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Biomedical subjects

M Krieger

Publications and source records attributed to M Krieger.

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

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis.

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.

Acyl Coenzyme A↗
PubMed1983

Complementation of mutations in the LDL pathway of receptor-mediated endocytosis by cocultivation of LDL receptor-defective hamster cell mutants.

We have previously isolated Chinese hamster ovary (CHO) cell mutants that do not express low density lipoprotein (LDL) receptors. When one mutant clone was cocultivated with other receptor-defective clones, it was induced to express receptors that could mediate normal endocytosis. These LDL receptor-defective clones defined two classes of mutations: cbc (complemented by cocultivation) and icc (inducer cells in cocultivation). The induction and short-term (18 hr) stability of LDL receptors in cbc cells did not require protein synthesis by icc cells. Receptor activity could not be induced by DMSO, 5-azacytidine, phosphatidylcholine liposomes, dibutyryl cAMP, compactin, soybean trypsin inhibitor, low temperature (30 degrees C), or conditioned medium, but could be induced by cocultivation with parental CHO cells and normal and LDL receptor-negative human fibroblasts. Complementation by cocultivation only occurred when the cbc and inducing cells were in close proximity, suggesting that an unstable diffusible factor or intimate cell-to-cell association was required for complementation.

Animals↗
PubMed1983

Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis.

This paper describes a rapid and efficient two-step procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of two fungal metabolites, compactin (ML236B), a potent inhibitor of cholesterol biosynthesis, and amphotericin B, a polyene antibiotic that forms toxic complexes with sterols in membranes. Mutagen-treated Chinese hamster ovary cells were preincubated overnight in a medium containing mevalonate, low density lipoprotein (LDL), and compactin (Mev/LDL/Com). At the end of the preincubation period, wild-type cells were cholesterol replete while mutant cells that could not utilize the cholesterol in LDL were cholesterol deficient. Subsequent incubation with amphotericin B for 6 hr killed most of the wild-type cells. After a second round of Mev/LDL/Com-amphotericin B selection, endocytosis-defective clones appeared at a frequency of approximately equal to 2.6 X 10(-5). Some of these clones expressed LDL receptor-defective phenotypes and fell into one of two previously defined classes of mutation. Sensitivity of the mutants to infection by vesicular stomatitis virus suggested that the mutations do not disrupt the coated pit-coated vesicle pathway of endocytosis. Minor modifications in the Mev/LDL/Com-amphotericin B selection permit the isolation of cholesterol auxotrophs and might allow the isolation of conditional-lethal mutations. Because LDL can be coupled to ligands that bind to receptors other than the LDL receptor, Mev/LDL/Com-amphotericin B selection may permit the isolation of mutant cells with defects that specifically disrupt other endocytic pathways.

Amphotericin B↗
PubMed1983

Reconstitution of LDL with lipophilic fluorescein derivatives: quantitative analysis of the receptor activity of human lymphocytes.

Low density lipoprotein (LDL), the major cholesterol transport protein in human plasma, consists of an apolar core of cholesteryl esters surrounded by a polar shell containing phospholipids, unesterified cholesterol and protein. In the current paper we report the absorption and fluorescence spectra of members of a new class of lipophilic fluorescein derivatives which were designed to be reconstituted into the core of LDL in place of the native cholesteryl esters. One of these derivates, cholesteryl 12-O-[methyl 3-O-methyl-5' (6')-carboxyfluorescein]ricinoleyl carbonate (MMC) was reconstituted into the core of LDL. The resultant fluorescent reconstituted LDL was used in conjunction with flow cytometry to quantify the LDL receptor activity of fresh blood lymphocytes derived from normal individuals and from patients with the heterozygous and homozygous forms of familial hypercholesterolemia (FH). The LDL receptor activities of the heterozygous and homozygous FH lymphocytes were approximately 37% and 1% of normal, respectively. LDL reconstituted with these lipophilic fluorescein derivatives will be valuable in studying LDL metabolism and may be useful for the diagnosis of FH.

Fluoresceins↗
PubMed1982

Violent Behavior among hospitalized medical and surgical patients.

To characterize violent behavior in hospitalized medical and surgical patients, we reviewed documented violent incidents at the San Francisco General Hospital during a two-year period. Twenty-nine incidents of verbal and physical violence occurred. One patient was gravely ill and three were delirious. All the incidents were associated with increased levels of tension and loss of impulse control. In most cases, contention with the staff regarding pain medication or ward regulations was a precipitating event. Of the 28 patients with mental disorders, 19 were substance abusers, six had organic brain syndrome, tw had neurosis, and one had schizophrenia. The findings suggest that physicians should be more sensitive to patient characteristics and to the situational characteristics of the violent incident. Explicit measures that anticipate and reduce violent behavior are reviewed.

Adult↗
PubMed1981

[Use of health services and drugs by old people of Montreal receiving home care].

A survey was conducted among 160 persons aged 64 year or more in Montreal who were receiving home care. They answered at home a questionnaire on their use of health care services and drugs, and showed the interviewer all the drugs they were taking. In comparison with similar data from elsewhere, the use of health care services (an average of 8.0 encounters with a physician per person per year) and of drugs (an average of 5.3 per person) by this group seems high. Perhaps this group of people was obviously sicker than others of the same age, but this remains to be shown. Moreover, despite the reported frequency of health problems, it is uncertain whether such use of services and drugs was necessary. The question is raised whether the home care system is doing for the patient what it was intended to do.

Aged↗
PubMed1981

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells.

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using 125I-labeled canine plasinogen. Throughout a 3-day period, 125I-plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate-sized macromolecules that were the same size as the large (74,600-dalton) and small (25,000-dalton) chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. Binding to SV3T3 cells was independent of the protease-dependent morphological change (PDMC) characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3-iodo-L-tyrosine, a terminal lysozymal digestion product. The results of a sublethal cell-surface trypsinization assay suggest that the cell-associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of 125I plasminogen degradation exhibited by SV3T3 cells was approximately two time greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC. In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95-100% of plasmin covalently bound to a 47,000-dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95-100% of the plasmin was inactive to reaction with DF32P. Thus the serum component is a plasmin inhibitor. The plasmin-containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin-containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (approximately 10,000 and 12,000 daltons) by as-yet uncharacterized serum factors.

Animals↗
PubMed1980

beta-Carotene as a probe of lipid domains of reconstituted human plasma low-density lipoprotein: induced circular dichroism.

Mixtures of neutral lipids containing cholesteryl esters and beta-carotene were used to reconstitute the lipid core of heptane-extracted low-density lipoproteins (LDL). The resulting preparations of reconstituted LDL, referred to as r-[cholesteryl ester + beta-carotene]LDL, exhibited temperature-dependent circular dichroism (CD) in the visible region similar to that of native LDL. Since beta-carotene lacks intrinsic optical asymmetry, the observed CD must be induced by environmental constraint. LDL reconstituted with cholesteryl oleate, cholesteryl linoleate, or cholesteryl linolenate in the presence of beta-carotene exhibited thermotropic transitions at approximately 40, 35, and 30 degrees C, respectively. These temperatures are similar to the liquid-crystalline to isotropic liquid phase transition temperatures observed for pure cholesteryl ester model systems. The addition of triolein to r-[cholesteryl linoleate + beta-carotene]LDL lowered the transition temperature and decreased the specific ellipticity of the visible CD bands. When triolein accounted for 70% or more of the total neutral lipid content of r-[cholesteryl linoleate + triolein + beta-carotene]LDL, specific ellipticity was zero. The addition of low levels of unesterified cholesterol to r-[cholesteryl linoleate + beta-carotene]LDL did not alter the transition temperature but did reduce the specific ellipticity by 40%. The current data indicate that the organization of the core of neutral lipids in reconstituted LDL resembles that of native LDL with respect to environmental constraint on the beta-carotene molecule and that the helicity of the protein moiety resembles that of native LDL. In addition, the data are consistent with the possibility that the core cholesteryl esters of reconstituted LDL undergo phase transitions similar to their transitions in the free state.

Absorption↗
PubMed1979

Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells.

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3 beta-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37 degrees C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.

Cells, Cultured↗
PubMed1979

Demonstration of low density lipoprotein receptors in mouse teratocarcinoma stem cells and description of a method for producing receptor-deficient mutant mice.

Familial hypercholesterolemia, a widespread human genetic disorder implicated in vascular and coronary disease, has had no laboratory animal counterpart that would enable the pathogenesis to be analyzed and drugs to be tested in vivo. The primary lesion in some patients is known to occur in the cells' initial handling of the major cholesterol-carrying lipoprotein of plasma. It entails a deficiency in the specific cell surface receptor that binds low density lipoprotein (LDL), with a consequent alteration in the control of cholesterol metabolism. The present study was undertaken to devise a practical scheme for producing, from developmentally versatile mouse teratocarcinoma stem cells, whole-animal models with a comparable genetic lesion. This requires first learning whether the tumor stem cells in culture express LDL receptors, and next establishing a selection or screening procedure to identify receptor-deficient mutants in mutagenized cell cultures. The results show that the teratocarcinoma cells do in fact have specific high-affinity LDL receptors which are similar to those reported for fibroblasts and the parenchymal cells of specialized tissues and different from those of phagocytic cells. Sterols suppressed the otherwise efficient binding, internalization, and degradation of LDL ((125)I-labeled) by the cells. Acetylation of LDL blocked the binding. Only LDL and not high density lipoprotein (HDL) was bound. After LDL uptake and degradation, the liberated cholesterol led, as expected, to increased cholesteryl ester formation; it also suppressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMG CoA reductase; mevalonate: NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate-limiting step in cholesterol biosynthesis. Cells with LDL receptors were readily visualized by administering a fluorescent derivative of LDL; in the fluorescence microscope, labeling was seen in all cells. Cells with experimentally depressed receptors, yielding little fluorescence, were separable from those with normal fluorescence in the fluorescence-activated cell sorter. Thus, two methods for isolating receptor-deficient cells from mutagenized cultures are now available, either by visual recognition of low-fluorescing or nonfluorescing colonies in culture plates or by electronic cell sorting. Such mutants in an appropriate line of teratocarcinoma cells can then be passaged into blastocysts for full somatic tissue differentiation and germ-line development into mice.

Animals↗
PubMed1979

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.

Acetylation↗
PubMed1979

Fantasy and reality in patients reports of incest.

This paper explores some of the complex difficulties facing clinicians trying to assess whether a patient's report of incest is fantasy or reality. The role of sexualized family interactions, the age of the child, the nature of the act, and the quality of the reports are discussed as relevant variables. Cases are used to illustrate various points. In conclusion, we provide 8 questions the clinician should review before making any judgments about a report of incest being reality, especially when legal procedures are involved.

Adolescent↗