Biomedical subjects

M J Poznansky

Publications and source records attributed to M J Poznansky.

At least 19 recordsLinked to original sources

PubMed1992

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)-loaded lipid vesicles and SOD-loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O2.- while SOD inside the lipid bilayers has no effect. These results confirm that O2.- is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti-oxidant therapy is to be approached relative to the site of oxygen free radical production.

Cell Membrane Permeability↗

PubMed1992

The effects of ischemia and ischemia-reperfusion on bacterial translocation, lipid peroxidation, and gut histology: studies on hemorrhagic shock in pigs.

The bacterial translocation hypothesis was tested in two studies (acute and subacute) in a porcine model of hemorrhagic shock. Male pigs (30-40 kg each) under general anesthesia had their femoral vein, femoral artery, and portal vein catheterized. After stabilization (1 hour) they were bled (40% of blood volume) over 30 minutes, then maintained in the hypotensive state (MAP = 30-40 mm Hg) for 2 hours, following which, according to randomization, they entered the control group or were resuscitated with whole blood (WB group) or with lactated Ringer's solution (LR group). In the acute study, the mesenteric efferent lymphatic was also cannulated, the control group was not resuscitated, and the animals remained under general anesthesia to the end of the experiment (8.5 hours), when gut tissue was obtained for histologic study and measurement of lipid peroxidation. In the subacute study, the control group was not bled, the animals were awakened at 6.5 hours, and the portal vein catheter remained in situ until 48 hours. In both studies, samples of portal blood were obtained for culture at regular intervals and on completion, samples from mesenteric lymph nodes (MLNs) for culture were taken in the acute study, and in the subacute study samples from MLNs, spleen, and liver were obtained. In the acute study significant bacterial translocation to the MLNs and portal blood did not occur among the controls (n = 3), the LR group (n = 5), and the WB group (n = 6). Significant evidence of lipid peroxidation was found in both the LR and WB groups. Histologic assessment showed no difference among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effect of intrathecal superoxide dismutase and catalase on oxyhemoglobin-induced vasospasm in monkeys.

A gel consisting of agarose and oxyhemoglobin (OxyHb) was developed so that, when placed in the subarachnoid space, OxyHb would be slowly released, simulating lysis of erythocytes after subarachnoid hemorrhage. The system was used to investigate the importance of reactions mediated by free radicals in the genesis of OxyHb-induced vasospasm in monkeys. Seventeen monkeys were randomly assigned to have subarachnoid placement, on Day 0, of one of the following: 1) agarose gel alone (n = 2); 2) agarose plus OxyHb (n = 3); 3) agarose plus OxyHb plus intrathecal administration of superoxide dismutase and catalase (n = 6); and 4) agarose plus OxyHb plus intrathecal administration of placebo (n = 6). Vasospasm was assessed by comparison of angiograms performed on Day 0 and 7 days after subarachnoid placement of compounds, and by electron microscopy. OxyHb alone caused significant reduction in the diameter of the middle cerebral artery (40 +/- 8%, P less than 0.005, paired t test), which was associated with ultrastructural damage to smooth muscle. Treatment with superoxide dismutase plus catalase or with placebo attenuated vasospasm of the middle cerebral artery, although significant narrowing persisted in both groups (27 +/- 12% and 26 +/- 13%, respectively, P less than 0.05, paired t test). Analysis of variance showed no difference in the degree of vasospasm between groups exposed to subarachnoid placement of OxyHb. Cerebrospinal fluid aspirated from the cisterna magna on Day 7 contained elevated activity of superoxide dismutase in animals that received treatment. Malondialdehyde was undetectable in cerebrospinal fluid after subarachnoid placement of agarose alone, although it was present in similar amounts in all groups that received subarachnoid placement of OxyHb. Since intrathecal superoxide dismutase and catalase failed to protect against OxyHb-induced vasospasm, mechanisms mediated by free radicals may not be important in its genesis. As only one combination of doses of superoxide dismutase and catalase was administered, however, it may be that other dosage schedules might be efficacious.

Animals↗

PubMed1992

Neonatal jaundice and bilirubin encephalopathy: a clinical and experimental reappraisal.

Hyperbilirubinemia and bilirubin encephalopathy are well-known occurrences in the neonatal period. The fact that bilirubin might be toxic to neural cells stems from the neurological picture that has emerged and the associated hyperbilirubinemia. Studies of the relationship between hyperbilirubinemia in healthy full-term infants and the long-term consequences showed little or no effect of bilirubin. Nevertheless, bilirubin is still considered toxic to premature infants and infants with hemolysis. In addition, in spite of a fairly detailed understanding of the chemistry of bilirubin and the biochemistry of bilirubin metabolism, the primary target of its toxic effect is still not known. The difficulties in analyzing the results are due to the variation in experimental designs, the use of different animal models and cell systems and the use of unstable bilirubin solutions. We review some of the problems related to clinical and experimental studies of bilirubin toxicity.

Animals↗

PubMed1991

Low density lipoprotein receptors on epithelial cell (Madin-Darby canine kidney) monolayers. Asymmetric distribution correlates with functional difference.

Low density lipoprotein (LDL) receptors are present on both the apical and basal surfaces of confluent monolayers of Madin-Darby canine kidney (MDCK) epithelial cells grown on gelatin-coated polycarbonate filters. Although there is only a single species of receptor protein present, as shown by immunoblotting, the receptors on the two surfaces were found to behave differently. LDL receptors on the basal surface show all of the characteristics of the LDL receptor described in fibroblasts in that their number is dependent upon the sterol (or LDL) content of the medium; however, regulation is only affected by LDL in the medium in contact with the basal side. In contrast, the apical surface LDL receptors are not regulated by the presence of LDL in the media on either the apical or basal surface. LDL particles can be transported across the monolayer in a temperature-dependent and -specific manner from the apical to the basal sides of the cell, but not in the opposite direction. The binding of 125I-LDL to both surfaces can be effectively inhibited not only by unlabeled LDL and very low density lipoprotein, but also by an antibody directed against the LDL receptor. The data suggest that the LDL receptors on the two aspects of the cell surface are biochemically identical, but differ in function. Thus, the basal surface receptor is involved in the control of cell cholesterol homeostasis, while that on the apical surface is responsible for the transport of LDL to the basal side.

Animals↗

PubMed1990

Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells.

The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.

Animals↗

PubMed1990

Bilirubin-neural cell interaction: characterization of initial cell surface binding leading to toxicity in the neuroblastoma cell line N-115.

The toxicity of bilirubin to the nervous system might be due to its effect on several key enzyme reactions occurring in the intracellular compartment as suggested by in vitro studies. The question of how bilirubin, a molecule with poor solubility in water and organic solvents, interacts with the plasma membrane and reaches intracellular targets is unclear. In an attempt to get some insight into this problem, we have measured the uptake of bilirubin from bilirubin-albumin solutions by the murine neuroblastoma cell line N-115. At a constant total concentration of bilirubin, the initial rate, as well as the extent of uptake, increases with increasing bilirubin to albumin molar ratio (B/A). The binding is reversible, at least partially, as indicated by the ability of albumin to extract cell-bound bilirubin. The cellular uptake of bilirubin was found to depend also on the concentration of bilirubin, on temperature and on pH. The results are not consistent with either a carrier-mediated transport or passive diffusion across the plasma membrane. The data, however, seem to fit a multistep binding of bilirubin to the plasma membrane proposed for the interaction of bilirubin with synaptosomal plasma membrane vesicles, erythrocyte ghosts and lipid vesicles. These studies, thus, reveal the complexity of the binding interaction at the level of the plasma membrane and leave open the question of transport across the membrane.

Albumins↗

PubMed1990

Lipid peroxidation inactivates rat liver microsomal glycerol-3-phosphate acyl transferase. Effect of iron and copper salts and carbon tetrachloride.

Lipid peroxidation is known to affect the activity of several enzymes including microsomal enzymes such as glucose-6-phosphatase; but its effect on the enzymes of lipid biosynthesis has not been investigated. Glycerol-3-phosphate acyltransferase (GPAT) represents the first committed step and probably the rate limiting step in glycerolipid synthesis and thus may be a good candidate for study. Rat liver microsomal GPAT was assayed after preincubating the microsomes under conditions known to induce peroxidation. In 30 min, 10 microM Fe2+ can diminish the activity by as much as 80%. The inactivating effect can be blocked to different extents by several antioxidants, while ascorbic acid enhances it. These effects, along with the concomitant measurement of lipid peroxidation, indicate that microsomal GPAT activity is inactivated by lipid peroxidation in a sensitive and rapid fashion. This is further confirmed by the inactivating effect of carbon tetrachloride, which is known to induce lipid peroxidation in microsomes. Fe3+ also inactivates the enzyme, but at a higher concentration. Copper salts inactivate GPAT by a mechanism apparently different from that of iron. The mechanism might involve a direct sulfhydryl modification by copper and lipid peroxidation apparently different from that induced by iron. It is suggested that the inactivation of GPAT by lipid peroxidation could accelerate the process of membrane disintegration caused by lipid peroxidation in pathological conditions involving free radical-mediated tissue injury.

Acyltransferases↗

PubMed1990

A modified tetramethylbenzidine method for measuring lipid hydroperoxides.

A simple and sensitive spectrophotometric method for measuring lipid peroxides and peroxides in general is described. The method was developed by modifying an existing method based on the peroxidase activity of hemoglobin with tetramethylbenzidine as the electron donor. The modifications resulted in much improved sensitivity and reproducibility. With the modified method lipid peroxides as low as 2 nmol can be measured, a high sensitivity compared with other spectrophotometric methods. The absorbance is linear over a wide range of concentrations. It is suggested that this modified method in combination with the commonly used thiobarbituric acid method will give a better quantitation of lipid peroxidation.

Animals↗

PubMed1990

Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.

Animals↗

PubMed1990

Characterization of the ZO-1 protein in endothelial and other cell lines.

A high molecular weight tight junction-associated protein, ZO-1, has been demonstrated in liver (hepatocytes) and in both epithelium and endothelium. We carried out studies to examine the presence of the protein in vascular endothelial cell cultures and several other types of cultured cells, and the relationship between the ZO-1 protein content and confluency of endothelial cell monolayers. Immunofluorescence labelling of endothelial monolayers and two types of epithelial monolayers, IEC-6 and MDCK, with monoclonal antibody against ZO-1 protein localized the protein to the cell peripheries. Its association with the cell periphery only occurred when cells had contact with one another as demonstrated in endothelial cells. We have been able to show a positive correlation between the ZO-1 content of the cells and the extent of monolayer confluency in the endothelial cells by immunoblotting. The protein is much less expressed in nonconfluent endothelial cell monolayers and totally absent from mouse myeloma cultures. The presence and confluence-related expression of the protein in endothelium give support to the hypothesis that tight junctions exist in confluent endothelial cells and that the ZO-1 protein is expressed under the conditions where tight junction interactions occur.

Animals↗

PubMed1989

Curvature and composition-dependent lipid asymmetry in phosphatidylcholine vesicles containing phosphatidylethanolamine and gangliosides.

The effect of curvature on transbilayer lipid asymmetry in vesicles is investigated using vesicles of different sizes (30-140 nm) prepared by sonication and polycarbonate filter extrusion techniques. The transbilayer distributions of phosphatidylethanolamine and gangliosides are measured using 2,4,6-trinitrobenzenesulphonic acid and Clostridium perfringens neuraminidase as non-penetrating probes, respectively. The distribution of phosphatidylethanolamine in a phosphatidylcholine/phosphatidylethanolamine (4:1, molar ratio) system is more or less symmetric and curvature seems to have little effect. However, the distribution of gangliosides in a phosphatidylcholine/ganglioside (10:1, molar ratio) system is asymmetric in favour of the outer layer in smaller vesicles, the asymmetry disappearing as the degree of curvature decreases. In a phosphatidylcholine/phosphatidylethanolamine/ganglioside (8:2:1, molar ratio) system, both phosphatidylethanolamine and gangliosides distribute asymmetrically, indicating a composition-dependent asymmetric distribution of phosphatidylethanolamine. In this system asymmetry also increases with increasing curvature. The asymmetric distribution of gangliosides in vesicles of low curvature may be due to their long headgroup and larger headgroup surface area in accordance with the theoretical predictions of Israelachvili et al. (Biochim. Biophys. Acta 470 (1977) 185-201).

Chromatography, Gel↗

PubMed1989

Enzyme replacement therapy in fibroblasts from a patient with cholesteryl ester storage disease.

Enzyme replacement has long been considered only a remote possibility in the treatment of a wide range of genetic disorders, many manifested as lysosomal storage diseases. The complexity of having a particular enzyme gain access to the lysosomal compartment in a specific cell seemed insurmountable. We report here on an attempt to introduce the enzyme cholesteryl esterase into fibroblasts from a patient with cholesteryl ester storage disease (CESD). The enzyme gains access to the lysosomal compartment and the accumulating cholesteryl ester by virtue of being carried into the cell conjugated to a ligand (insulin or apoprotein B [apoB]) that binds to its own specific receptor and is internalized by the well-described process of receptor-mediated endocytosis. Regardless of whether the enzyme enters the cell via the insulin receptor or via the low-density lipoprotein (ApoB) receptor, it can be found associated with a lysosomal fraction and is effective in lowering levels of accumulated substrate, cholesteryl ester. The time course of the substrate degradation and the dependence on the receptor density and receptor density and receptor-ligand interaction indicate that the enzyme is simply being carried to the site of substrate accumulation by virtue of the fact that that is the destination of the ligand (along with its conjugated enzyme) following internalization.

Apolipoproteins B↗

PubMed1989

Bilirubin toxicity in a neuroblastoma cell line N-115: I. Effects on Na+K+ ATPase, [3H]-thymidine uptake, L-[35S]-methionine incorporation, and mitochondrial function.

Though bilirubin is reported to affect a variety of cellular functions, the primary target of its toxic effect is still not known. A major problem in understanding this is the wide variation in results reported by different groups. This is probably due to the differences in stability of bilirubin solutions arising from large differences in bilirubin:albumin molar ratios used in experiments. Hence in studying the toxic effects of bilirubin in tissue culture systems, it is important to be certain that the bilirubin is maintained in solution throughout the time of the exposure to bilirubin. Spectrophotometric measurements have shown that bilirubin is stable in Dulbecco's modified Eagle medium solution at bilirubin:albumin molar ratios up to 3. Under these defined conditions, bilirubin was found to affect Na+K+ ATPase, [3H]-thymidine uptake, L-[35S]methionine incorporation into protein and mitochondrial function at bilirubin concentrations up to 125 microM and bilirubin:albumin molar ratio of 1.5. Toxic effects on all parameters measured were evident at bilirubin:albumin molar ratio of 1.5 after a minimum of 2 h of exposure. No effect was evident at a bilirubin:albumin molar ratio below 1. Although it is not possible to identify with certainty the primary target, the effect on mitochondrial function appeared earlier and was more profound than that seen with the other assessed functions.

Albumins↗

PubMed1989

Bilirubin toxicity in a neuroblastoma cell line N-115: II. Delayed effects and recovery.

Clinical studies have suggested that the early stages of bilirubin encephalopathy are reversible. These phenomena are investigated at the cellular level using the neuroblastoma cell line N-115 as a model system. To determine whether the cell line N-115 can recover from bilirubin toxicity, and whether the cellular function remains intact after a short period of bilirubin exposure during which time no toxic effects are manifest, the cells are exposed to bilirubin at varying concentrations and varying bilirubin:albumin ratios for 1 and 2 h. The bilirubin is then washed out, and the cells are reincubated in fresh media with appropriate amounts of albumin. Mitochondrial function, [3H]thymidine uptake and L-[35S]methionine uptake are assessed at 2, 8, and 24 h of reincubation after the bilirubin washout. After the short-term exposure, the cells begin to demonstrate evidence of toxicity in all parameters measured 8-24 h after the bilirubin washout. After the 2-h exposure to bilirubin, the cells demonstrate significant toxicity within 2 h of the bilirubin washout. The degree of toxicity seems to depend on the bilirubin:albumin ratio and bilirubin concentration. In general, after bilirubin exposure of 1 h or longer, the N-115 cells develop evidence of toxicity which is progressive and irreversible.

Albumins↗

PubMed1989

Superoxide dismutase: improving its pharmacological properties by conjugation with human serum albumin.

Superoxide Dismutase has been reported to offer important pharmacological advantages in modifying oxygen toxicity as a result of its ability to scavenge oxygen free radicals. This has proven most exciting in reducing damage associated with post-reperfusion damage following myocardial ischemia. Unfortunately Superoxide Dismutase has a circulation life time of only a few minutes making the exact time of its administration crucial and somewhat impractical. We report here on the production of SOD-Albumin conjugates which have important advantages over free SOD in terms of stability, extended circulation time and reduced immunogenicity.

Animals↗

PubMed1988

Growth hormone-albumin conjugates. Reduced renal toxicity and altered plasma clearance.

The effective therapeutic use of many small peptides such as growth hormone has been limited by their small molecular masses and rapid clearance by the kidneys. Moreover, various degrees of nephrotoxicity have been reported for small proteins which are readily filtered at the level of the glomerulus. We have attempted to circumvent this drawback by conjugating growth hormone (somatotropin) to serum albumin in an effort to alter the peptide's pharmacokinetics while retaining its biological activity.

Adipose Tissue↗

PubMed1988

Effect of surface curvature on the rate of cholesterol transfer between lipid vesicles.

The effect of surface curvature on the spontaneous movement of cholesterol between membranes was investigated by measuring the rates of cholesterol transfer from donor vesicles of various sizes to a common acceptor vesicle. Donor vesicles of size in the range 40-240 nm were prepared by extruding multilamellar dispersions through polycarbonate filters of different pore sizes under pressure. The smallest donor vesicle and the acceptor vesicles were obtained by the normal sonication procedures. The rate of cholesterol transfer, as measured by the movement of [3H]cholesterol, decreases with increasing size of the donor vesicle in an almost linear fashion. The extrapolation of the results gave a half-time (t1/2) of 16-20 h of the desorption of cholesterol from a planar bilayer, and this can be considered as a reference value for most cellular membranes which are characterized by very low curvatures. Our earlier studies have shown that the t1/2 for cholesterol efflux is influenced by the presence of gangliosides and phosphatidylethanolamine, and the asymmetric distribution of these lipids in the plasma membrane could partially account for the large difference in the rates of cholesterol movement from the two sides of the plasma membrane. The small differences in rates arising from asymmetric distribution will be magnified by the longer t1/2 obtained here for membranes of low curvatures, so that the large difference in rates might be a coupled effect of lipid asymmetry and low curvature of the plasma membrane. This, in turn, may have a role in maintaining the large differences in cholesterol/phospholipid molar ratios observed between plasma membrane and intracellular membranes.

Biological Transport↗