Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.     

Positive anemotaxis by Varroa mites: responses to bee odour plumes and single clean‐air puffs

Abstract.The stimuli and mechanisms mediating host location and host choice by the bee mite, Varroa jacobsoni (Oudemans), are currently unknown. It is shown that Varroa can use single clean‐air puffs and bee‐odour plumes in a wind tunnel as directional cues. Varroa turned nearly straight upwind in response to single 0.1‐s puffs of clean air directed at 90° to the their anterior‐posterior axis. They turned significantly further to their left side (104°) than to their right (76°), but showed no difference in latency to initiation of the turns (means of 63.3 ms vs. 62.6 ms, respectively). They also followed bee‐odour plumes in a wind tunnel. When released in odour and control plumes mid‐way between the plume's origin and the downwind end of the tunnel, mites responding to bee‐odour walked upwind in, or along the edge of, the odour plume with 38% making contact with the odour delivery tube; mites in clean air did not walk upwind along the air stream, and none made contact with the air delivery tube. Walking speeds were not different between the bee‐odour and control groups (0.28 vs. 0.29 cm s^–1); there were also no differences in the turning rates (96.85 vs. 97.16 deg s^–1 and 388.08 vs. 379.18 deg cm^–1, respectively). Under all conditions, mites walked in a zigzag fashion.     

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

A nuclear magnetic resonance study of the glucose metabolism of Hymenolepis diminuta exposed to histamine and serotonin in vitro.

The direct effects of the inflammatory mediators, histamine (HI) and serotonin (SE), on the glucose metabolism of Hymenolepis diminuta in vitro were studied by analyzing the excretory products from culture media, containing D-1-13C-glucose and various concentrations of HI and/or SE, by 1H-nuclear magnetic resonance (n.m.r.) spectroscopy. The results revealed that HI markedly accelerated the glycolysis process by increasing the amount of lactate production. The increased glycolytic activity was reflected in a concentration-dependent increase in glucose uptake. Excretion of acetate was also stimulated by HI. A low concentration of SE significantly increased succinate, acetate and lactate excretions, whereas a high concentration had little effect on lactate production and significantly decreased succinate and acetate excretions. A combination of HI and SE treatment at a low concentration had no significant effect, but at a high concentration showed an additive effect, with an increase in lactate production, a decrease in succinate production and an increase in glucose uptake. Thus this work confirms that HI and SE directly influence, albeit differently, energy metabolism of the tapeworm H. diminuta.     

Current aspects of the short-term prognosis of meningococcal infection morbidity for the sake of epidemiological surveillance

The short-term prognosis of the epidemic situation requires the establishment of the system of constant surveillance, which takes into account the data on morbidity rates for several years, both total and in various age groups, the data on the morbidity level and its changes, induced by meningococci of the epidemic group, in the IV quarter of the year, the data on the level of carriership among the indicator groups of the population in this period, as well as the data on changes in antibody titers in the sera of donor blood samples taken in summer and in December.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.