DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchinStrongylocentrotus purpuratus

Summary The general organization and primary amino acid sequences of theS. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26–27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage.The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.     

Tree and Liana Enumeration and Diversity on a One-Hectare Plot in Papua New Guinea1

Enumeration of a one hectare plot at 900 m a.s.l in Papua New Guinea revealed 693 individuals of 228 tree and liana species ≥ 10 cm DBH. A 0.1 hectare subplot contained 302 individuals of 106 species 2.5 ≥ DBH < 10 cm. Lauraceae, Moraceae, and Myristicaceae were the most important families in both size classes. This site is very diverse compared with other tropical forests, and like other species-rich sites worldwide, it has high aseasonal rainfall and high rates of natural disturbance.     

Inhibition of thecal androstenedione production by exogenous progesterone in the cyclic hamster

Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6.0 +/- 0.7 ng/ml, N = 8; sesame oil controls, less than 0.5 ng/ml, N = 6) and a decline in serum levels of LH (5.3 +/- 0.4 ng/ml; controls 12 +/- 2 ng/ml) and oestradiol (10 +/- 2 pg/ml; controls 69 +/- 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 micrograms ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.     

Telomere length analysis in residents of a community exposed to arsenic

Differentiated cells telomere length is an indicator of senescence or lifespan; however, in peripheral blood leukocytes the relative shortening of the telomere has been considered as a biological marker of aging, and lengthening telomere as an associated risk to cancer. Individual’s age, type of tissue, lifestyle, and environmental factors make telomere length variable. The presence of environmental carcinogens such as arsenic (As) influence as causal agents of these alterations, the main modes of action for As described are oxidative stress, reduction in DNA repair capacity, overexpression of genes, alteration of telomerase activity, and damage to telomeres. The telomeres of leukocytes resulting a finite capacity of replication due to the low or no activity of the telomerase enzyme, therefore, elongation telomere in this kind of cells is a potential biological marker associated with the development of chronic diseases and carcinogenesis.     

A Clp/Hsp100 chaperone functions in Myxococcus xanthus sporulation and self-organization

The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.     

Complete genome sequence of a sucrose-nonfermenting epidemic strain of Vibrio cholerae O1 from Brazil

We report the genome sequence of Vibrio cholerae strain IEC224, which fails to ferment sucrose. It was isolated from a cholera outbreak in the Amazon. The defective sucrose phenotype was determined to be due to a frameshift mutation, and a molecular marker of the Latin American main epidemic lineage was identified.     

Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase

A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.     

Evidence for folate-salvage reactions in plants

Folates in vivo undergo oxidative cleavage, giving pterin and p -aminobenzoylglutamate ( p ABAGlu) moieties. These breakdown products are excreted in animals, but their fate is unclear in microorganisms and unknown in plants. As indirect evidence from this and previous studies strongly suggests that plants can have high folate-breakdown rates (approximately 10% per day), salvage of the cleavage products seems likely. Four sets of observations support this possibility. First, cleavage products do not normally accumulate: pools of p ABAGlu (including its polyglutamyl forms) are equivalent to, at most, 4–14% of typical total folate pools in Arabidopsis thaliana , Lycopersicon esculentum and Pisum sativum tissues. Pools of the pterin oxidation end-product pterin-6-carboxylate are, likewise, fairly small (3–37%) relative to total folate pools. Second, little p ABAGlu built up in A. thaliana plantlets when net folate breakdown was induced by blocking folate synthesis with sulfanilamide. Third, A. thaliana and L. esculentum tissues readily converted supplied breakdown products to folate synthesis precursors: p ABAGlu was hydrolysed to p -aminobenzoate and glutamate, and dihydropterin-6-aldehyde was reduced to 6-hydroxymethyldihydropterin. Fourth, both these reactions were detected in vitro ; the reduction used NADPH as cofactor. An alternative salvage route for p ABAGlu, direct reincorporation into dihydrofolate via the action of dihydropteroate synthase, appears implausible from the properties of this enzyme. We conclude that plants are excellent organisms in which to explore the biochemistry of folate salvage.     

2,3-diphenyl-1,4-naphthoquinone: a potential chemotherapeutic agent against Trypanosoma cruzi

Chagas disease, caused by Trypanosoma cruzi, is a widespread infection in Latin America. Currently, only 2 partially effective and highly toxic drugs, i.e., benznidazole and nifurtimox, are available for the treatment of this disease, and several efforts are underway in the search for better chemotherapeutic agents. Here, we have determined the trypanocidal activity of 2,3-diphenyl-1 ,4-naphthoquinone (DPNQ), a novel quinone derivative. In vitro, DPNQ was highly cytotoxic at a low, micromolar concentration (LD50 = 2.5 microM) against epimastigote, cell-derived trypomastigote, and intracellular amastigote forms of T. cruzi, but not against mammalian cells (LD50 = 130 microM). In vivo studies on the murine model of Chagas disease revealed that DPNQ-treated animals (3 doses of 10 mg/kg/day) showed a significant delay in parasitemia peak and higher (up to 60%) survival rate 70 days post-infection, when compared with the control group (infected, untreated). We also observed a 2-fold decrease in parasitemia between the control group (infected, untreated) and the treated group (infected, treated). No apparent drug toxicity effects were noticed in the control group (uninfected, treated). In addition, we determined that DPNQ is the first competitive inhibitor of T. cruzi lipoamide dehydrogenase (TcLipDH) thus far described. Our results indicate that DPNQ is a promising chemotherapeutic agent against T. cruzi.