Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.     

Tribal and intergeneric relationships of Mesechiteae (Apocynoideae, Apocynaceae): evidence from three noncoding plastid DNA regions and morphology

The Neotropical tribe Mesechiteae (Apocynaceae) is currently considered to include nine genera: Allomarkgrafia, Galactophora, Macrosiphonia, Mandevilla, Mesechites, Quiotania, Secondatia, Telosiphonia, and Tintinnabularia. Tribal and intergeneric relationships, however, are in dispute. To test the monophyly of the tribe and evaluate intratribal relationships, a maximum parsimony analysis was conducted based on DNA sequences from the plastid rpl16 intron, rps16 intron, and trnS-G intergenic spacer region as well as morphological data for 23 taxa of Mesechiteae and 11 taxa from other tribes of Apocynoideae. Mesechiteae, as currently circumscribed, was found to be polyphyletic. Only removal of Secondatia and Galactophora and inclusion of Forsteronia rendered the tribe monophyletic. Thus defined, Mesechiteae forms a strongly supported clade including seven genera in three subclades: the Mesechites subclade (comprising Tintinnabularia, Allomarkgrafia, and Mesechites), the Forsteronia subclade (containing only Forsteronia) and the Mandevilla subclade (comprising Macrosiphonia, Mandevilla, and Telosiphonia). Allomarkgrafia is nested in Mesechites. Macrosiphonia and Telosiphonia form two distinct monophyletic clades. Both, however, are nested in Mandevilla. Results suggest upholding the following genera in Mesechiteae: Allomarkgrafia, Forsteronia, Mandevilla, Mesechites, and Tintinnabularia. The status of Quiotania could not be evaluated.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.     

Comparison of PCR fingerprinting techniques for the discrimination of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subsp. <Emphasis Type="Italic">enterica</Emphasis> serovar Weltevreden isolated from indigenous vegetables in Malaysia

Salmonella enterica subsp. enterica (S.) serovar Weltevreden has emerged as a public health problem in many countries. Genomic DNA of S. Weltevreden from indigenous vegetables namely ‘selom’ (Oenanthe stolonifera), ‘pegaga’ (Centella asiatica), ‘kesum’ (Polygonum minus) and ‘kangkong’ (Ipomoea aquatica) were characterized by duplex-polymerase chain reaction (duplex-PCR), multiplex-polymerase chain reaction (multiplex-PCR), random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results demonstrated that a total of four clusters and three single isolates were generated from ERIC-PCR with primers ERIC-1 and ERIC-2 whereas RAPD with arbitrary primers OPAR2, OPAR17 and OPAR19 discriminated the S. Weltevreden into nine clusters and eight single isolates at a common 65% similarity level with discriminatory index (D) of 0.7443 and 0.9394 respectively. Composite analysis of banding profiles generated from RAPD-PCR and ERIC-PCR showed eight clusters and six single isolates at 65% similarity level with the highest D value that is 0.9508. On the other hand, PCR-RFLP and duplex PCR data exhibited a consistent profile for S. Weltevreden. Multiplex-PCR targeting three different antibiotic resistance genes and a common Salmonella specific gene segment produced two distinguishing profiles among the S. Weltevreden examined. These results demonstrated that the combined analysis of RAPD-PCR and ERIC-PCR is a better tool for characterizing S. Weltevreden than individual methods.     

High cell density and latent membrane protein 1 expression induce cleavage of the mixed lineage leukemia gene at 11q23 in nasopharyngeal carcinoma cell line

Background  

Nasopharyngeal carcinoma (NPC) is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV) infection is well associated with NPC and has been implicated in its pathogenesis. Moreover, various chromosome rearrangements were reported in NPC. However, the underlying mechanism of chromosome rearrangement remains unclear. Furthermore, the relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. We hypothesize that during virus- or stress-induced apoptosis, chromosomes are initially cleaved at the base of the chromatin loop domain structure. Upon DNA repair, cell may survive with rearranged chromosomes.     

Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.     

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABA_B receptor agonist; 1–10 μg/5 μl) or bicuculline (a GABA_A receptor antagonist; 1–10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABA_A receptor agonist; 1–5 μg/5 μl) or phaclofen (a GABA_B receptor antagonist; 5–10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen–induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABA_B receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABA_A receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.     

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.