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Biomedical subjects

W Gevers

Publications and source records attributed to W Gevers.

At least 19 recordsLinked to original sources
PubMed1992

Low-density lipoprotein receptor point mutation results in expression of both active and inactive surface forms of the same mutant receptor.

LDL receptors, expressed in cultured fibroblasts from patients homozygous for the FH Afrikaner-1 (FH1) mutation (Asp206 to Glu), are transported from the endoplasmic reticulum (ER) to the Golgi apparatus more slowly than in normal cells. In the present study, binding characteristics of FH1 cells for lipoprotein ligands (LDL and beta VLDL) and for receptor-specific monoclonal antibodies pointed to the existence of two surface forms of the same mutant receptor. One of these forms bound lipoproteins with normal high affinity whereas another did not. Binding studies of transfected hamster cells expressing only the mutant human gene confirmed the single-gene origin of the different forms. The existence of functionally distinct forms of the receptor protein was supported by the observation that only lipoprotein-binding receptor molecules were trapped intracellularly and degraded following ammonium chloride treatment of cells in the presence of ligand. The lipoprotein-binding receptor population was indistinguishable from normal receptors with respect to its affinity for LDL and beta VLDL, uptake and degradation of lipoprotein, and receptor recycling. Ligand blotting versus immunoblotting of receptors revealed normal-sized mutant receptors that were not recognized by lipoprotein ligand. Despite these differences, both mutant forms of the receptor were degraded at rates similar to those of normal receptors. We propose that the single amino acid substitution in this receptor interferes with the folding and/or posttranslational processing of precursor molecules in such a way that receptors adopt alternative stable structures.

Ammonium Chloride↗
PubMed1991

The genetic basis of cancer.

Cancer is essentially a genetic disease resulting from congenital or acquired alterations in some cells of the patient. Such changes may occur in particular oncogenes and are responsible for the tumour phenotype of the affected population of cells. Oncogenes function by continuous positive action in the mitogenic pathway, and may become activated by point mutations, chromosomal rearrangements, gene amplification or viral insertion events. In contrast, unaltered tumour-suppressor genes are responsible for suppressing the neoplastic phenotype, and their inactivation by deletion or mutation permits cancerous development in the affected cells. The genetic model of carcinogenesis is thus based on the idea that mutations at the DNA level create a functional imbalance between the oncogenes and the tumour-suppressor genes, resulting in uncontrolled clonal proliferation. It is likely that the clinical importance of these recent findings will soon be realised and utilised in the development of therapies and diagnostic procedures that will directly benefit the patient.

Colorectal Neoplasms↗
PubMed1991

Low density lipoprotein receptor founder mutations in Afrikaner familial hypercholesterolaemic patients: a comparison of two geographical areas.

Afrikaners with familial hypercholesterolaemia (FH) were screened for the presence of three point mutations in the low density lipoprotein receptor gene that were previously described as being relatively common in this population. The prevalence and distribution of the mutations were compared in 27 unrelated homozygous and 79 unrelated heterozygous FH Afrikaner patients from two regions in South Africa, the Transvaal and Cape Provinces. The relative distribution of the three mutations was similar in the two regions, with the FH1 mutation being the most prevalent (66%), followed by the FH2 mutation (27%) and the FH3 mutation (7%). Interestingly, defects other than the three common mutations are more common in the Cape than in the Transvaal; thus the three known mutations account for 98% of FH alleles in the Transvaal and only 74% in the Cape Province. None of the patients carried the recently described familial defective apolipoprotein B100 mutation. These results establish that three "founder" mutant genes occur amongst the Afrikaner and are responsible for the overall high prevalence of FH in this population.

Alleles↗
PubMed1990

Auto-ubiquitination of ubiquitin-activating enzymes from chicken breast muscle.

A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane ('TLCK'), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions.

Adenosine Triphosphate↗
PubMed1990

Ammonium chloride causes reversible inhibition of low density lipoprotein receptor recycling and accelerates receptor degradation.

The effects of the acidotropic agent, NH4Cl, on the recycling and turnover of low density lipoprotein (LDL) receptors were analyzed in human skin fibroblasts using ligand binding assays, [35S]methionine pulse-chase experiments, and electron microscopy. NH4Cl did not prevent receptor internalization but caused a marked redistribution of LDL receptors to intracellular sites (endosomes) that was completely dependent on the presence of apolipoprotein-B- or -E-containing ligands. Maximal inhibition of recycling was observed at LDL concentrations that only partially saturated receptors, suggesting that the receptors function as oligomers. In contrast, full receptor occupancy by the multivalent, apolipoprotein-E-containing beta-very low density lipoprotein was required for the same effect. The intracellular accumulation was reversible and the majority of receptors returned to the cell surface when NH4Cl was removed after short treatments. The rate of degradation of LDL receptors was greatly accelerated in the presence of NH4Cl and ligand, with a t1/2 of about 2 h (approximately 6 times faster than receptor degradation in the absence of NH4Cl). Neither the redistribution nor the accelerated loss of immunoprecipitable LDL receptors was observed in an LDL receptor internalization-defective mutant cell line. We conclude that NH4Cl inhibited the recycling specifically of occupied receptors, thereby accelerating their degradation, probably in endosomes.

Ammonium Chloride↗
PubMed1990

Chylomicron remnant clearance from the plasma is normal in familial hypercholesterolemic homozygotes with defined receptor defects.

The retinyl palmitate fat tolerance test was used to measure chylomicron remnant clearance in 10 normal subjects (apolipoprotein E [apo E] isotypes 3 or 4 only), 6 normolipidemic apo E2/2 homozygotes and 5 familial hypercholesterolemic homozygotes. Skin fibroblasts with fully upregulated LDL receptors from the latter subjects degraded rabbit 125I-beta VLDL in vitro at rates ranging from less than 10-48% of normal. Experiments in vivo revealed no significant differences between the normal and homozygous familial hypercholesterolemic (FHH) subjects in chylomicron remnant clearance assessed on the basis of "areas under the curves" for retinyl palmitate levels present in post-prandial serum, chylomicron remnants (Sf. less than 1,000), or chylomicrons (Sf. greater than 1,000). Remnant clearance was greatly decreased at all times in the apo E2/2 homozygotes, indicative of an important degree of flux control exerted by a receptor-mediated step involving apo E as ligand. The absence of any excess remnant accumulation in FHH subjects with varying "impairment" of LDL receptor-mediated degradation of apo E-containing lipoproteins, permits the conclusion that chylomicron remnants are initially cleared from the plasma by apo E-recognizing receptors which are genetically distinct from LDL receptors.

Adolescent↗
PubMed1989

Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes.

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.

Cells, Cultured↗
PubMed1989

Absence of alpha 2(1) procollagen synthesis in a clone of SV40-transformed WI-38 human fibroblasts.

Normal diploid human embryonic lung fibroblasts (WI-38) produce type I collagen of chain composition [alpha 1(1)]2.alpha 2(1) together with small amounts of type III collagen. We have examined the synthesis and secretion of type I collagen in a clone of SV40-transformed WI-38 fibroblasts (SVWI-38). These cells produced only 20-25% of the total collagen synthesized by their normal counterparts, with no detectable synthesis of alpha 2(1) chains and deposited a type I trimer consisting of overmodified alpha 1(1) chains. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cyanogen bromide peptides of collagens isolated from cells cultured either in the presence or absence of alpha,alpha-dipyridyl revealed that this overmodification occurred along the entire length of the alpha 1(1) chains. Analysis of poly(A+) RNA by Northern blot analysis and total RNA by slot blot analysis using cloned type I procollagen cDNA probes revealed that no alpha 2(1) mRNA was present in the SVWI-38 cells. Primer extension of the RNA confirmed this finding. The SVWI-38 cells had a normal chromosome number, but contained 28 normal and 18 abnormal and marker chromosomes. Restriction mapping of the entire alpha 2(1) procollagen gene did not reveal any gross deletions or insertions within this gene, nor was the gene hypermethylated in the transformed cells, when compared with their normal counterparts. One interesting feature, however, was the fact that certain CpG dinucleotides in the alpha 2(1) gene were methylated in the normal as well as the transformed cells. These SV40-transformed WI-38 fibroblasts therefore do not produce any alpha 2(1) collagen chains due to transcriptional inactivation of their genes.

Cell Line, Transformed↗
PubMed1989

Activation and demethylation of the intracisternal A particle genes by 5-azacytidine.

Treatment of C3H 10T1/2 mouse embryo fibroblasts with the cytidine analogues 5-azacytidine and 5-aza-2-deoxycytidine causes altered gene expression and results in the manifestation of phenotypic changes and altered cell morphology. This includes the conversion of these cells to adipocytes, chondrocytes and myotubes. The effects of these analogues on intracisternal A particle (IAP) gene expression in mouse C3H 10T1/2 cells have been examined. Treatment with either 3 microM 5-azacytidine or 0.3 microM 5-aza-2-deoxycytidine for 24 h was associated with an immediate increase in IAP gene transcription, and with the subsequent appearance of IAPs in the cisternae of the cells 24 h after removal of the drug. Control cells contained no, or very few, IAPs and IAP mRNA. Analysis of the methylation status of the IAP genes, using the restriction endonucleases HpaII, MspI and HhaI, showed that these genes were already demethylated at the end of the 24-h treatment period. IAP gene transcripts were detectable even after a 16-h drug treatment period, at which stage the genes were not yet fully demethylated. After further growth in fresh medium for 90 h, the levels of IAP RNA started to decline, but the demethylated CpG sites were not yet remethylated. These results suggest the involvement of other factors, in addition to methylation, in the regulation of IAP gene expression. These drugs were found to have no stimulatory effect on several oncogenes examined in this study.

Animals↗
PubMed1989

Early appearance of dispersed low density lipoprotein receptors on the fibroblast surface during recycling.

We have examined the distributions of recycling low density lipoprotein receptors (LDL-Rs) as they emerge onto and cluster on the surfaces of cultured cells. Surface LDL-Rs were labeled with colloidal gold-LDL conjugates (AuLDL) and cells viewed as whole-mounts in the transmission electron microscope. The steady-state distribution of LDL-Rs on the cell surface, labeled with AuLDL at 4 degrees C, comprised ring-shaped clusters of receptors with dispersed receptors scattered amongst them. After 12 min of incubation at 37 degrees C, virtually all AuLDL probes were internalized. Electron microscopy of thin sections revealed clustered receptors in coated pits and the progressive accumulation of AuLDL in endosomes, multivesicular bodies and lysosomes. By initially blocking all surface LDL-Rs, either with unconjugated LDL or AuLDL of one size, the clustering behavior of newly emerged receptors which recycled to the cell surface was selectively visualized with an AuLDL probe of a second size over a defined time-course. Release of the blocking ligand during the time-course was found to be negligible. Newly appearing dispersed LDL-Rs were detected as early as 2 min and these were often concentrated at the cell margins. The newly labeled and preblocked LDL-Rs did not cocluster before 6 min. By 8 to 12 min, ring-shaped clusters of newly emerged receptors had formed and these were often seen associated with pre-blocked LDL-Rs. The clustering of LDL-Rs on the cell surface was independent of the presence of ligand, AuLDL. Our results indicate that LDL-Rs recycle to the cell surface where they form a dispersed population which gives rise to the ring-shaped clusters of cell surface LDL-Rs associated with coated pits.

Cell Line↗
PubMed1988

Two mutant low-density-lipoprotein receptors in Afrikaners slowly processed to surface forms exhibiting rapid degradation or functional heterogeneity.

Two distinct mutant low-density-lipoprotein receptors in South African Afrikaners exhibit retarded posttranslational processing to mature forms. One mutation gives rise to cell-surface receptors that are subject to abnormally rapid degradation, whereas the other is associated with a functionally heterogeneous surface population degraded at a normal rate.

Ethnicity↗
PubMed1988

Action limits for serum total cholesterol. A statement for the medical profession by an ad hoc committee of the Heart Foundation of Southern Africa.

Hypercholesterolaemia is common in many segments of the South African population, both by virtue of high mean population serum total cholesterol (TC) values and of a high prevalence of familial hypercholesterolaemia (FH). Age-specific action limits for TC are proposed in order to remove the variation in 'normal values' used by different laboratories. The action limits are derived from epidemiological studies rather than purely statistical norms. They are used to designate individuals as falling into high, moderate and ideal TC ranges. The high-risk action limit has also proved to be useful for screening for FH. After an initial screening TC estimation, the further management of a patient will depend on the TC risk category and the presence or absence of other risk factors. Risk factors such as hypertension, a smoking habit, a low high-density lipoprotein cholesterol value, diabetes, evidence of existing coronary heart disease (CHD) or a family history of premature CHD multiply the risk conferred by elevated TC, and change the moderate-risk status of an individual with moderately elevated TC to a high-risk status. Intensity of investigation, treatment and follow-up depend on the overall risk status of an individual patient. Drug therapy is reserved for high-risk patients who have not responded to a reasonable trial of non-drug measures. Other reversible risk factors are treated in their own right. The guidelines embodied in this report are intended to facilitate and justify the clinical approach to individual patients with hypercholesterolaemia. They do not replace the need for a population strategy to reduce risk factors in the general population.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up- or down-regulation.

Treatment of cultured human skin fibroblasts with cycloheximide retarded the down-regulation of low density lipoprotein (LDL) receptor activity caused by 25-hydroxycholesterol. The rate of LDL receptor degradation, measured directly by means of [35S]methionine pulse-chase experiments, was also markedly inhibited by cycloheximide (or puromycin), suggesting that continuous synthesis of a short-lived mediator protein(s) was necessary for normal LDL receptor turnover. In the absence of cycloheximide, both the up- and down-regulation of LDL receptor activity took place with a half-time of approximately 12 hr. Pulse-chase measurements with [35S]methionine yielded a receptor half-life (t1/2) of 11.7 +/- 2.2 hr (n = 10) in up-regulated cells; the t1/2 in the partially down-regulated state was similar. The presence of LDL or 25-hydroxycholesterol did not alter this degradation rate. Regulation of LDL receptor activity under these various culture conditions therefore probably occurred solely as a result of changes in the rate of receptor synthesis. The cycloheximide-sensitive factor(s) that influences receptor turnover apparently did not play a regulatory role in the up- or down-regulation of the LDL receptor.

Antibodies↗
PubMed1987

Regulation of metabolite fluxes and concentrations in living cells and organisms.

A quantitative framework for the analysis of metabolic control depends on the recognition of input and output fluxes occurring in steady-state systems in which the concentrations of intermediates are constant--the fluxes and concentrations peculiar to a given steady state are determined by the catalytic components of the system. Control of the variables is a global function shared among the components in a complex pattern, for reasons which are evident when the detailed properties of such systems are analysed. Such properties include circuit design and a host of factors affecting the sensitivity of catalytic components to substrates and effectors.

Cell Membrane Permeability↗
PubMed1987

Mucus and mucins.

Mucus is a secretion which contains a number of components of which mucins (glycoproteins of varying structure) are the entities mainly responsible for the physical properties of a visco-elastic gel. Technical problems which have previously hindered the elucidation of the chemical structure of these molecules have recently been overcome sufficiently to permit the development of structural models which explain both the common and variable features of mucus, healthy or pathological, in different sites of the body.

Chemical Phenomena↗
PubMed1987

Disulfide-bonded heparan sulfate proteoglycans associated with the surfaces of cultured bovine vascular endothelial cells.

A predominant species of heparan sulfate proteoglycan that consisted of at least two subunits linked by disulfide bonding was isolated from cell layers of normal ('cobblestone') bovine vascular endothelial cells in culture. Treatment of the parent molecules with dithiothreitol caused their complete cleavage and permitted the subsequent separation of the larger and smaller subunits on Sepharose CL-4B columns. Removal of dithiothreitol by dialysis resulted in the reformation of large disulfide-bonded molecules but such recombination of the subunits was prevented by prior reductive alkylation using iodoacetamide. Buoyant density gradient analysis as well as gel chromatography on Sepharose CL-6B columns, following alkaline borohydride and nitrous acid treatment of individual carbohydrate-rich subunits, showed that the latter consisted of core proteins associated solely with heparan sulfate glycosaminoglycans. The sizes of the latter were estimated by chromatographic techniques to be approximately 50 kDa in the case of the larger and 14 kDa for the smaller subunit. This is the first description of disulfide-bonded proteoheparan sulfates in endothelial cells.

Animals↗