Biomedical subjects

F C Guma

Publications and source records attributed to F C Guma.

9 recordsLinked to original sources

PubMed2001

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells.

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.

Animals↗

PubMed2001

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Actinin↗

PubMed2001

Synthesis and transport of different sphingomyelin species in rat Sertoli cells.

Cellular phospholipids of Sertoli cells from immature rats were labeled with [14C]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [14C]-choline over a 45 h period of incubation demonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibility to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and SM2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease of about 60-70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-course experiments indicated a phosphatidylcholine --> SM precursor product relationship and differences in kinetic properties between SM1 and SM2. Resynthesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated that the resynthesized fraction reached the outer leaflet of the plasma membrane. The 60-70% inhibition of SM synthesis by monensin showed that the trans-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/medial Golgi apparatus. Additionally the results indicated that plasma membrane SM synthase activity could be the site of about 15% of SM synthesis in Sertoli cells.

Animals↗

PubMed1999

An Mn2+-stimulated neutral-sphingomyelinase in seminiferous tubules of immature Wistar rats.

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. The seminiferous tubules of immature (19 day-old) Wistar rats have at least three types of sphingomyelinases, a lysosomal one and two microsomal ones. One of the microsomal sphingomyelinases is active at pH 6.5 and is stimulated by Mn2+ > Co2+ > Mg2+, and the other is active at pH 7.4 and is stimulated by Mn2+ > Mg2+ and inhibited by Co2+. The two microsomal enzymes are only slightly inhibited by EDTA and at pH 7.4 the stimulatory effects of Mn2+ and Mg2+ are additive. These data characterize the existence of two different membrane-bound sphingomyelinases in the seminiferous tubules of the rat.

Age Factors↗

PubMed1997

Influence of the biomatrix on the response of Sertoli cells to FSH.

Sertoli cell preparations isolated from 15-day-old Wistar rats were cultured on two different substrates, i.e., plastic and a biomatrix isolated from seminiferous tubules of rat testis. Sertoli cells cultured on a biomatrix acquired a phenotype and morphology more characteristic of in vivo differentiated cells. In order to determine the influence of a biomatrix on the response of Sertoli cells to FSH, on the 7th day of culture, untreated cells, or cells pretreated for 12 h with FSH (1 microgram/ml), were incubated with [U-14C] leucine or [2-3H] mannose. Cells cultured on the biomatrix showed higher [U-14C] leucine and [2-3H] mannose incorporation into proteins and glycoproteins. FSH increased these activities in cells cultured on both substrates, although its stimulating effect was higher on cells cultured on the biomatrix. These results demonstrate that the biomatrix increases protein and glycoprotein synthesis and secretion, and also influences the response of Sertoli cells to FSH.

Animals↗

PubMed1997

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats.

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 micrograms/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed microgram protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2-14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

ATP Citrate (pro-S)-Lyase↗

PubMed1994

Effects of retinol on glycoprotein synthesis by Sertoli cells in culture: dolichyl phosphomannose synthase activation.

Sertoli cells were isolated from Wistar rats aged 19 days and cultured for 48 h. The addition of retinol (10 microM) to the culture medium significantly stimulated the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into cellular and secreted glycoproteins. Incorporation of [U-14C] leucine into proteins and of [5, 6-3H] uridine into RNA was unaffected by retinol treatment. Incubation of microsomal fractions of retinol-treated cells showed an increase in mannose incorporation into dolichyl phosphomannose, into dolichyl pyrophosphoryl oligosaccharide and into proteins. Chromatographic analysis of the fraction soluble in chloroform/methanol (2:1 v/v) did not show the presence of retinyl phosphomannose either in control or in retinol-treated cells. When the formation of dolichyl phosphomannose was studied in microsomes isolated from control cells and from cells treated with 10 microM retinol for 48 h in the presence of exogenous dolichyl phosphate, the results showed that the retinol effect was due to stimulation of dolichyl phosphomannose synthase.

Animals↗

PubMed1994

Biomatrix effect on Sertoli cells phospholipids.

In order to investigate the influence of biomatrix on Sertoli cell morphology and on the phospholipids content, these cells were isolated from testes of 15-day old Wistar rats and plated onto plastic coated with extracellular matrix extracted from seminiferous tubules, here denoted biomatrix. When the Sertoli cells were cultured on biomatrix they did not form a monolayer until day 7 of culture, while cells plated onto plastic did so 48 h after plating. On day 5 of culture, Sertoli cells were incubated for 48 h with 5 microCi/ml 32P. There was no difference in 32P incorporation into lipids of cells plated onto biomatrix or plastic. However, there was a larger amount of phospholipid phosphate in cells plated onto biomatrix than onto plastic. When the phospholipids were analyzed by bidimensional thin-layer chromatography, no differences were detected in their distribution; however, there was a significant decrease in the percentage of sphingomyelin in cells plated onto biomatrix when compared to plastic. These results showed that the cells cultured on biomatrix change their phospholipids content, but not their distribution. The importance of a small reduction in sphingomyelin content remains to be investigated.

Animals↗

PubMed1990

Glycoprotein biosynthesis by testes of 40-day-old rats subjected to protein malnutrition.

The testes of 40-day-old rats subjected to protein malnutrition show a marked delay in maturation of the seminiferous epithelium, as well as greater mannose incorporation into glycoprotein than observed in normal animals of the same age. Testes were incubated for 1 h with [2-3H]mannose and germ cells were then separated by the Staput method. Mannose incorporation occurred in the same cell fraction, i.e. the spermatocytes, both in normally fed and protein-undernourished animals. These data were confirmed by incubating the cells previously isolated on the gradient with [2-3H]mannose. Comparison of these data with results obtained in previous studies on 20-day-old animals in which mannose incorporation was lower in undernourished rats suggests that the differences observed in the present study between the experimental groups are due to alterations in the germ cells.

Animals↗