The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits?

Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes.     

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

The effect of caffeine on some mouse brain free amino acid levels

Changes in free amino acids were examined in the central nervous system of mice treated with caffeine for three weeks. Caffeine was administered in the drinking water, and at the end of three weeks the level of caffeine in the cerebral cortex was 113±19 g/g. When amino acid levels in cerebral hemispheres, midbrain, pons and medulla, and cerebellum were measured a significant increase in glutamine levels was found in all four regions. Glycine, alanine, serine, threonine, and GABA were significantly reduced in some regions. Caffeine appears to alter some of the metabolic or transport processes regulating amino acid pools in the brain. The decrease of GABA found in pons and medulla may contribute to the observed increase in reflex excitability after caffeine.Special issue dedicated to Dr. Elling Kvamme     

Segregation analysis detects a major gene controlling blood infection levels in human malaria.

The profound influence that the genetic makeup of the host has on resistance to malaria infection has been established in numerous animal studies. This genetic heterogeneity is one of the main causes of the difficulties in developing an effective malaria vaccine. Segregation analysis is the first step in identifying the nature of genetic factors involved in the expression of human complex diseases, as infectious diseases. To assess the role of host genes in human malaria, we performed segregation analysis of blood parasite densities in 42 Cameroonian families by using both the unified mixed model and the class D regressive model of analysis. The results provide clear evidence for the presence of a recessive major gene controlling the degree of infection in human malaria. Parameter estimates show a frequency of .44-.48 for the deleterious allele, indicating that about 21% of the population is predisposed to high levels of infection.     

Differential regulation of thyroid cell-cell and cell-substrate adhesion by thyrotropin

Preservation of cell aggregation is necessary for thyroid follicular differentiation in vitro and requires stimulation by thyrotropin (TSH). We have tested the hypothesis that TSH preferentially increases thyroid cell-cell adhesion relative to cell-substrate adhesion. Cell-cell adhesion was measured in short-term suspension cultures by the decrease in the fraction of single cells remaining in culture (free cell ratio, FCR). When incubated in medium alone freshly isolated cells showed a progressive fall in FCR but this was accelerated by TSH and the cyclic AMP analog, 8-(4-chlorophenylthio)cyclic AMP. Aggregation was dependent upon extracellular Ca2+ and also promoted by a cell-free membrane extract. In contrast, attachment of cells to plastic dishes treated for tissue culture was not affected by TSH. We conclude that thyroid cells possess a TSH-sensitive cell adhesion system. The preferential increase in cell-cell adhesion may be one mechanism by which TSH stimulates the formation and preservation of follicles in vitro.     

pH-dependent phase transition of chlorpromazine micellar solutions in the physiological range

The effects of pH and drug concentration on aggregation properties of chlorpromazine-HCl (CPZ) are examined. The critical micelle concentration (cmc) changes from 0.2 mM at pH 7.3 to 2 mM at pH 5.6 as estimated from the stearic acid spin label solubility measurements. For concentrations above the cmc CPZ micelles undergo a concentration-, temperature- and pH-dependent transition leading to phase separation. This phase transition is followed by a sudden increase of light scattering. The phase diagram pH vs. concentration is obtained by observation of the cloud point for concentrations ranging from 0.01 to 10 mM. The intramicellar environment is probed at pH ranging from 5.5 to 8.0 using a stearic acid spin label. The intramicellar compactness increases smoothly with increasing pH suggesting the weakening of polar heads repulsion due to charge decrease. The reported results indicate that pH effects are relevant and should be properly taken into account in the performance and interpretation of experiments with CPZ.     

Close physical linkage of the murine Ren-1 and Ren-2 loci

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.     

Studies on Halimeda

Large areas of the inter-reefal seabed in the Great Barrier Reef are carpeted with vegetation composed almost entirely of the green calcareous alga Halineda. These meadows occur principally in the northern sections between 11°30 and 15°35S at depths of 20 to 40 m, but there are also some in the central and southern sections, where they have been found at depths down to 96 m. The vegetation is dominated by the same sprawling Halimeda species that are common on coral reefs in this region. However, on reefs these species grow on solid substrata, not soft sediments like the Halimeda-rich gravels that underlie the meadows. A total of 12 Halimeda species, together with two Udotea and one Penicillus species, are characteristic components of the shallow meadows. Below 50 m depth, species composition is restricted to only two major components. One, H. copiosa, is also important shallower, but the other is an unusually large and heavily calcified form of H. fragilis, a species that is normally a minor, fragile component of the shallow meadows. The maximum biomass found in these meadows was 4637 gm² of calcareous algae, although the thean for vegetated areas was 525 gm². These meadows are confined to the nutrient-depleted waters of the outer continental shelf just inside the outer barrier reefs, and are usually associated with distinct shoaling of the seabed caused by accumulation of thick deposits of calcareous Halimeda segments. The meadows are probably supported by very localized upwelling of nutrients from the adjacent Coral Sea onto the shelf, where they enrich the otherwise nutrient-depleted waters.Contribution No.367 from the Australian Institute of Marine Science     

Similarities and Differences Between High-Affinity Binding Sites for Cocaine and Imipramine in Mouse Cerebral Cortex

Previously we found close similarities between high-affinity binding sites for [3H]cocaine and those for [3H]imipramine in the mouse cerebral cortex in regard to their association with neuronal uptake of serotonin. In the present study we investigated whether the two ligands bind to the same site. The two ligands had the following high-affinity binding properties in common: localization in both synaptosomal and microsomal fractions; vulnerability to treatment with N-ethylmaleimide, trypsin, and phospholipase A2; and resistance to exposure to dithiothreitol. In contrast, cocaine binding in the cerebral cortex was more sensitive to heat inactivation than imipramine binding. In addition, the mechanism by which cocaine inhibited [3H]imipramine binding differed from that by which imipramine inhibited [3H]cocaine binding. These data suggest that the high-affinity binding sites for [3H]cocaine and [3H]imipramine in the cerebral cortex are distinct entities.