Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆ ¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅ ¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.     

Complementary Sex Determination in the Parasitic Wasp Diachasmimorpha longicaudata

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.     

Expression and function of the testis-predominant protein LYAR in mice

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.     

Prevalence of antibodies against human respiratory viruses potentially involving anthropozoonoses in wild bonobos

Primates - One of the current threats to the bonobo (Pan paniscus), a highly endangered ape species only found in the Democratic Republic of the Congo, are anthropozoonoses caused by human...     

Molecular Requirements for T Cell Recognition of N-Myristoylated Peptides Derived from the Simian Immunodeficiency Virus Nef Protein

We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C₁₄-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, functional studies indicated that the putative antigen-presenting molecule for 2N5.1 was likely to have two separate antigen-binding sites, one for interaction with a C₁₄-saturated acyl chain and the other for anchorage of the C-terminal serine residue. Mutants with alanine substitutions for the second glycine residue and the fourth isoleucine residue were not recognized by 2N5.1 but interfered with the presentation of C14nef5 to 2N5.1, indicating that these structural analogues retained the ability to interact with the antigen-presenting molecules. In contrast to the highly specific recognition of C14nef5 by 2N5.1, an additional cytotoxic T cell line, SN45, established independently from a C14nef5-stimulated T cell culture, showed superb reactivity to both C14nef5 and an N-myristoylated Nef 4-mer peptide, and therefore, the C-terminal serine residue was dispensable for the recognition of lipopeptides by the SN45 T cells. Furthermore, the mutants with alanine substitutions were indeed recognized by the SN45 T cells. Given that N-myristoylation of the Nef protein occurs in the conserved motifs and is critical for viral pathogenesis, these observations predict that the lipopeptide-specific T cell response is difficult for viruses to avoid by simply introducing amino acid mutations.     

Two new species and new records of biting midges of the genus Culicoides from northwestern Argentina (Diptera: Ceratopogonidae)

The following two new species of Culicoides from the Argentinean Yungas are described, illustrated and placed to subgenus or species group and compared with related congeners: Culicoides calchaqui Spinelli & Veggiani Aybar and Culicoides willinki Spinelli & Veggiani Aybar. Culicoides daedaloides Wirth & Blanton is recorded for the first time for Argentina and Culicoides pseudoheliconiae Felippe-Bauer is firstly mentioned from the northwestern region of the country.     

Roseoflavin Formation by Streptomyces davawensis

We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled ¹⁵N to identify its corresponding ¹⁵NO in vivo. The properties were examined with an ESR spectrometer. To synthesize ¹⁵N-PBN, the starting material, ammonium-¹⁵N chloride, was converted to 2-amino-¹⁵N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-¹⁵N, and finally reacted with benzaldehyde to give ¹⁵N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)₂, as a trapping agent to measure the NO levels of ¹⁵N-PBN or ¹⁴N-PBN in vitro, the peak intensity of ¹⁵NO[Fe(MGD)₂] was over 50% stronger than that of ¹⁴NO[Fe(MGD)₂], and that ¹⁵NO and ¹⁴NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of ¹⁵NO derived from ¹⁵N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish ¹⁵NO from PBN and ¹⁴NO generated from cells. These results suggested that ¹⁵N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.     

Orally available and efficacious α4β1/α4β7 integrin inhibitors

A series of potent α4β1/α4β7 integrin inhibitors is reported, including an inhibitor 12d with remarkable oral exposure and efficacy in rat models of rheumatoid arthritis and Crohn’s disease.