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Biomedical subjects

M J Poznansky

Publications and source records attributed to M J Poznansky.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

Cholesterol transfer between lipid vesicles. Effect of phospholipids and gangliosides.

The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.

Biological Transport↗
PubMed1987

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by changes in microsomal cholesterol content or phospholipid composition.

Microsomal preparations from normal human skin fibroblasts were used to investigate the role of free cholesterol in the endoplasmic reticulum in the control of cholesterol biosynthesis by regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. Controlled changes in the cholesterol/phospholipid ratio of microsomes were introduced either in intact cells by incubating fibroblast cultures with whole serum or lipoprotein-deficient serum or by enrichment or depletion of cholesterol in microsomal preparations by incubating microsomes with cholesterol-rich or cholesterol-poor egg phosphatidylcholine unilamellar vesicles. Cholesterol enrichment resulted in suppression of reductase activity and increased ESR order parameters for 12-nitroxystearate in the microsomal preparations. Conversely, cholesterol depletion caused an activation of reductase and a decrease in order parameter. Enrichment of microsomal preparations with a "nonfluid" lipid, dipalmitoyl phosphatidylcholine, also suppressed enzyme activity and increased membrane order. The effect was reversed by subsequent enrichment of the microsomes with fluid egg phosphatidylcholine. Our findings suggest that cholesterol may regulate its own biosynthesis, at least in part, by suppression of hydroxymethylglutaryl-CoA reductase mediated through changes in membrane fluidity as measured by ESR order parameter.

Cells, Cultured↗
PubMed1986

Bilirubin diffusion through lipid membranes.

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.

Bilirubin↗
PubMed1985

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.

Cells, Cultured↗
PubMed1985

Bilirubin toxicity in neural cell lines N115 and NBR10A.

The toxicity of bilirubin was investigated in 2 neural cell lines NBR10A and N115 using a quantitative dye assay 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [3H]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and [3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 mM or higher, whereas lower bilirubin levels at this molar ratio showed no deleterious effect. The effect of bilirubin is more pronounced at a molar ratio of 1.5 with longer incubation periods. The MTT assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBR10A cells, a finding which was not obtained from [3H]thymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the MTT assay estimates the number of viable cells at the end of the experiment by measuring mitochondrial function whereas the [3H]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the [3H]-thymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 mM or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]thymidine incorporation (decrease in specific activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

alpha-Glucosidase-albumin conjugates: effect of chronic administration in mice.

Enzyme albumin conjugates have been proposed as a means of increasing the efficacy of enzyme use in vivo and decreasing immune response to the enzyme. Particulate drug carriers, however, have a pronounced tendency to localize in the mononuclear phagocyte (reticuloendothelial) system. We have examined in mice the effect on phagocytic index, tissue distribution and organ size of continued administration of conjugates of alpha-glucosidase with either homologous or heterologous albumin. Mice received 10 X 2-mg injections of bovine serum albumin (BSA) or mouse serum albumin (MSA), either free, polymerized or conjugated with alpha-glucosidase. Experiments involving BSA had to be terminated before the end of the experiment because of anaphylaxis, but these reactions were less severe to the polymerized albumin than to free albumin. Free BSA, BSA polymer and BSA-enzyme conjugates all caused a decrease in phagocytic index after six injections. Mice receiving MSA showed no evidence of anaphylaxis, but mice receiving six or more injections of free MSA, MSA polymer or MSA-enzyme conjugate had significantly decreased phagocytic indices as compared to controls. Phagocytic indices had returned to normal by 7 days after the final injection. Tissue distribution of 125-I-labeled albumin preparations was determined in either naive or chronically injected mice.

Animals↗
PubMed1984

Insulin: carrier potential for enzyme and drug therapy.

Several carrier systems and targeting agents have been considered as means of delivering enzymes and drugs to specific tissues or cells. In this report insulin is shown to be effective in delivering enzyme-albumin conjugates to cells and tissues rich in insulin receptors. The complex is transported into cells by a process that resembles receptor-mediated endocytosis and can be identified in a lysosomal fraction. The enzyme-albumin-insulin complex retains its enzymatic activity and its ability to bind antibodies to insulin. It also has a hypoglycemic effect; however, plasma glucose concentrations can be maintained by glucose administration.

Albumins↗
PubMed1984

Enzyme-albumin polymers. New approaches to the use of enzymes in medicine.

The widespread use of enzymes as drugs or therapeutic agents has been limited by (a) enzyme availability, (b) biodegradation of administered enzyme, (c) immunogenecity of the enzyme as a foreign protein, and (d) accessibility of the enzyme to the appropriate site of action. It has become obvious that due to these limitations, the administration of free or native enzyme is not likely to be effective. Various mechanisms of protecting or packaging enzymes to offset some of these drawbacks have been described. We have been successful in producing conjugates of a number of different enzymes with a molar excess of homologous albumin. The resulting enzyme-albumin complex is resistant to proteolytic and heat inactivation and is apparently non-immunogenic. Using specific ligands crosslinked to the enzyme-albumin conjugate we have been able to target these conjugates to specific receptor sites and specific tissues. Ligands including cell surface-recognizing antibodies and hormones such as insulin have been used. These approaches offer new possibilities for the increased use of enzymes in medicine.

Albumins↗
PubMed1984

Membrane electrical properties of developing fast-twitch and slow-tonic muscle fibres of the chick.

Isolated single fibres from the anterior (a.l.d.) and the posterior (p.l.d.) lattissimus dorsi muscles of embryonic and young chicks were used to study in vivo development of membrane electrical properties. Isolated fibres were obtained by an enzymatic dissociation procedure. Intracellular micro-electrode recordings from isolated fibres and from fibres in intact muscles showed that the dissociation procedure did not significantly alter resting membrane potentials, input resistances or membrane time constants (tau m). The 14 day embryonic fibres of a.l.d. and p.l.d. did not have a measurable resting conductance to Cl-. At hatching, about 70% of the resting conductance in p.l.d. fibres was due to Cl-. Membrane electrical properties were estimated from the analysis of voltage responses to intracellular injection of rectangular pulses of current. At 14 days in ovo, membrane resistance (Rm) was approximately 20 k omega cm2 and membrane capacitance (Cm) was 1-2 microF/cm2 for both a.l.d. and p.l.d. The mean membrane length constants (lambda) were 1.7 mm for a.l.d. and 1.5 mm for p.l.d. For p.l.d., the values of Rm, tau m and lambda decreased as development proceeded. For a.l.d., there was no change in these values by the time of hatching (21 days). The decreases in the electrical constants for p.l.d. fibres were partly explained by the appearance of a resting Cl- conductance during the last week of embryonic development.

Animals↗