Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

To the intranucleolar translocation of AgNORs in leukemic early granulocytic and plasmacytic precursors

Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.     

Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs

The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%–91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.     

Human immunodeficiency virus (HIV) vaccine trials: a novel assay for differential diagnosis of HIV infections in the face of vaccine-generated antibodies

All current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and elicit antibodies that react positively in licensed HIV diagnostic tests, which contain similar viral products. Thus, vaccine trial participants could be falsely diagnosed as infected with HIV. Additionally, uninfected, seropositive vaccinees may encounter long-term social and economic harms. Moreover, this also interferes with early detection of true HIV infections during preventive HIV vaccine trials. An HIV-seropositive test result among uninfected vaccine trial participants is a major public health concern for volunteers who want to participate in future HIV vaccine trials. Based on the increased number of HIV vaccines being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Using a whole-HIV-genome phage display library, we identified conserved sequences in Env-gp41 and Gag-p6 which are recognized soon after infection, do not contain protective epitopes, and are not part of most current HIV vaccines. We established a new HIV serodetection assay based on these peptides. To date, this assay, termed HIV-SELECTEST, demonstrates >99% specificity and sensitivity. Importantly, in testing of plasma samples from multiple HIV vaccine trials, uninfected trial participants scored negative, while all intercurrent infections were detected within 1 to 3 months of HIV infection. The new HIV-SELECTEST is a simple but robust diagnostic tool for easy implementation in HIV vaccine trials and blood banks worldwide.     

Anaphylactic Reactions to Oligosaccharides in Red Meat: a Syndrome in Evolution

Background

Human mast cells are capable of a wide variety of inflammatory responses and play a vital role in the pathogenesis of inflammatory diseases such as allergy, asthma, and atherosclerosis. We have reported that cigarette smoke extract (CSE) significantly increased IL-6 and IL-8 production in IL-1??-activated human mast cell line (HMC-1). Baicalein (BAI) has anti-inflammatory properties and inhibits IL-1??- and TNF-??-induced inflammatory cytokine production from HMC-1. The goal of the present study was to examine the effect of BAI on IL-6 and IL-8 production from CSE-treated and IL-1??-activated HMC-1.

Methods

Main-stream (Ms) and Side-stream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI-1640 medium. Two ml of HMC-1 at 1 × 10⁶ cells/mL were cultured with CSE in the presence or absence of IL-1?? (10 ng/mL) for 24 hrs. A group of HMC-1 cells stimulated with both IL-1?? (10 ng/ml) and CSE was also treated with BAI. The expression of IL-6 and IL-8 was assessed by ELISA and RT-PCR. NF-??B activation was measured by electrophoretic mobility shift assay (EMSA) and I??B?? degradation by Western blot.

Results

Both Ms and Ss CSE significantly increased IL-6 and IL-8 production (p < 0.001) in IL-1??-activated HMC-1. CSE increased NF-??B activation and decreased cytoplasmic I??B?? proteins in IL-1??-activated HMC-1. BAI (1.8 to 30 ??M) significantly inhibited production of IL-6 and IL-8 in a dose-dependent manner in IL-1??-activated HMC-1 with the optimal inhibition concentration at 30 ??M, which also significantly inhibited the enhancing effect of CSE on IL-6 and IL-8 production in IL-1??-activated HMC-1. BAI inhibited NF-??B activation and increased cytoplasmic I??B?? proteins in CSE-treated and IL-1??-activated HMC-1.

Conclusions

Our results showed that CSE significantly increased inflammatory cytokines IL-6 and IL-8 production in IL-1??-activated HMC-1. It may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. BAI inhibited the production of inflammatory cytokines through inhibition of NF-??B activation and I??B?? phosphorylation and degradation. This inhibitory effect of BAI on the expression of inflammatory cytokines induced by CSE suggests its usefulness in the development of novel anti-inflammatory therapies.     

Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.     

Comparison of the influence of small GTPases Arl1 and Ypt6 on yeast cells' tolerance to various stress factors

The GTPases Arl1 and Ypt6 are involved in the intracellular transport of vesicles and their fusion with the trans-Golgi network. This work is focused on comparing the roles of these GTPases in the tolerance of Saccharomyces cerevisiae cells to an increased concentration of alkali metal cations and other stress factors. We studied the phenotypes of arl1 or ypt6 deletions in combination with the deletions of genes encoding alkali-metal-cation transporters (ena1-4, nha1, nhx1, and kha1). Salt sensitivity of the arl1 and ypt6 mutants was shown to be independent of the tested cation transporters and electrochemical membrane potential. Phenotype manifestations of ypt6 deletion were usually more prominent than those of arl1 (cells were more sensitive to KCl, NaCl, LiCl, hygromycin B, increased temperature, and increased pH). At suboptimal temperature, the growth inhibition of arl1 and ypt6 mutants was approximately the same, and low pH was the only condition where arl1 mutants grew even worse than ypt6 mutants. Overexpression of the ARL1 gene suppressed the phenotypes of ypt6 deletion; however, this did not work vice versa (additional copies of YPT6 could not replace ARL1). Our results suggest partially overlapping functions of the GTPases in resistance to various stress factors, with Ypt6 being more efficient under physiological conditions and Arl1 more versatile when overexpressed.     

Platycleis vittata (Orthoptera: Tettigoniidae) in the northwestern part of its range is close to extinction: is this the result of landscape changes?

The northwestern distributions of several steppe species of Orthoptera extend to the southeastern part of the Czech Republic (Pannonia) and occupy more or less isolated fragments of optimal habitats. Their distributional limits are not conditioned by macroclimate in most cases but reflect landscape development (physical structure, plant community type and microclimate) and the insects’ dispersal abilities. These species prefer permanent grassland, and assessment of land use records indicates that the area occupied by permanent grassland has been greatly reduced by agriculture over the last two centuries. The area occupied by permanent grasslands in Pannonia was highest in the nineteenth century, declined until the second half of the twentieth century, and slightly increased at the beginning of the twenty first century. These changes in the area of permanent grassland generally reflect economic and political processes; in particular, consolidation of land plots and expropriation of private property brought an end to grazing. The distribution of a model Orthoptera specialist, Platycleis vittata, on steppe habitats was surveyed at 48 potentially suitable locations from 2004 to 2010. The survey determined that the area with suitable habitat is currently very limited at all 48 locations. Occurrence was confirmed in just two locations that are 3 km apart. The research suggests that Platycleis vittata in the Czech Republic is on the brink of extinction because of extreme fragmentation of suitable biotopes and degradation of optimal habitats.