Effect of vitamin on 2-deoxy-d-glucose uptake in human fibroblast cultures

Summary 2-Deoxy-d-glucose (2-DOG) uptake was tested in human fibroblast cultures in the presence and absence of vitamin E. Addition of 10 μg/ml vitamin E to the culture medium significantly reduced this uptake for 2-DOG concentrations of 0.005, to 10 mmol/liter (P≤0.01). The decrease of 2-DOG uptake was inversely proportional to the rise in 2-DOG concentration (P≤0.01). The presence of vitamin E reduced by 71% the average cellular level of lipid peroxides (expressed as thiobarbituric acid reactive substances) and caused a small but significant decrease in the cholesterol concentration (P≤0.01). These last results might explain the decrease in 2-DOG uptake observed in the presence of vitamin E.     

A unique genomic sequence in the Wolf-Hirschhorn syndrome [WHS] region of humans is conserved in the great apes

The Wolf-Hirschhorn syndrome (WHS) is caused by a partial deletion in the short arm of chromosome 4 band 16.3 (4p16.3). A unique-sequence human DNA probe (39 kb) localized within this region has been used to search for sequence homology in the apes' equivalent chromosome 3 by FISH-technique. The WHS loci are conserved in higher primates at the expected position. Nevertheless, a control probe, which detects alphoid sequences of the pericentromeric region of humans, is diverged in chimpanzee, gorilla, and orangutan. The conservation of WHS loci and divergence of DNA alphoid sequences have further added to the controversy concerning human descent.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells

A major 27 kDa particulate and a minor 24 kDa cytosolic GTP-binding protein was detected in HEL cells upon incubation with [-³²P]GTP of nitrocellulose blots containing polypeptides separated using SDS-PAGE. Addition of lovastatin (30 M) to HEL cells in culture inhibited protein synthesis by 35%. However, this treatment resulted in a 5-fold increase, as quantitated by [-³²P]GTP binding, in the amount of cytosolic 24 kDa GTP-binding protein. Addition of cycloheximide plus lovastatin to cells in culture abolished the observed increase in 24 kDa GTP-binding protein. Incubation of cells with lovastatin plus [R,S]-[5-³H]mevalonolactone resulted in the incorporation of radioactivity into several polypeptides in both the cytosolic and particulate fractions including a polypeptide of molecular mass of 24 kDa in the cytosol. The mobility of this 24 kDa isoprenylated protein on SDS-PAGE was identical to that of the GTP-binding protein increased in response to lovastatin. However, the 24 kDa protein remained in the cytosol after undergoing isoprenylation. The 24 kDa protein was distinct from the HEL cell, G25K/CDC42Hs GTP-binding protein and the GTP-binding protein that was a substrate for botulinum toxin C3 catalyzed ADP-ribosylation. Results demonstrate that lovastatin specifically increases the expression of a 24 kDa GTP-binding protein in HEL cells and that, isoprenylation of low molecular mass GTP-binding protein(s) may have function(s) in addition to its role in the targetting of these proteins to cell membrane.     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Biological evaluation of trace element data in human ovaries by statistical analysis

Analysis of variance, analysis of covariance, correlation coefficient, multiple correlation, and partial correlation coefficient statistical tests were applied to Cs, Cr, Co, Fe, Rb, Sc, Se, and Zn content in human ovaries in order to evaluate statistically the possible relationships between these trace elements at: the ovary as an organ, each ovarian phase separately, each morphological part independent of the ovarian phase, and between cortex and medulla within the ovarian phases. The element Cs seems to have a homogenous distribution between cortex and medulla within reproductive and menopausal phase. Zinc shows a trend to have an antagonistic relation with Cs, Cr, Co, and Fe during fetal and reproductive phases and not during menopausal phase. The relationship between Zn and Cs when Fe is kept constant could be used as a tool for the decontamination of the ovary from an abnormal Cs content or for the inhibition of the accumulation of the same element to the ovarian tissue.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

A cellular model for drug interactions on hematopoiesis: The use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis

A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3-azido-3-deoxythymidine, acetylsalicyclic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC₅₀) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.Abbreviations ARA-CC cytosine arabinoside - AS acetylsalicylic acid - AZTT 3-azido-3-deoxythymidine - BFUU burst-forming units - BM bone marrow - CFU colony-forming units - DOXX doxorubicin - FU 5-fluorouracil - glyAA glyAcophorin A - HCB human umbilical cord blood - IU international units - PCMEM human placenta-conditioned medium - VA sodium valproate     

Assay of free-radical toxicity and antioxidant effect on the Hep 3B cell line: a test survey using lindane

The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS atomic absorption spectrometry - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle medium - GPx glutathione peroxidase - G.Red glutathione reductase - GSH reduced glutathione - GSSG glutathione disulfide - GST glutathione S-transferases - Prot proteins - SOD superoxide dismutase