Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5–96 h) with [³H]galactose, [³H]glucosamine, or [³H]myoinositol and treatment of the purified [³H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([³H]galactose and [³H]myoinositol) or 72 h ([³H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([³H]galactosamine, [³H]mannose, and [³H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5–72 hr) with [¹⁴C]linoleate, [³H]myristate, [³H]-oleate, [³H]palmitate, or [³H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [³H]palmitate and [³H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA₂ treatment of the dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [³H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 μg/ml), or the membrane permeable cAMP analog (but)₂ cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)₂ cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [³H]glucosamine in the presence of (but)₂cAMP (1 mM), TPA (10^?7 M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [³H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no efect of the hormone on undifferentiated granulosa cells could be observed. The rapid effect elicited by hCG on GPI content and turnover may be an early transduction mechanism involved in the biological effects of LH/hCG in differentiated granulosa cells. © 1993 Wiley-Liss, Inc.     

Histo-cytological study of the liver of the cabrilla sea bass, Serranus cabrilla (Teleostei, Serranidae), an available model for marine fish experimental studies

Livers of juvenile cabrilla sea basses ( Serranus cabrilla ) were subjected to light and electron transmission microscopy following different periods of maintenance in an aquarium. Since this fish is easy to feed in captivity and the hepatic structure was found to be comparable at the four periods tested (0, 5, 10 and 20 days), both at the histological and ultrastructural level, the liver of S. cabrilla could be an available model for marine contamination experimental studies. As in other fish species, it is not possible to distinguish the portal lobules and the triads. The Melano-Macrophage Centres contain tipofuscins, ceroids and haemosiderin, but they do not contain any melanin. The hepatocytes are arranged in cords (two cells thick), and, at the ultrastructural level, they show numerous microvilli in the perisinusoidal and canalicular areas. The hyaloplasm includes a considerable amount of glycogen and some lipid droplets are occasionally observed. Mitochondria, rough endoplasmic reticulum and the Golgi apparatus are relatively scarce.     

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered. (c) 1993 John Wiley & Sons, Inc.     

Relationships between cell surface protease and acid phosphatase activities ofLeishmania promastigote

A correlation between the ratio of the cell surface protease activity to phosphatase activity and the complexity of the pattern of cell surface exposed polypeptides ofLeishmania promastigotes was demonstrated for various strains grown under similar conditions. The ratio of the cell surface protease activity to acid phosphatase activity was high forL. major andL.b. panamensis and it correlates with the expression of a single polypeptide of 63 KDa on their cell surface. Intermediate and lower ratios of these enzymatic activites relate with more complex radio-iodinated patterns: two main bands inL.b. guyanensis (70 and 58 KDa) andL.b. braziliensis (72 and 60 KDa) and three main bands 65, 50, 27 KDa in allL.m. mexicana strains tested. Evidence is presented that the acid phosphatase located on theL.m. mexicana cell surface is not an artifact due to a secondary absorption of the secreted acid phosphatase from the culture medium. These results confirm theLeishmania antigen cell surface heterogeneity. The implications on the biology ofLeishmania and the clinical manifestation of leishmaniasis are discussed.     

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells

Journal of Physiology and Biochemistry - We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells....     

In vitro characterization of estrogen induced syrian hamster renal tumors: Comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney

Summary Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro. This study was supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC, and by grant CA-22008 from the National Cancer Institute, NIH, DHHS, Bethesda, MD.     

Observaciones de campo sobre la biologia deMetriona elatior (Col.: Chrysomelidae) enSolanum elaeagnifolium (Solanaceae) del uruguay

Field observations on the biology ofMetriona elatior (Col.: Chrysomelidae) onSolanum elaeagnifolium (Solanaceae) from Uruguay. Specimens ofMetriona elatior were found feeding on leaves of the silverleaf nightshade,Solanum elaeagnifolium, from the eastern of Uruguay since 1984. According to a field study the young leaves were skeletonized since spring to early autumn by larvae and adults. Oothecae and pupae were found mainly on old leaves. The egg mean volume was of 0.614 mm³. Young plants of the weed were always killed after massive natural infections ofM. elatior. Therefore, this tortoise leaf-beetle may be studied as a potential biocontrol agent on populations ofS. elaeagnifolium.

     

Effect of stress,adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.