Role of calcium in the regulation of theca cell androstenedione production in the domestic hen

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.     

Nucleotide-activated chloride channels in lysosomal membranes.

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Movement of thallous ion across the ascites cell membrane

Summary The movement of thallous ion (Tl⁺ across the ascites cell membrane has been characterized. Analogous to previous findings for⁸⁶Rb⁺ (used as a tracer for K⁺),²⁰⁴Tl⁺-influx could be resolved into three components: a ouabain-inhibitable pump flux, a passive flux, and a furosemide- or NO ₃ ^– -sensitive exchange flux. Although Tl⁺ moved approximately nine times faster across the membrane than K⁺, the pump/leak ratio was equal for the two ions. This suggests that the pump- and leak-pathways share a common rate-limiting step. The exchange mechanism was shown to provide close coupling between the Tl⁺- and K⁺-gradients.     

Biogeographic variation in coral species diversity on coral reefs in three regions of Indonesia

Abstract . This paper assesses variation in coral species diversity within the Indonesian archipelago, and the influence of regional species pools, geomorphology and anthropogenic pollution on coral species diversity and occurrence. We obtained transects from 33 sites on 14 reefs in three regions of Indonesia: Ambon (Moluccas), South Sulawesi and the Java Sea. We determined the within‐site species richness by using species‐sampling curves. Cluster analysis and multi‐dimensional scaling showed that land‐based pollution was the primary determinant of coral species diversity and species occurrence on reefs. Relatively unaffected reference sites in eastern Indonesia were approximately 20% more diverse than Java Sea reference sites. Rare species formed a higher proportion of the coral fauna on eastern Indonesian sites, and eastern Indonesian apparent endemic species contributed approximately 25% of the total species pool sampled. Between‐site variation in species occurrence was lower on Java Sea reefs than on eastern Indonesian reefs. A larger species pool in eastern Indonesia than in the Java Sea probably accounted for most of the difference in within‐site species diversity between eastern Indonesian and Java Sea reference sites. High fishing intensity in the Java Sea, including destructive fishing practices, may have also contributed to reduced within‐site species diversity on Java Sea reference reefs. Despite the fact that the Java Sea was exposed during Pleistocene lowstands, and was recolonized by marine organisms only within the last 10 000 years, coral species diversity and assemblage composition on the Java Sea reefs was largely similar to open ocean reefs in eastern Indonesia.     

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.     

Immunomodulation after allogeneic bone marrow transplantation by CD4+CD25+ regulatory T cells

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation (BMT) caused by donor T cells. Immunosuppression mediated by CD4(+)CD25(+) regulatory T cells has been shown to ameliorate such pathogenic immune responses in animal models. Here, we summarize recent findings from experimental and clinical studies and propose a model for peripheral tolerance induction after BMT.