Biomedical subjects

A Verkerk

Publications and source records attributed to A Verkerk.

At least 37 records · Page 2Linked to original sources

PubMed1988

Reversible inhibition of DNA and protein synthesis by cumene hydroperoxide and 4-hydroxy-nonenal.

To test the possible role of lipid peroxidation in the process of in vitro ageing, human diploid skin fibroblasts were cultured with the lipophilic hydroperoxide cumene hydroperoxide (Chp) or the breakdown product of lipid peroxidation 4-hydroxy-2,3-trans-nonenal (HNE). Both compounds inhibited cellular DNA and protein synthesis in a dose-dependent way. Cells exposed to Chp or to HNE during growth inhibition recovered DNA and protein synthesis within 24 h upon removal of Chp or HNE from the culture medium. Continuously proliferating cells showed only a partial recovery of DNA and protein synthesis. Pre-culturing cells with the lipophilic free radical scavenger vitamin E did not abolish the effect of Chp upon DNA synthesis. Cellular levels of reduced glutathione (GSH) rose slightly during 1 week of culture with HNE, but remained unaltered with Chp. Neither ATP levels nor cellular energy charges were affected during culture with Chp or HNE. So, DNA synthesis is not impaired due to a shortage of nucleotides nor does GSH protect DNA synthesis against the effects of Chp or HNE. These results suggest that oxygen free-radical induced lipid peroxidation is not the cause of the irreversible loss of proliferation occurring during in vitro ageing.

Aging↗

PubMed1988

Characterization of oxygen-tolerant Chinese hamster ovary cells. II. Energy metabolism and antioxidant status.

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O2/1% CO2, and O2 level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O2 tolerance we compared CHO-20 cells growing at 20% O2 (normoxia) and CHO-99 cells at 99% O2 (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP+) levels were elevated 2.7-fold, while the ratio of NADPH to NADP+ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.

Animals↗

PubMed1987

Influence of cumene hydroperoxide and 4-hydroxynonenal on the glutathione metabolism during in vitro ageing of human skin fibroblasts.

Cumene hydroperoxide (Chp) and 4-hydroxynonenal (HNE) were used to investigate the effect of peroxidative challenge upon the glutathione (GSH) metabolism of human skin fibroblasts. Cellular GSH contents decreased during short-term incubations with Chp and oxidised glutathione (GSSG) was formed concomitantly. During longer incubations the GSH level was restored and the substrate flux through the pentose phosphate shunt increased. So in the presence of hydroperoxides the GSH level is maintained by reduction of GSSG. HNE caused a strong decrease in cellular GSH contents. Prolonged incubation with HNE lead to a rise in GSH contents above the basal level. The flux through the pentose phosphate shunt did not change during exposure to HNE. Hence, during incubation with HNE the cell maintains its GSH content by de novo synthesis of GSH. This conclusion is further substantiated by the findings with a cell strain deficient in GSH synthetase. These cells survived if incubated with Chp but not if exposed to HNE. GSH contents of normal cells from phase II (young) cultures and from phase III (aged) cultures responded similarly to Chp during short-term incubations and during a week of culture with the test compound. The flux through the pentose phosphate shunt rose much more in phase III than in phase II cells when incubated with the same concentration series of Chp. We conclude that during in vitro ageing the amount of NADPH needed to maintain cellular GSH levels in the presence of hydroperoxides increases, while the capacity to respond to such a challenge is not affected.

Adult↗

PubMed1987

Localization of a gene involved in complementation of the defect in xeroderma pigmentosum group A cells on human chromosome 1.

Human, Chinese hamster or Chinese hamster/human hybrid cytoplasts were fused with UV-irradiated xeroderma pigmentosum group A (XP-A) cells. Unscheduled DNA synthesis (UDS) of the XP-A nucleus was measured 0-2 and 2-4 h after seeding of the fused population. Human cytoplasts did correct the defect in the XP-A nucleus immediately after fusion, whereas the chinese hamster cytoplasts did not show this rapid increase in excision repair. The results obtained after fusion of cytoplasts isolated from a panel of 26 Chinese hamster-human hybrids showed that chromosome 1 bears genetic information that is necessary for the rapid correction of the XP-A defect. Furthermore, this genetic information was regionally assigned to 1q42-qter by analysing hybrid cell lines having retained various segments of chromosome 1. Cytoplasts from a Chinese hamster/XP-A hybrid containing chromosome 1 of XP-A origin corrected also the defect with fast kinetics. This result indicate that the correcting factor consists of human and Chinese hamster components. As a consequence, the gene mapped on chromosome 1 may not be the gene which is mutated in XP-A cells.

Cell Fusion↗

PubMed1987

Glucose flux through the hexose monophosphate shunt and NADP(H) levels during in vitro ageing of human skin fibroblasts.

In cultured human skin fibroblasts the glucose flux through the hexose monophosphate shunt (HMS) amounts to 4% of the glucose flux through the glycolytic pathway. Upon in vitro ageing the rate of glucose utilization through the HMS is decreased more than 50%. This decrease in HMS was not caused by a limiting enzymatic capacity since glucose utilization through the HMS could be raised at least 30-fold in both 'young' and 'aged' fibroblasts upon stimulation with phenazine methosulphate. This effect of in vitro ageing upon glucose metabolism was also not due to differences in proliferation rate between 'young' and 'aged' human fibroblasts, since there was no difference in glucose utilization between proliferating and growth-inhibited (confluently cultured) fibroblasts. The NADPH/NADP ratio was found to be decreased by 12% in 'aged' cells.

Adult↗

PubMed1986

De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method.

A flow cytometric method to determine cellular GSH contents has been developed. This method is fast and simple and enables the determination of GSH contents in intact cells. Results obtained with the new method correlate well with the results obtained by a specific biochemical assay for GSH (r = 0.9984; n = 7). The method has been used to determine GSH recovery rates in cultured fibroblasts from healthy subjects and from patients with Werner's syndrome, Spielmeyer-Vogt syndrome and Fanconi's anemia. No obvious differences in GSH recovery rates were observed. GSH recovery rates were also not affected after in vitro ageing. Experiments with cells deficient in GSH synthetase revealed that the observed GSH recovery is exclusively due to de novo synthesis.

Adolescent↗

PubMed1986

Detection of acid-beta-galactosidase activity in viable human fibroblasts by flow cytometry.

The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was used to detect acid beta-galactosidase in intact cultured human fibroblasts. The accumulation of intracellular fluorescein, as measured by flow cytophotometry was linear with the incubation time in three control strains. The two fibroblast strains from patients with acid beta-galactosidase deficiency did not show an accumulation of intracellular fluorescence. Within one control cell population there was a positive correlation between the amount of accumulated intracellular fluorescein fluorescence and the specific acid beta-galactosidase activity as measured biochemically on sorted cells from different zones of the fluorescence distribution. No correlation was found between the specific acid beta-galactosidase activity and the fluorescein fluorescence of three different control cell strains.

Cells, Cultured↗

PubMed1986

Accumulation of a high molecular weight glycoprotein during in vitro ageing and contact inhibition of growth.

A 240 000 molecular weight protein was found to accumulate in sorted autofluorescent (AF) cells, and during growth inhibition and in vitro ageing of cultures of human skin fibroblasts. Vitamin E, a lipophilic free radical scavenger which suppressed completely the formation of cellular autofluorescence, did not affect the accumulation of this protein. So, this accumulation is not related to cellular autofluorescence and lipid peroxidation, the major cause of this autofluorescence. This protein was also found in cells from a patient with the Spielmeyer-Vogt syndrome with a high percentage of maximal lifespan (MLS), while it was completely absent from all cells of a patient with Werner's syndrome. On two-dimensional gel electrophoresis the protein showed a heterogeneous acidic isoelectric point (IEP) of around 5.3. Neuraminidase treatment caused the IEP of this protein to shift towards a less acidic pH value (5.85). Upon differential centrifugation of a cell homogenate the protein was found to be located in the microsomal pellet and the cytosol. Chromatography on gelatin-sepharose revealed that the protein was not fibronectin. It is concluded that in human skin fibroblasts a high molecular weight glycoprotein accumulates as a result of impaired proliferation and that this accumulation is not related to cellular lipid peroxidation.

Adult↗

PubMed1985

Autofluorescence of human skin fibroblasts during growth inhibition and in vitro ageing.

The increase in autofluorescence (AF) of human skin fibroblasts during their in vitro ageing and growth inhibition was investigated by means of flow cytophotometry. The cellular AF of in vitro ageing cultures increased while the relative number of (3H)-thymidine incorporating cells decreased. Therefore, the rate of accumulation of cellular AF during in vitro ageing of the cultures is inversely related to the proliferation rate of the culture. The rates of increase of AF varied widely among the cell strains, being the highest in cells from patients with Werner's syndrome. Upon growth inhibition in a confluent culture the net rates of increase of cellular AF were found to vary widely among the cell strains. The respective net rates of increase of AF of the cells from patients with Werner's syndrome and the Spielmeyer-Vogt syndrome were within the range covered by the normal cell strains. The ultrastructure of the bright AF cells from patients with Werner's syndrome and the Spielmeyer-Vogt syndrome differed from the ultrastructure of AF cells from control persons with regard to the morphology of their residual bodies, those from the patients contained more multilamellar and multivesicular structures. In sorted non-AF cells vitamin E was found to completely inhibit the accumulation of AF without affecting the formation of 'residual bodies'. We infer that cellular AF is caused by lipid peroxidative reactions and that the accumulation of AF is due to a decrease in cellular proliferation rate.

Adult↗

PubMed1984

Glutathione content of cultured human fibroblasts during in vitro ageing.

The glutathione level of cultured human fibroblasts was determined with a micromodification of a spectrophotometric glutathione cycling method. There was a slight increase in reduced glutathione (GSH) content during in vitro ageing of normal human fibroblasts. Fibroblasts from patients with Werner's syndrome or ceroid lipofuscinosis (Spielmeyer-Vogt syndrome) and healthy individuals exhibited similar patterns of GSH levels during in vitro ageing. The GSH content of non-proliferating confluent cultures of normal fibroblasts and of proliferating normal fibroblasts was identical. Moreover, autofluorescent "aged" cells isolated by cell sorting did not differ in GSH content from the non-autofluorescent cells in the same culture. It was concluded that the GSH content does not play a role in in vitro ageing, nor in the accumulation of autofluorescent material in human skin fibroblasts.

Cell Survival↗

PubMed1984

Cell sorting and microchemistry of cultured human fibroblasts: applications in genetics and aging research.

Cell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra-) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000-10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X-linked genetic disease. Moreover, microchemistry is used to study enzyme activities in sorted autofluorescent "aged" fibroblasts.

Aging↗

PubMed1984

Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry.

The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2 and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5-1%, whereas glutaraldehyde should be used at concentrations between 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.

Acrolein↗

PubMed1983

Flow sorting in the study of cell-cell interaction.

Undifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation. A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells. In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%. After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern. Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell-cell interaction in vitro.

Animals↗

PubMed1983

Detection of Fabry's disease heterozygotes by enzyme analysis in single fibroblasts after cell sorting.

Single cells were sorted from cultured fibroblasts of five carriers of Fabry's disease using a cell sorter (FACS II). The alpha-galactosidase A activity in the single fibroblasts was assayed in nanoliter droplets with the help of quantitative microfluorimetric techniques. Two populations of fibroblasts were present in the carriers, one showing an alpha-galactosidase-A activity comparable to that of Fabry patients, and another with normal alpha-galactosidase-A activity. This provides evidence of X-inactivation at the alpha-galactosidase-A locus. Since X-inactivation occurs at random, a high number of single cells has to be assayed to increase the clinical reliability for carrier detection. The methodology as presented enables such an approach.

Cell Separation↗

PubMed1982