Generation of free radicals induced by nifurtimox in mammalian tissues

Nifurtimox is reduced by rat liver microsomes to a nitro anion-free radical as indicated by ESR spectroscopy. This subcellular fraction gives a steady state radical concentration which is proportional to the square root of the protein concentration, suggesting that the nifurtimox anion radical is a necessary intermediate in the reduction and that the radical decays through a nonenzymatic second order process. The steady state concentration of the anion radical in the microsomal system is not decreased by superoxide dismutase or catalase, thus indicating that neither the superoxide anion nor hydrogen peroxide is an intermediary in the generation of the anion radical. The steady state concentration of the anion radical in the microsomal system is also not altered in the presence of metyrapone or CO and is decreased in the presence of NADP+ and p-chloromercuribenzoate. This observation suggests that the formation of nifurtimox anion radical is mediated through NADPH-cytochrome P-450 (c) reductase and not by the cytochrome P-450 system. In accordance with this interpretation, a model system consisting of NADPH and FMN-reduced nifurtimox to the nitro anion-free radical. Nifurtimox anion radical generation is significantly stimulated by rat brain and testes homogenates. The enhanced free radical formation may be the basic cause of nifurtimox toxicity in mammals.     

Usa1 Protein Facilitates Substrate Ubiquitylation through Two Separate Domains

Background

Defects in protein folding are recognized as the root of many neurodegenerative disorders. In the endoplasmic reticulum (ER), secretory proteins are subjected to a stringent quality control process to eliminate misfolded proteins by the ER-associated degradation (ERAD) pathway. A novel ERAD component Usa1 was recently identified. However, the specific role of Usa1 in ERAD remains obscure.

Methodology/Principal Findings

Here, we demonstrate that Usa1 is important for substrate ubiquitylation. Furthermore, we defined key cis-elements of Usa1 essential for its degradation function. Interestingly, a putative proteasome-binding motif is dispensable for the functioning of Usa1 in ERAD. We identify two separate cytosolic domains critical for Usa1 activity in ERAD, one of which is involved in binding to the Ub-protein ligase Hrd1/Hrd3. Usa1 may have another novel role in substrate ubiquitylation that is separate from the Hrd1 association.

Conclusions/Significance

We conclude that Usa1 has two important roles in ERAD substrate ubiquitylation.     

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins--eIF5A(K56A) and eIF5A(Q22H,L93F)--and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.     

Sugar cane buds as an efficient explant for plantlet regeneration

An efficient and reproducible protocol for regeneration of plantlets at a high frequency was developed by using sugar cane buds. Disinfected buds were firstly submerged in ethanol sodium hypochlorite solution with 0.1 % polyvinylpyrrolidone, 1.5 % ascorbic acid and 1.75 % citric acid as antioxidants and subsequently treated with solution of agrimicin:captan (1:1). The upper stalk segment was better to obtain bud in vitro culture compared to lower segments. The medium for induction of multiple shoots consisted of Murashige and Skoog basal medium (MS) supplemented with 2 mg dm⁻³ thidiazuron and 1 mg dm⁻³ naphthalene acetic acid. An average of 24 shoots per bud was obtained for cv. Mex 68-P23 within four weeks and 29 shoots for cv. MY 55-14 within six weeks. Indole-3-butyric acid induced more roots in both cultivars compared to the untreated plantlets. Plantlets transferred to soil showed normal growth with up to four axilliary buds in each node. It was concluded that the germplasm obtained through the above mentioned technique generated stalks with more buds in each node which would give farmers more vegetative material for plantations in field with 100 % germination.This research was funded by Fundacion Produce Chiapas A.C. (Mexico).     

Molecular phylogeny of ateline new world monkeys (Platyrrhini, atelinae) based on gamma-globin gene sequences: evidence that brachyteles is the sister group of lagothrix

Nucleotide sequences, each spanning approximately 7 kb of the contiguous gamma1 and gamma2 globin genomic loci, were determined for seven species representing all extant genera (Ateles, Lagothrix, Brachyteles, and Alouatta) of the New World monkey subfamily Atelinae. After aligning these seven ateline sequences with outgroup sequences from several other primate (non-ateline) genera, they were analyzed by maximum parsimony, maximum likelihood, and neighbor-joining algorithms. All three analyzes estimated the same phylogenetic relationships: [Alouatta [Ateles (Brachyteles, Lagothrix)]]. Brachyteles and Lagothrix are sister-groups supported by 100% of bootstrap replications in the parsimony analyses. Ateles joins this clade, followed by the basal genus Alouatta; these joinings were strongly supported, again with 100% bootstrap values. This cladistic pattern for the four ateline genera is congruent with that obtained in previous studies utilizing epsilon-globin, IRBP, and G6PD nuclear genomic sequences as well as mitochondrial COII sequences. Because the number of aligned nucleotide positions is much larger in the present datasetoff than in any of these other datasets, much stronger support was obtained for the cladistic classification that divides subfamily Atelinae into tribes Alouattini (Alouatta) and Atelini, while the latter divides into subtribes Atelina (Ateles) and Brachytelina (Brachyteles and Lagothrix).     

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.     

House dust mites in Brazil--an annotated bibliography.

House dust mites have been reported to be the most important allergen in human dwellings. Several articles had already shown the presence of different mite species at homes in Brazil, being Pyroglyphidae, Glycyphagidae and Cheyletidae the most important families found. This paper is an annotated bibliography that will lead to a better knowledge of house dust mite fauna in Brazil.     

Proposed chromosomal phylogeny for the South American primates of the Callitrichidae family (Platyrrhini)

Cytogenetic and cytotaxonomic studies (G, C, sequential G/C, and NOR banding) were performed on 110 specimens representing the four genera of South American primates of the family Callitrichidae: Cebuella (C. pygmaea), Callithrix, groups argentata (C. argentata, C. emiliae, C. chrysoleuca, C. humeralifera, C. mauesi), and jacchus (C. aurita, C. geoffroyi, C. jacchus, C. kuhli, C. penicillata), Leontopithecus (L. chrysomelas, L. rosalia), and Saguinus (S. midas midas, S. m. niger). Mitotic chromosomes are characterized, and the rearrangements distinguishing the karyotypes of the taxa are inferred from arm homologies. The results were then converted into numerical data and submitted to cladistic analysis. The following conclusions were achieved: 1) Five karyotypic classes were observed, which correspond to the five taxa studied. Differences between them are as follows: a) Cebuella (2n = 44, 10 acrocentrics, A + 32 bi‐armed autosomes, bi) and the argentata group (2n = 44, 10A + 32bi) are different from each other due to a reciprocal translocation; b) both can be distinguished from the jacchus group (2n = 46, 14A + 30bi) by a centric fusion/fission rearrangement and a paracentric inversion; c) Leontopithecus (2n = 46, 14A + 30bi) and Saguinus (2n = 46, 14A + 30bi) differ from the jacchus group by a reciprocal translocation and three paracentric inversions; and d) Saguinus is different from the others by one paracentric inversion and pericentric inversions in at least four pairs of acrocentric autosomes. 2) The cladistic analysis separates Cebus (used as an outgroup) from the Callitrichidae groups, which forms a clade. Among the Callitrichidae, marmosets (Cebuella and Callithrix) form a sub‐clade, Cebuella and the argentata group being more closely related to each other than both are to the jacchus group. Tamarins (Leontopithecus and Saguinus) are also quite close, so that if one was not derived from the other, they with the marmosets share a common ancestor. Among the tamarins, Leontopithecus is karyotypically closest to the marmosets, specifically to the jacchus group. 3) Based on the chromosome information and considering the possible direction of the evolutionary changes (primitivity or phyletic dwarfism hypothesis, previously advanced by other authors), it was possible to propose the ancestral karyotypes and to develop two alternatives for the origin, differentiation and dispersion of the callitrichid. Both proposals are plausible, but when the geographical distribution is considered, the phyletic dwarfism hypothesis seems to be the most probable. Am. J. Primatol. 49:133–152, 1999. © 1999 Wiley‐Liss, Inc.