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.     

Models for Trypanosoma evansi (surra), its control and economic impact on small-hold livestock owners in the Philippines

Simple demographic and infectious disease models of buffaloes and other domestic hosts for animal trypanosomosis (surra) caused by Trypanosoma evansi were developed. The animal models contained deterministic and stochastic elements and were linked to simulate the benefit of control regimes for surra in village domestic animal populations in Mindanao, Philippines. The impact of the disease on host fertility and mortality were key factors in determining the economic losses and net-benefit from the control regimes. If using a high (99%) efficacy drug in surra-moderate to high risk areas, then treating all animals twice each year yielded low prevalence in 2 years; targeted treatment of clinically sick animals, constantly monitored (monthly), required 75% fewer treatments but took longer to reach a low prevalence than treating all animals twice each year. At high drug efficacy both of these treatment strategies increased the benefit over untreated animals by 81%. If drug efficacy declined then the benefit obtained from twice yearly treatment of all animals declined rapidly compared with regular monitoring and targeting treatment to clinically sick animals. The current control regimen applied in the Philippines of annual sero-testing for surra and only treating sero-positive animals provided the lowest net-benefit of all the control options simulated and would not be regarded as effective control. The total net-benefit from effective surra control for a typical village in a moderate/high risk area was 7.9 million pesos per annum (US $158,000). The value added to buffaloes, cattle, horses, goats/sheep and pigs as a result of this control was US $88, $84, $151, $7, $114 per animal/year, respectively.     

Microsatellite markers for the common bean Phaseolus vulgaris

Efforts to develop molecular tools for genetic analysis and breeding of common bean in the tropics are still limited. The number of microsatellite markers available for the crop is small compared to other crops of similar social and economic importance. As part of a project to broaden the use of molecular tools in bean breeding, a genomic library enriched for AG/TC repeat sequences was constructed for Phaseolus vulgaris. Twenty microsatellite markers were initially developed and 10 were characterized using a panel of 85 representative accessions of the bean gene bank. The number of alleles per marker ranged from three to 10. The polymorphism information content (PIC) varied from 0.23 to 0.80. The results indicate that the new markers can be readily used in genetically analysis of common bean.     

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.     

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.