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Biomedical subjects

D M Stocco

Publications and source records attributed to D M Stocco.

At least 127 records · Page 7Linked to original sources
PubMed1988

Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin.

The side-chain cleavage of cholesterol is the rate-limiting enzymic step in steroidogenesis and occurs in the mitochondria of steroid-producing tissues. In these studies, the effect of acute stimulation of MA-10 Leydig-tumour cells on the synthesis of mitochondrial proteins was investigated. Cells were incubated in the presence of stimulating levels of human choriogonadotropin (hCG) and [35S]methionine for 2 h periods. Mitochondria were isolated and their proteins analysed by two-dimensional polyacrylamide-gel electrophoresis and fluorography. At least three mitochondrial-specific proteins were found in cells exposed to gonadotropin, and these proteins were also found in cells treated with dibutyryl cyclic AMP (db cyclic AMP). The appearance of these proteins was prevented by the addition of cycloheximide to the cells, as was the production of progesterone, the major steroid produced in MA-10 cells. In addition, mitochondria isolated from cells stimulated with db cyclic AMP produced progesterone at a rate 3-fold greater than mitochondria isolated from control cells during 3 h of incubation. Lastly, mixing experiments demonstrated that sonicated mitochondria isolated from db cyclic AMP-treated cells stimulated progesterone production in control mitochondria 6-fold. These studies show that hCG and db cyclic AMP stimulation of MA-10 cells results in the rapid induction of cycloheximide-sensitive proteins located in the mitochondria which may be instrumental in the acute regulation of steroidogenesis.

Animals↗
PubMed1988

Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells.

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Stimulation of cholesterol side-chain cleavage enzyme activity by cAMP and hCG in MA-10 Leydig tumor cells.

Using a cloned Leydig tumor cell line (designated MA-10), we have studied the activity of cholesterol side-chain (CSCC) enzyme, the rate-determining step in steroidogenesis, in mitochondria isolated from cells pretreated either with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Results showed a slight but significant increase in CSCC activity with treatment by cAMP (25% increase) and hCG (60% increase), as compared to mitochondria isolated from nontreated control cells. However, this stimulation of CSCC activity appears to be of limited significance when compared to the approximately 1000-fold or greater increase observed in progesterone production in the presence of hCG or dbcAMP. On the other hand, unstimulated MA-10 cells or isolated mitochondria efficiently converted 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone, and this conversion was not affected by cycloheximide. The addition of cholesterol to intact cells or to isolated mitochondria did not affect progesterone production. Our observations clearly indicate that given the proper hydroxy substrates (22R-hydroxycholesterol or 25-hydroxycholesterol), MA-10 Leydig cells are able to convert them into progesterone without any stimulation by steroidogenic stimuli, i.e. cAMP or hCG. Since MA-10 Leydig cells can efficiently convert 22R-hydroxycholesterol--an intermediate in CSCC reaction--into progesterone, these results suggest that the key regulatory step in the mechanism of trophic hormone-stimulated steroid production is the first hydroxylation step of the 3 sequential monooxygenation reactions involved in the conversion of cholesterol to pregnenolone.

Allosteric Regulation↗
PubMed1984

Effect of cesium and potassium salts on survival of rats bearing Novikoff hepatoma.

The effect of CsCl on the life span of female Sprague-Dawley rats innoculated with Novikoff's hepatoma was studied as a function of both pre- and post-treatment with CsCl and as a function of the inoculant dose. The effect of KCl on the CsCl treatment was also studied. Rats treated with CsCl for 12 consecutive days prior to or immediately after inoculation with 1.0 ml of viable hepatoma cell suspension showed an increase in mortality score from corresponding controls. Conversely, increases in the dose of the inoculant resulted in delaying the onset of toxicity in rats receiving the Cs-treatment after inoculation as evidenced by a decrease in mortality. Availability of KCl in drinking water ad lib further decreased total mortality when given alone but not when combined with CsCl. The results indicate a dose-dependent paradoxical effect of CsCl on Novikoff hepatoma cell toxicity and suggest a critical intercellular balance requirement between Cs+ and K+ on the effect studied.

Animals↗
PubMed1983

Rapid, quantitative isolation of mitochondria from rat liver using Ficoll gradients in vertical rotors.

A method for the rapid, quantitative isolation of rat liver mitochondria has been developed. The method consists of placing portions of whole liver homogenate on linear Ficoll gradients followed by reorienting rate-zonal centrifugation in a vertical rotor. Fractions collected from the gradient were analyzed for protein, cytochrome oxidase, and a number of marker enzymes to determine mitochondrial location and the extent of their contamination with other cellular organelles. In the fraction designated as the mitochondrial peak, 85% of the total cytochrome oxidase activity was recovered. Contamination with other cellular organelles ranged from 7.07 to 14.05%. The respiratory control ratio of the isolated mitochondria was 8.10, indicating they were well coupled.

Animals↗
PubMed1983

Cell wall proteins of Candida albicans.

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.

Candida albicans↗
PubMed1983

Sertoli cell function in diabetic, insulin-treated diabetic, and semi-starved rats.

It is well documented that long-term diabetes mellitus results in numerous deleterious consequences. However, considerable controversy exists concerning male reproductive function in diabetes. The purpose of this investigation was to study several endocrine parameters in diabetic male rats with emphasis on Sertoli cell function. Male Wistar rats were injected with streptozotocin and then either left untreated for 30 days or injected with insulin so as to prevent spillover of glucose into the urine. These two groups were compared with control animals that had only been injected with the vehicle for streptozotocin. Semi-starved control animals were included to determine if any of the potential endocrine alterations were related to body weight changes which occur in streptozotocin-injected rats. It was found that FSH, LH, PRL, and GH serum levels were reduced in diabetic animals. Only FSH was restored to normal by insulin injections. The testis, seminal vesicle, and epididymis weights were all reduced in diabetic animals. Insulin injections raised all organ weights; however, only testis weights were fully restored. Levels of epididymal ABP activity were found to be higher in diabetic animals when expressed per mg protein. Similar patterns of organ weight loss and hormonal alterations were observed in semi-starved rats. However, epididymal levels of ABP activity were unaffected by the semi-starved condition. While weight loss should be taken into consideration when interpreting cause and effect relationships in streptozotocin-treated animals, epididymal ABP levels appear to be well correlated with the altered metabolic state characteristic of diabetes.

Androgen-Binding Protein↗
PubMed1981

Peritubular cell influence on the efficiency of androgen-binding protein secretion by Sertoli cells in culture.

The efficiency of androgen-binding protein (ABP) secretion into the medium of cultured Sertoli cells was increased by the addition of peritubular cells into the culture system. There was a positive correlation between the number of peritubular cells added to the cultures and the amount of ABP secreted. These responses were observed in cultures treated with highly purified FSH or in untreated cultures. Medium that had been exposed to peritubular cells before exposure to Sertoli cells did not mimic the effect observed when peritubular cells were added directly to Sertoli cells. The efficiency of ABP secretion by Sertoli cells was not a function of plating density, since the amount of ABP per mg cellular protein did not vary significantly among cultures of Sertoli cells which contained a wide variation in the numbers of cells present. These results suggest that peritubular cells play a role in regulating ABP secretion and thus support the hypothesis that cell to cell communication exists between these two cell types of the seminiferous tubule.

Androgen-Binding Protein↗
PubMed1980

Effects of serum components on the morphology of Sertoli cells in culture.

Studies of Sertoli cell structure, maturation, and function have been aided by the use of in vitro systems. Although numerous papers have appeared that utilize the Sertoli cell culture model, few papers have dealt with the characterization of these cells under various culture environments. Recently, it has been reported that the addition of serum to the culture medium prevents induction of long cytoplasmic appendages in cultured Sertoli cells that have been treated with FSH, TSH, or c-AMP. The purpose of this investigation was to determine which serum components, obtained by gel filtration, are capable of inhibiting the morphological response induced by FSH, TSH, or c-AMP. Sertoli cell-enriched cultures were prepared using collagenase and trypsin digestion, each followed by gravity sedimentation. Untreated cells grown on plastic or glass substrates assumed an epithelioid appearance after several days. Cells treated with FSH, TSH, or c-AMP formed long cytoplasmic appendages after 1-2 days. This response was prevented or reversed by the addition of fetal calf serum (10%), crystallized bovine serum albumin (0.25%-2%), or purified albumin obtained by gel filtration of whole serum (0.25%). It was also found that fractions that elute between the void volume and the initial albumin fractions (molecular weights of approximately 50,000 and greater) mimic the hormone-induced response after only 10-12 hours. The results of this investigation indicate that albumin is the primary serum component responsible for inhibiting morphological alterations induced by FSH, TSH, and c-AMP. Furthermore, it is apparent that the production of long filamentous cytoplasmic appendages in Sertoli cells can be induced by a wide variety of substances.

Animals↗
PubMed1980

Sialic acid: regulation of electrogenesis in cultured heart cells.

Removal of up to 50% of the sialic acid from aggregates of 7-day chick embryo heart cells during incubation in a highly purified preparation of neuraminidase resulted in hyperpolarization of the maximum diastolic potential, reduction in the slope of diastolic depolarization leading to slowing of beating, negative shifts of threshold potential and the voltage at which upstroke velocity was maximal, and an initial increase in the action potential overshoot. These effects were opposite to those induced by phospholipase C, a possible contaminant. The modification of electrical properties by neuraminidase is suggested to result from an enhanced influx of calcium ions that might "screen" or bind specifically to internal negative fixed charges and thereby shift the voltage dependence of conductance and kinetic parameters to more negative potentials. This hypothesis is supported by the results above and by greater uptake of 45Ca following release of sialic acid, augmentation of enzyme-induced changes in elevated Ca2+, and the similarity of effects produced by A23187, and inophore which also increased 45Ca uptake. However, other mechanisms cannot be ruled out at the present time.

Action Potentials↗
PubMed1980

Characteristics of mitochondria isolated by rate zonal centrifugation from normal liver and Novikoff hepatomas.

Mitochondria were isolated from whole homogenates of normal liver and Novikoff hepatomas using reorienting rate zonal centrifugation on sucrose gradients. The activities of several mitochondrial-specific enzymes and ultrastructure were compared in the two tissues. Our results indicate that cytochrome oxidase, lipoamide dehydrogenase, malate dehydrogenase, and succinate dehydrogenase activities are all higher in liver homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepatoma than in liver. The activity of these enzymes in isolated mitochondria displayed a much different pattern. Both cytochrome oxidase and succinate dehydrogenase activities were higher in hepatoma mitochondria than in liver mitochondria. Lipoamide dehydrogenase and malate dehydrogenase, conversely, were higher in liver mitochondria. Hexokinase was found to be virtually absent in liver mitochondria but plentiful in hepatoma mitochondria. Ultrastructural studies have shown that the hepatoma mitochondria are much smaller in size, which results in a decreased rate of migration into the gradient. These studies have also shown that normal liver consists of predominantly "condensed" forms of mitochondria, whereas hepatoma contained a majority of "twisted" species. Experiments using 1% bovine serum albumin in the homogenization procedures and in the gradient have confirmed earlier observations that bovine serum albumin is essential for optimal isolation of neoplastic mitochondria.

Animals↗
PubMed1979

The requirement of DNA synthesis for the induction of alkaline phosphatase by bromodeoxyuridine in a derivative of the HeLa cell line.

Non-lethal concentrations of bromodeoxyuridine induce a 2- to 5-fold increase in the specific activity of alkaline phosphatase in a HeLa subclone, S3G. Experiments employing 10-hour pulses of BRdU showed that 48 hours were required before induction commenced, and that maximal induction was attained by 96 hours. Under conditions in which DNA synthesis was prevented with hydroxyurea induction did not occur. Upon removal of hydroxyurea both DNA synthesis and induction were rapidly reestablished. Furthermore, experiments employing radiolabelled BRdU demonstrated that the kinetics of the induction process paralleled the incorporation of the analogue into cellular DNA. These results indicate that DNA synthesis, or some process intimately linked to DNA synthesis, is required for the induction of alkaline phosphatase, and suggest that the mode of the induction may be through the incorporation of the analogue into cellular DNA.

Alkaline Phosphatase↗
PubMed1978

Quantitation of mitochondrial DNA and protein in the liver of Fischer 344 rats during aging.

Mitochondria from liver whole homogenates of Fischer 344 rats, 3,6, 12 and 24 mo of age were separated by reorienting rate-zonal centrifugation on sucrose gradients. Employing this method, mitochondria can be harvested nearly quantitatively and subjected to a number of biochemical analyses. Separation and recovery of the mitochondria from young and old rats was monitored by assaying each fraction from the gradient for cytochrome oxidase and lipoamide dehydrogenase, two mitochondrial specific enzymes. In addition, several regions of the gradient were studied by transmission electron microscopy. In this study, mitochondrial DNA and protein were quantitated from the livers of the animals at each age group. It was demonstrated that between the ages of 12 and 24 mo these macromolecules decreased significantly. These data would seem to indicate that the number of mitochondria is decreased in this tissue in aged animals.

Aging↗
PubMed1977

Quantitation of mitochondrial DNA, RNA, and protein in starved and starved-refed rat liver.

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved-5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of 3H-TTP into into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA sythesis.

Animals↗
PubMed1975

Adenine nucleotide metabolism in heat-synchronized Tetrahymena.

The metabolism of acid soluble adenine nucleotides in heat-synchronized Tetrahymena pyriformis GL has been studied. In addition, the effect of the synchronizing temperature (34 degrees C) on adenine nucleotide metabolism in heat-synchronized cells has been determined. In cells induced to divide synchronously through heat treatment (cyclic pulses of 34 degrees C for 30 min alternating with a 30 min recovery period at 28 degrees C) variations occurred in the levels of adenine nucleotides when samples of cells were analysed at the end of the last thermal period and at various time preceding the first synchronous cell division. The specific activities of the adenine nucleotides were found to be significantly higher during a pulse label period performed at the end of the last thermal period than at any time during the subsequent synchronous division cycle. The synchronizing temperature was found to partially deplete the intracellular stores at ATP in heat synchronized cells. This decrease was reversible with ATP levels recovering after 15 min of incubation at 28 degrees C. Fluctuations in the levels and specific activities of the adenine nucleotides are discussed in their relation to macromolecular synthesis and the cell cycle in Tetrahymena.

Adenine Nucleotides↗