/- 0.5) x 10(-3) and (2.6 +2- 0.8) x 10(-3) h-1, were obtained for the release of vinblastine and fluconazole, respectively, from collagen-ULV. (b) For a given drug, that rate constant can be shifted (up or down) by the choice of liposome type and collagen-surface density and the latter, if high enough, lead to the formation of an additional liposome-associated drug reservoir. (c) Using monolayers of the A431 cell line to model the in vivo targets, the bioadhesive (but not the regular) liposomes were found to bind with high affinity to the monolayers. For example, equilibrium dissociation constants of 6.3(+/- 3) microM and 2.7(+/- 0.5) microM were determined for bioadhesive MLV and ULV, respectively, with corresponding saturation occupancies of 3.7(+/- 1) and 4.0(+/- 0.2) pmoles liposomal collagen/monolayer of 10(5) cells. (d) Following the retention of bioadhesive MLV at A431 monolayers for 24 h, it was found that: at 4 degrees C, 24 h did not suffice to reach equilibrium, but at 37 degrees C equilibrium binding was obtained within 3-5 h and there was quantitative liposome retention (per viable monolayer) thereafter. It is concluded that these liposomes are bioadhesive sustained-release carriers, as desired, meriting further cellular and in vivo studies.

Binding Sites↗

PubMed1994

Molecular and cellular studies of hyaluronic acid-modified liposomes as bioadhesive carriers for topical drug delivery in wound healing.

Liposomes, modified into bioadhesive systems by the covalent anchoring of hyaluronic acid (HA) to their surface, were investigated for their abilities to act as site-adherent and sustained-release carriers of drugs for the topical therapy of wounds and burns. Epidermal growth factor (EGF) served as the test drug for growth factors and monolayers of the A431 cell line served as models of the in vivo designated targets. The hyaluronic acid was radiolabeled to carry 3H, through a deacetylation-reacetylation procedure, yielding specific activities in the range of 1 x 10(6) dpm/mg. The HA was bound to the surface of the liposomes, using carbodiimide to crosslink its carboxyl residues to amine residues at the liposomal surface. The latter was provided through the inclusion of phosphatidylethanolamine in the liposome formulation. Varying the initial HA/lipid ratio over the range 1-500 mg HA/mmol lipid, it was found that the increase in bound HA was linear, providing up to 80 mg HA/mmol lipid. The HA-modified, but not the nonmodified, liposomes were found to bind with high affinity (delta G degree in the range of -7 to -9 Kcal/mol) to monolayers of the A431 cell line. The HA-modified liposomes were found to encapsulate EGF at high yields (% encapsulation, > 87) and to act as sustained-release depots, the rate constant for the release of the encapsulated EGF in the range of 1.4 x 10(-3) h-1. Taking all of these findings together, it is concluded that these liposomes are bioadhesive sustained-release carriers of drugs, as desired, meriting further cellular and in vivo studies.

Cell Line↗

PubMed1994

Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Intravenous gammaglobulin treatment in multiple sclerosis and experimental autoimmune encephalomyelitis: delineation of usage and mode of action.

Multiple sclerosis (MS) is a central nervous system demyelinating disease of implicated autoimmune aetiology. The effect was evaluated of intravenous gammaglobulin (IVIg), a successful therapy in various autoimmune diseases, in relapsing-remitting MS patients treated for three years. IVIg treatment significantly reduced the number and severity of acute exacerbations and resulted in a lesser neurological disability. There were no significant short or long-term adverse effects to IVIg treatment. To clarify the putative therapeutic effects of IVIg, this treatment was examined in the animal model of experimental autoimmune encephalomyelitis (EAE) in the rat. IVIg suppressed active EAE in relation to disease severity and duration, despite the presence of T-cell reactivity to specific antigens, while the treatment had no effect on passive EAE induced by adoptive transfer of myelin basic protein specific CD4 + T-cells. It is concluded that IVIg treatment may be a promising treatment in relapsing-remitting MS as it can alter the natural course of the disease.

Adult↗

PubMed1994

Intravenous immunoglobulin treatment of experimental T cell-mediated autoimmune disease. Upregulation of T cell proliferation and downregulation of tumor necrosis factor alpha secretion.

It has been reported previously that intravenous administration of normal human immunoglobulins (IVIg) to human patients can suppress the clinical signs of certain autoimmune diseases. However, the mechanism(s) by which normal Ig interferes with the various disorders and the scheduling of treatment have been poorly delineated. To study these questions, we examined IVIg treatment of two experimentally induced T cell autoimmune diseases in rats: experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA). We now report that IVIg treatment (0.4 g/kg) inhibited the active induction of both EAE and AA, and that this treatment did not affect the acquisition of resistance to reinduction of EAE. The importance of the site of administration and schedule of treatment were studied in the AA model. Ig was effective when given intravenously, but not when administrated subcutaneously or intraperitoneally. IVIg treatment was effective when given daily from immunization to outbreak of disease; but it was also effective when given once at the time of immunization or once 2 wk after induction of AA, just at the clinical outbreak of disease. Administration of IVIg between immunization and outbreak of AA was less effective. Prevention of disease by IVIg occurred despite the presence of T cell reactivity to the specific antigens in the disease. In fact, IVIg administrated to naive rats activated T cell reactivity to some self-antigens. Nevertheless, IVIg treatment led to decreased production of the inflammatory cytokine TNF alpha. Thus, IVIg treatment may exert its therapeutic power not by inhibiting T cell recognition of self-antigens, but by inhibiting the biological consequences of T cell recognition.

Animals↗

PubMed1993

Attachments of pentaammineruthenium(III) to Trichoderma reesei cellobiohydrolase I increases its catalytic activity.

Pentaammineurthenium(III) was covalently attached to cellobiohydrolase I (CBH I, EC 3.2.1.91), the major component of Trichoderma reesei cellulase, resulting in 0.7 mol ruthenium/mol CBH I and an electrode potential of +95 mV. Fractionation of modified CBH I by chromatofocusing resulted in the separation of fractions with a 1.4- to 3.2-fold increase in specific activity toward p-nitrophenylcellobioside, depending on the assay conditions, over that of native enzyme. The extent of the hydrolysis of insoluble cellulosic substrates (Avicel and newsprint) to glucose by modified CBH I was also greater than that observed by the native enzyme.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1993

Thermodynamics of the binding of hematoporphyrin ester, a hematoporphyrin derivative-like photosensitizer, and its components to human serum albumin, human high-density lipoprotein and human low-density lipoprotein.

The phenomena of the high affinity of porphyrins to the human serum proteins, albumin, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) is well established. Yet, evaluation of the activities of these proteins as endogenous porphyrin carriers, especially with respect to receptor-mediated porphyrin uptake into tumor cells, the merits of which are still in dispute, requires more quantitative protein-porphyrin binding data. As a continuation of previous studies on this issue, the binding of several porphyrin systems to each of the three proteins, employing previously developed spectral methodologies, was studied. The specific systems reported here are hematoporphyrin ester (HPE), which is a novel hematoporphyrin derivative (HPD)-like system, two porphyrin trimers (denoted O1 and O2) and a porphyrin dimer (denoted O3) isolated from HPE. Human serum albumin (HSA) was found to have a single high-affinity site for the monomeric components of HPE, with an equilibrium binding constant of 3.6 x 10(6). The equilibrium parameters determined for the binding of the three HPE-isolated oligomers to each of the serum proteins are: (1) Binding constants (Kb') of 2.3 x 10(6), 6.9 x 10(4) and 1.5 x 10(4) and number of sites per protein molecule (n) of 3, 1 and 5, for the binding of O1, O2 and O3, respectively, to HSA. (2) Kb' values of 15.5 x 10(3), 15.3 x 10(3) and 6.6 x 10(3) and n values of 1, 2 and 2, for the binding of O1, O2 and O3, respectively, to HDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Esters↗

PubMed1992

Structure-activity relationships among noncyclic dicarboxamide Li(+)-selective carriers studied in lipid bilayer membranes.

The structure-activity relationships among three noncyclic diimide ionophores, designed to be Li+ carriers, were studied in lipid bilayer membranes. These ionophores (ETH1644, ETH1810, and ETH1811) vary in their N-imide substituents, going from two isobutyls to one isobutyl and one cyclohexyl to two cyclohexyls, respectively. ETH1811 was found to form two types of complexes with 1:1 and 1:2 ion-carrier stoichiometries, the former type dominant over most of the ionophore (0.1-10 microM) and salt (0.01-1.0 M) concentration ranges studied. In contrast, ETH1644 and ETH1810 were previously found to form a single type of complex with the 1:2 stoichiometry. The alkali cations selectivity sequence induced by ETH1811 is Li+ (1) greater than Na+ (0.08) greater than K+ (0.02) greater than Cs+ (0.008). These ETH1811-induced ionic selectivites as well as its relative potency in ion transport were found to be inferior to those of ETH1644 and ETH1810 (the latter being the best in this series). The conductance-voltage relationships reported here, for all three ionophores transporting alkali cations, were found to fit with a transport mechanism in which the diffusion of the ion-ionophore complex across the membrane is the single rate-limiting step, with the following exceptions: The addition of the dissociation of the ion-carrier complex as a second rate limiting step, for the Li(+)-ETH1810 and the Li(+)-ETH1811 systems. For ETH1810 the kinetics of the dissociation step is a minor, whereas for ETH1811 case it is a significant, addition (the ratio of the diffusion to dissociation rate constants being 0.08 and 0.2, respectively). The implications of the effects of the continuous structural change of these ionophores on their performance as ion carriers and on the design and synthesis of improved Li(+)-selective ionophores are discussed.

Amides↗

PubMed1992

Tamoxifen induced changes in MCF7 human breast cancer: in vitro and in vivo studies using nuclear magnetic resonance spectroscopy and imaging.

The effects of 17 beta-estradiol versus tamoxifen on the growth and metabolism of MCF7 human breast cancer cells, in culture and in tumors implanted in nude mice, were studied by 31P and 13C nuclear magnetic resonance spectroscopy and by proton magnetic resonance imaging. In culture, the content of the phosphate metabolites including nucleoside triphosphates (NTP), phosphomonoesters, phosphodiesters and inorganic phosphate (Pi) were not affected by tamoxifen treatment. However, in the presence of estrogen the rate of glucose consumption and lactate production via glycolysis (270 and 280 fmol/cell.h, respectively) were twice that of tamoxifen treated cells. Estrogen rescue of tamoxifen treated cells indicated that glycolysis induction occurs at the early stages of the hormonal response. The in vivo studies included recording of proton images that provided an accurate measure of tumor size and distribution of tumor cells, necrotic regions and stromal tissue. Tamoxifen caused enhanced necrosis extending from the center of the tumor during the first two days of treatment (12 h to 6 days). This was followed by growth of reparative tissue along with tumor regression. Tamoxifen also modified the content of the phosphate metabolites, increasing markedly (P less than 0.0002) the ratio of NTP to Pi from 0.41 before treatment to 1.75 9-19 days after treatment. This change was attributed to the enhanced growth of repair tissue. The results provide new information regarding the response of human breast cancer to hormonal treatment and suggest a mechanism for the induction of tumor regression by tamoxifen.

Animals↗

PubMed1991

Lithium-selective permeation through lipid bilayer membranes mediated by a di-imide ionophore with nonsymmetrical imide substituents (ETH1810).

The neutral, noncyclic Li(+)-selective ionophore ETH1810, which is a di-imide, differs structurally from previous similar ionophores by removal of the intramolecular symmetry of the N-imide substituents. Properties of this ionophore, as a potential carrier of lithium, were probed through studies of ionophore-induced changes in electrical properties of lipid bilayer membranes. ETH1810 was found capable of transporting lithium and other monovalent cations, across lipid bilayer membranes, forming 2:1 ionophore:ion membrane-permeating species. It was found to be 10-fold more potent than ETH1644, which was the previous best ionophore of this type. The selectivity sequence among alkali cations was found to be: Li+ (1) greater than Na+ (0.009) greater than K+ (0.004) greater than Cs+ (0.0035). Among the physiological alkali cations, it constitutes a 40 (vs. Na+) to 160% (vs. K+) improvement over ETH1644. ETH1810 was also found to be capable of acting as a carrier of biogenic amines and related molecules, with the following selectivity sequence:tryptamine (20) greater than phenylethylamine (7.8) greater than tyramine (4.3) greater than serotonin (2.5) greater than Li+ (1) greater than NH+4 (0.013) greater than dopamine (0.012). It was found that protons, at physiological concentrations, do not interfere with the lithium transport mediated by ETH1810. The relationship between the improvements in ionic selectivity and potency vs. the differences in structural features is discussed.

Biogenic Amines↗

PubMed1991

In vivo studies by magnetic resonance imaging and spectroscopy of the response to tamoxifen of MCF7 human breast cancer implanted in nude mice.

Magnetic resonance 1H-imaging and 31P-localized spectroscopy were utilized to monitor, noninvasively, MCF7 human breast cancer tumors implanted in immunodeficient mice. The tumors were followed during estrogen dependent growth and tamoxifen induced remission. Early after tamoxifen administration enhanced necrosis developed, extending to most of the tumor volume. This was followed by growth of repair tissue along with tumor regression. The short-term tamoxifen treatment also modified the content of the phosphate metabolites, increasing the nucleoside triphosphate to inorganic phosphate ratio from 0.41 +/- 0.15 (n = 14) before treatment to 1.10 +/- 0.70 (n = 8, P less than 0.025) and to 1.75 +/- 0.66 (n = 9, P less than 0.0002) 4-7 days and 9-19 days, respectively, after treatment. This change was attributed to the growth of reparative tissue. The results provide new information regarding the response and the mechanism of action of tamoxifen.

Animals↗

PubMed1990

Binding of porphyrin to human serum albumin. Structure-activity relationships.

The equilibrium binding of hydroxyethyl vinyl deuteroporphyrin (HVD) and of irreversible porphyrin aggregates to human serum albumin was studied at the molecular level. This protein may function as an endogenous drug carrier for porphyrins in photodynamic therapy of tumours. HVD-protein binding studies revealed two types of binding sites, which are attributed to the two HVD isomers. The binding constant for the high-affinity isomer, 2.1 (+/- 0.3) x 10(8) M-1, is similar to that previously determined for protoporphyrin. At the same time the binding constant for the lower-affinity HVD isomer, 1.8(+/- 0.3) x 10(6) M-1, is similar to that previously determined for haematoporphyrin. Irreversible porphyrin aggregates were purified from the haematoporphyrin derivative and from Photofrin and are defined by spectral and chromatographic data. Gel-exclusion studies indicate that the dominant size of these aggregates is ten porphyrin monomeric units. The protein-binding constant of these aggregates is 1.7(+/- 0.2) x 10(5) M-1, with four binding sites per protein molecule. The distinction between the HVD isomers along the porphyrin-protein affinity sequence gives insight into the relationship of porphyrin structure to porphyrin-albumin binding. On the basis of this study an evaluation of human serum albumin as an endogenous carrier for porphyrins (at various aggregation states) in photodynamic therapy of tumours is presented.

Binding Sites↗

PubMed1990

Determination of protein orientation on surfaces with X-ray photoelectron spectroscopy.

The use of X-ray photoelectron spectroscopy (XPS) for determination of the preferred orientation of proteins on metal surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium (III) group is attached to a specific histidine (residue 81) is used for this purpose, with the Ru and Fe acting as a double marker. The relative intensities of the XPS photoelectron spectra for these two markers are shown to be a sensitive measure of orientation of the protein film. On both aluminum and indium-tin-oxide surfaces, XPS indicates that the Ru-Fe axis of the myoglobin derivative is preferentially oriented with the Ru furthest from the substrate.

Electron Probe Microanalysis↗