Development and characterization of novel microsatellite markers from the olive ridley sea turtle (Lepidochelys olivacea)

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery‐related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat‐enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross‐species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species.     

Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1)

Background

The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Methodology/Principal Findings

Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.

Conclusions/Significance

The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.     

Phylogenetic relationships among Oryza species revealed by AFLP markers

 The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta ^* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms. Received: 1 July 1998 / Accepted: 2 November 1998     

Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

Essential oils extracted from 10 medicinal plants were evaluated for larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species; Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. The essential oils of Juniperus macropoda and Pimpinella anisum were highly effective as both larvicidal and ovicidal. The essential oil of P. anisum showed toxicity against 4th instar larvae of A. stephensi and A. aegypti with equivalent LD95 values of 115.7 microg/ml, whereas it was 149.7 microg/ml against C. quinquefasciatus larvae. Essential oils of Zingiber officinale and Rosmarinus officinalis were found to be ovicidal and repellent, respectively towards the three mosquito species. The essential oil of Cinnamomum zeylanicum resulted into highest repellent (RD95) values of 49.6, 53.9 and 44.2 mg/mat against A. stephensi, A. aegypti and C. quinquefasciatus, respectively apart from oviposition-deterrent potential.     

Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17

Interleukin (IL)-17 is a pro-inflammatory cytokine that is produced by activated T cells. Despite increasing evidence that high levels of IL-17 are associated with several chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and multiple sclerosis, the regulation of its expression is not well characterized. We observe that IL-17 production is increased in response to the recently described cytokine IL-23. We present evidence that murine IL-23, which is produced by activated dendritic cells, acts on memory T cells, resulting in elevated IL-17 secretion. IL-23 also induced expression of the related cytokine IL-17F. IL-23 is a heterodimeric cytokine and shares a subunit, p40, with IL-12. In contrast to IL-23, IL-12 had only marginal effects on IL-17 production. These data suggest that during a secondary immune response, IL-23 can promote an activation state with features distinct from the well characterized Th1 and Th2 profiles.     

Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases

Although DNA polymerase eta (Pol eta) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Pol eta of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.     

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.     

Triggering of apoptosis is not sufficient to induce human immunodeficiency virus gene expression

We examined whether there is any causative link between apoptosis and HIV gene expression elicited in response to ultraviolet light (UV) and ionizing radiation (IR). We found that both UV and IR activate HIV gene expression in human T lymphoblastoid 1G5 (HIVluc) cells, but with different kinetics and magnitudes. Treatment with either type of radiation resulted in increased apoptosis, which correlated closely with HIV gene expression. The involvement of caspases in the IR response was demonstrated by using zVAD-FMK and zDEVD-FMK caspase inhibitors; both apoptosis and HIV gene expression were inhibited to similar extent. Surprisingly, treatment of 1G5 cells with FAS antibody triggered apoptosis but did not increase HIV gene expression. A correlation between increased apoptosis and gene expression was also demonstrated in human carcinoma HIVcat/A549 cells with UV whereas IR triggered apoptosis but did not activate HIV gene expression. Most significantly, UV activation of HIV gene expression, and NF-kappa-B and p38 MAP kinase, both important for efficient HIV gene expression, were not affected by treatment with the zVAD-FMK and zDEVD-FMK inhibitors. Treatment of HIVcat/A549 cells with staurosporine or scrape-loading of cells with cytochrome c resulted in apoptosis but no increase in HIV gene expression. Altogether, a direct correlation exists between apoptosis and HIV gene expression in T-cells in response to both UV and IR but this is not the case in carcinoma cells. Triggering of apoptosis per se in either cell type does not necessarily result in increased HIV gene expression. Most importantly, the apoptotic and HIV gene expression responses elicited by UV are different to some extent and can be separated.