Horse microsatellites and their amenability to comparative equid genetics.

We investigated the applicability of microsatellite primers, designed in horses, for use in plains and mountain zebras. Fifteen of the 20 tested horse-isolated primer pairs reliably amplified polymorphic loci in two wild equid species. We used this information to assess whether levels of genetic variation and repeat size differed in species from which microsatellites were isolated and in closely related target species. Target equid species exhibited similar levels of genetic variation to the horse, the species from which primers were originally isolated. We show that ascertainment bias results in lower mean and modal repeat size in target species. The data also provide evidence for a bi-directional mutational constraint in allele size across three equid species.

Radiometric assessment of hexose monophosphate shunt capacity in erythrocytes of rhinoceroses.

OBJECTIVES: To measure metabolic rates of the hexose monophosphate shunt (HMPS) in erythrocytes of rhinoceroses, and to test the hypothesis that low concentrations of endogenous ATP in erythrocytes impair HMPS capacity, thereby increasing susceptibility to oxidant-induced hemolysis. ANIMALS: 13 black and 3 white rhinoceroses, free-ranging in several regions of southern Africa, and 1 Sumatran rhinoceros in US captivity. PROCEDURE: HMPS fluxes were measured in rhinoceros erythrocytes with carbon-labeled glucose in the presence and absence of known HMPS activators. RESULTS: Compared with values for human erythrocytes, mean basal state HMPS fluxes were appreciably lower (22 to 46%) in all 3 rhinoceros species studied. Shunt activators increased HMPS rates approximately 5-fold over basal rates in rhinoceros erythrocytes, compared with increases in humans of 10-fold with ascorbate and 15-fold with methylene blue. Stimulated HMPS rates in human erythrocytes were quantitatively 5- to 10-times greater than those observed in rhinoceros erythrocytes. Overall HMPS catabolic rates were completely independent of intracellular ATP concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: HMPS glycolytic and recycling rates and responses to activators are inherently low in erythrocytes from 3 species of rhinoceros, likely contributing to (but not solely responsible for) the high susceptibility of black rhinoceroses to oxidant-induced hemolysis. Slow erythrocyte HMPS capacities were independent of intracellular ATP concentrations, invalidating a current hypothesis regarding the pathogenesis of hemolytic anemia in captive black rhinoceroses. Limitations in HMPS capacities emphasize the importance of protecting rhinoceroses from exposure to drugs, chemicals, toxins, foodstuffs, and other conditions known to increase production of oxidizing metabolites, reactive oxygen species, and free radicals.

Uptake of purine substrates shows major physiological variations in mammalian skin fibroblasts.

Uptake of the purine bases hypoxanthine and adenine were studied in skin fibroblast cultures from three different mammalian species. Marked variation in uptake was observed, and using an analysis of variance, the main component of this variation was found to be at the between experiment level as opposed to the between replicate or between individual level. After allowing for this variation, significant between species differences in uptake were found. Uptake of both purines decreased proportionately with passage number, and increased markedly after viral transformation of the fibroblast cultures. These results demonstrate significant metabolic differences in purine anabolic pathway fluxes between mammalian species but also have serious implications for the use of these methods in a diagnostic context.

Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships.

Glycyrrhetinic acid was shown previously to inhibit intercellular gap-junctional communication between human fibroblasts. In the present study 31 derivatives of glycyrrhetinic acid were tested for their ability to inhibit communication. Eight of the compounds inhibited communication with high potency (IC50 less than 3 microM) and showed low toxicity, properties which suggest they may be useful pharmacological probes for studies of gap-junction function.

Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid.

Intercellular gap-junctional communication was measured using metabolic co-operation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. 18-alpha-glycyrrhetinic acid (AGA) was found to inhibit communication by more than 95% at concentrations as low as 2 microM. Concentrations up to 100 microM were not cytotoxic over a period of 2 hours. Communication inhibition was of rapid onset and was readily reversible. Communication remained continuously yet reversibly blocked in cells cultured in the presence of AGA for 20 days. The related compounds 18-beta-glycyrrhetinic acid and carbenoxolone also caused communication inhibition. The effect is probably not mediated via mineralocorticoid or glucocorticoid receptors since aldosterone and glucocorticoids had no effect on communication. AGA thus has properties of a useful inhibitor in the study of intercellular junctional communication.

Inhibition of intercellular junctional communication in human fibroblasts by triphenylmethane, triphenylmethylchloride, tetraphenylboron and related compounds.

Intercellular junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. Triphenylmethane, triphenylmethylchloride and tetraphenylboron inhibited communication at concentrations at least 12-fold lower than cytotoxic concentrations. This inhibition was of rapid onset and was rapidly reversible by washing the co-cultures. Refractoriness to inhibition did not develop after prolonged exposure. Several other compounds also induced communication inhibition, but only at concentrations slightly below cytotoxic concentrations. Treatment of co-cultures with calcium ionophore A23187 or cycloheximide did not cause communication inhibition. It is suggested that triphenylmethane, triphenylchloride and tetraphenylboron may be useful inhibitors for studying the roles of intercellular junctional communication in some biological systems.

Studies on the mechanism of phorbol ester-induced inhibition of intercellular junctional communication.

Intercellular gap-junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate lyase-deficient human fibroblasts and argininosuccinate synthetase-deficient Chinese Hamster V79 cells. As previously shown, in this system junctional communication is completely inhibited by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the absence of extracellular calcium, TPA inhibition was less pronounced. However, synergism with calcium ionophore A23187 could not be demonstrated. Chlorpromazine, trifluoperazine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester partially antagonised the effect of TPA. No antagonism was demonstrable between calmidazolium and TPA. Treatment of co-cultures with exogenous phospholipase C or 1-oleoyl-2-acetylglycerol (OAG) resulted in communication inhibition, suggesting that protein kinase C activation is involved in the mechanism of phorbol ester-mediated communication inhibition. However co-cultures which had been made refractory to TPA by prolonged exposure to high concentrations remained sensitive to inhibition by phospholipase C and OAG. These results suggest either that diacylglycerol can produce other effects independent of protein kinase C activation, or that refractoriness to phorbol esters is not simply due to a decrease in the amount of protein kinase C.

Effects of 12-O-tetradecanoylphorbol-13-acetate and retinoids on intercellular junctional communication measured with a citrulline incorporation assay.

Inhibition of intercellular junctional communication by 12-O-tetradecanoylphorbol-13-acetate (TPA) and retinoids was investigated using a citrulline incorporation assay. This new assay uses metabolic co-operation between argininosuccinate lyase-deficient human fibroblasts and arginosuccinate synthetase-deficient cells as a measure of junctional communication. Short-term exposure to TPA resulted in virtually complete inhibition of metabolic co-operation when V79 cells were used as the synthetase-deficient type. When synthetase-deficient human fibroblasts were used, inhibition by TPA was only partial. Exposure to high concentrations of TPA for prolonged periods resulted in partial reversal of communication inhibition and a refractory state in which cells were unresponsive to TPA. Retinoic acid and other retinoids also inhibited metabolic co-operation, but did not cause desensitisation of the type seen with TPA after prolonged exposure. Cultures which had been made refractory to TPA remained sensitive to inhibition by retinoic acid and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, indicating that these latter compounds inhibit junctional communication by a mechanism different from TPA. Simultaneous exposure of cultures to TPA and retinoic acid showed that the inhibitory effects on metabolic co-operation of these compounds were additive. Fluocinolone acetonide did not antagonise the effect of TPA. These results suggest that retinoic acid and fluocinolone acetonide exert their anti-tumor-promoting action by mechanisms which are not mediated by intercellular junctional communication.

Use of a new citrulline incorporation assay to investigate inhibition of intercellular communication by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane in human fibroblasts.

A citrulline incorporation assay has been developed to measure intercellular communication between argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts (J. S. Davidson, I. M. Baumgarten, and E. H. Harley, Exp. Cell Res., 150: 367-378, 1984). This method was used to investigate the effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) on intercellular junctional communication. DDT at a concentration of 20 micrograms/ml inhibited metabolic cooperation by 90 to 98%. This inhibition was of rapid onset and was rapidly reversed by washing the cells. Inhibition of metabolic cooperation by DDT was not dependent on the presence of extracellular free calcium, indicating that DDT does not act by increasing net calcium influx into cells. This system should prove useful in elucidating the relationship between tumor promotion and intercellular communication.

Effects of extracellular calcium and magnesium on junctional intercellular communication in human fibroblasts.

The role of extracellular calcium and magnesium in intercellular communication via permeable junctions was studied by measuring metabolic co-operation between ASS- and ASL- human fibroblasts. Communication through pre-existing junctions was not affected by the removal of extracellular calcium and magnesium from the medium. On the other hand, the establishment of communication through new permeable junctions, when ASS- and ASL- cells were mixed, was dependent on the presence of extracellular calcium and magnesium.

Metabolic cooperation between argininosuccinate synthetase and argininosuccinate lyase deficient human fibroblasts.

Human fibroblasts deficient in either argininosuccinate synthetase or argininosuccinate lyase show low levels of incorporation of [14C]citrulline into protein. However, when these two cell types are co-cultured [14C]citrulline incorporation is restored to the levels found in control cultures. This metabolic cooperation is cell-density-dependent and does not occur by diffusion of argininosuccinate into the medium. Our results indicate that argininosuccinate passes between the two cell types via intercellular junctions, and this system provides a simple and accurately quantifiable model for the study of intercellular communication.

HLA antigens in acute anterior uveitis in South African blacks.

Fifty-three black patients with acute nongranulomatous anterior uveitis (AAU) were tissue typed and the results compared to those from a panel of 200 healthy unrelated black volunteers. No statistically significant deviation from the norm with regard to the frequencies of 38 HLA antigens could be observed.

Histocompatibility antigens in patients with hepatocellular carcinoma and their relationship to chronic hepatitis B virus infection in these patients.

Although hepatocellular carcinoma is probably caused by one or more environmental carcinogens, a genetically determined susceptibility to the development of the tumor has not been excluded. In looking for such a predisposition, we have compared the histocompatibility antigens (HLA) of 102 southern African blacks with histologically proved HCC with those of 208 healthy blacks. The standard two-stage lymphocyte microcytotoxicity method was used to test for 40 antigens: 17 in the A locus, 20 in the B locus, and 3 in the C locus. None of the HLA antigens had a frequency that was significantly different in the patients and the controls. A close association undoubtedly exists between chronic hepatitis B virus infection and hepatocellular carcinoma. If this virus is proved to be oncogenic with respect to hepatocellular carcinoma, a genetic predisposition to the hepatitis B virus carrier state may have an indirect bearing on the etiology of the tumor. Sera from the hepatocellular carcinoma patients were therefore tested for hepatitis B virus markers (HBV surface antigen and antibody against HBV core antigen), and these were related to the patients' histocompatibility antigens. None of the HLA antigen frequencies was significantly different in the surface antigen-positive and the surface antigen-negative patients. As 88% of the patients were anticore positive, no meaningful correlation could be carried out with this marker. Analysis of histocompatibility antigens thus failed to show evidence of a genetic predisposition either to hepatocellular carcinoma or to chronic hepatitis B surface antigenemia in patients with this tumor.

HLA antigens in glaucoma and ocular hypertension.

Forty-five white patients (32 with open-angle glaucoma and 13 with ocular hypertension) and 63 black patients (41 with open-angle glaucoma and 22 with ocular hypertension) were tissue typed for a total of 38 HLA antigens and the results compared to normal, unrelated, panels of 248 white donors and 150 black volunteers, respectively. No statistically significant differences with regard to the frequencies of 38 HLA antigens were detected among the various groups.