A novel mechanism of action of corticotropin releasing factor in rat Leydig cells

We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.     

Effect of potassium humate on apple cv. ‘Golden Delicious’ cultured in vitro

The effects of humic substances on in vitro culture of Golden Delicious apple are reported. Potassium humate (KH) when used in proliferation showed a negative interaction with BA while it enhanced rooting when IBA was not present in the culture medium. In the presence of IBA, KH increased root number and reduced root growth. The highest concentration tested, 500 mg l^-1, caused a drastic reduction in root system development. 50 mg l^-1 KH hastened rooting and plants grew more rapidly when transferred to soil.     

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 andD-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: Role of Mg2+ ions

Agonists modulation of Mg²⁺-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg²⁺ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with higher and lower apparent affinity states, are extrapolated. In the presence ofD-Ala²-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E₂ increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg²⁺-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

Oncofetal antigen-I (OFA-I) relative antigenicity on melanoma cells

Summary The oncofetal antigen-I (OFA-I) has been defined as an immunogenic antigen that is expressed on human cancer cells and is cross reactive with fetal brain tissue. Quantitative variations in the expression of OFA-I among different cultured melanoma cell lines were determined by absorption techniques based on functionally monospecific anti-OFA-I serum. Allo-antibodies were removed by absorption with lymphoblasts autologous to an OFA-I-positive target cell. Functional monospecificity toward OFA-I was confirmed by complete absorption with a specimen of fetal brain but not by liver from the same fetus.Of 14 melanoma cell lines tested, two did not express OFA-I, whereas 12 expressed the antigen to varying degrees. Five of the cell lines were highly antigenic, and serum absorbed with 5×10⁵ of any of these cell lines could reduce the anti-OFA-I titer (1 : 96) at least four-fold. OFA-I was detected on biopsied melanomas autologous to the antigenic cultured cells. The ability to select highly antigenic cell lines could be useful in further attempts to characterize OFA-I and to monitor tumor immunity in vitro. Antigenic cell lines may improve the response of patients treated in trials of immunotherapy.     

Synthesis of dolichol derivatives in human erythrocyte membranes

Summary Human erythrocyte membranes contain the enzymes responsible for the synthesis of dolichol-P-glucose, dolichol-P-mannose, dolichol-PP-N-acetylglucosamine, dolichol-PP-NN diacetylchitobiose and of dolichol-PP-oligosaccharides containing NN diacetylchitobiose and mannose or the same sugar residues plus glucose. The transfer of the oligosaccharide moieties from the dolichol-PP-oligosaccharides to endogenous proteins could not be detected. These enzymes appeared to be integral membrane proteins.Abbreviation Dol dolichol Dedicated to ProfessorLuis f. Leloir on the occasion of his 70th birthday.     

Species composition,similarity, and structure of mayan homegardens in tixpeual and tixcacaltuyub,yucatan, Mexico

We studied species composition, similarity, and structure of homegardens in two Yucatecan Maya communities, Tixpeual and Tixcacaltuyub, Yucatan, Mexico. The number of gardens sampled per village was 20 and 22; total area sampled was very similar, 45,265 m² and 40,150 m²; the number of trees and shrubs present was 5651 and 5603; and number of species was 135 and 133, respectively. Diversity was low for both sites (H′= 1.6), as were the correlation coefficients (r) for the species-area and individuals-area correlations. The relatively low values obtained for the structural parameters reflect the random pattern of plant incorporation to the gardens, the variability in the proportion of constantly used and not constantly used garden area, and a certain uniformity in the number of species used and number of individuals present, and the relationship between these parameters and garden size. All these reflect the uniqueness of each homegarden, which depends upon the cultural background of the owner. We noticed a trend towards a change in homegarden structure and function in response to the modernization process. Homegardens in villages in the outskirts of cities tend to have more ornamental species and commercial fruit plants than homegardens in isolated villages.     

Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate

ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM.The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the P_i-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion.The addition of P_i induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP.Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.