Essentiality of the glnA gene in Haloferax mediterranei: gene conversion and transcriptional analysis.

Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.

Anaerobic Metabolism in Haloferax Genus: Denitrification as Case of Study.

A number of species of Haloferax genus (halophilic archaea) are able to grow microaerobically or even anaerobically using different alternative electron acceptors such as fumarate, nitrate, chlorate, dimethyl sulphoxide, sulphide and/or trimethylamine. This metabolic capability is also shown by other species of the Halobacteriaceae and Haloferacaceae families (Archaea domain) and it has been mainly tested by physiological studies where cell growth is observed under anaerobic conditions in the presence of the mentioned compounds. This work summarises the main reported features on anaerobic metabolism in the Haloferax, one of the better described haloarchaeal genus with significant potential uses in biotechnology and bioremediation. Special attention has been paid to denitrification, also called nitrate respiration. This pathway has been studied so far from Haloferax mediterranei and Haloferax denitrificans mainly from biochemical point of view (purification and characterisation of the enzymes catalysing the two first reactions). However, gene expression and gene regulation is far from known at the time of writing this chapter.

Morphological, molecular and phylogenetic analyses of the spirurid nematode Stegophorus macronectes (Johnston & Mawson, 1942).

Stegophorus macronectes (Johnston & Mawson, 1942) is a gastrointestinal parasite found in Antarctic seabirds. The original description of the species, which was based only on females, is poor and fragmented with some unclear diagnostic characters. This study provides new morphometric and molecular data on this previously poorly described parasite. Nuclear rDNA sequences (18S, 5.8S, 28S and internal transcribed spacer (ITS) regions) were isolated from S. macronectes specimens collected from the chinstrap penguin Pygoscelis antarctica Forster on Deception Island, Antarctica. Using 18S rDNA sequences, phylogenetic analyses (maximum likelihood, maximum parsimony and Bayesian inference) of the order Spirurida were performed to determine the phylogenetic location of this species. Primer pairs of the ITS regions were designed for genus-level identification of specimens, regardless of their cycle, as an alternative to coprological methods. The utility of this molecular method for identification of morphologically altered specimens is also discussed.

Concurrent interspecies and clonal dissemination of OXA-48 carbapenemase.

Several isolates of four different carbapenemase-producing Enterobacteriaceae species were recovered from a patient hospitalized for 4 months in a teaching hospital in Madrid. These species comprised seven Klebsiella pneumoniae belonging to ST15, four Escherichia coli belonging to ST2531, two Serratia marcescens and one Citrobacter freundii. This patient was the index case of a small outbreak of four patients infected and/or colonized by carbapenemase-producing K. pneumoniae. Molecular results identified the bla(OXA-48) gene in all Enterobacteriaceae isolates from the index case and in all isolates from the other three patients, suggesting intra- and interpatient dissemination. Our results highlight the great ability of OXA-48 carbapenemase to spread among different enterobacterial species by both clonal and nonclonal dissemination.

Transcriptional profiles of Haloferax mediterranei based on nitrogen availability.

The haloarchaeon Haloferax mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology.

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei.

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

Analysis of acidic surface of Haloferax mediterranei glucose dehydrogenase by site-directed mutagenesis.

Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of glucose dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.

Changes in vascularization in substantia nigra pars compacta of monkeys rendered parkinsonian.

The degeneration of nigral dopaminergic neurons in Parkinson's disease is believed to be associated with a glial reaction and inflammatory changes. In turn, local factors may induce changes in vascularization and contribute to neuronal vulnerability. Among these factors, Vascular Endothelial Growth Factor (VEGF) is released in adults under pathological conditions and is thought to induce angiogenesis. In order to determine whether changes in brain vasculature are observed in the affected brain regions in parkinsonism, we quantitatively analysed the VEGF-expressing cells and blood vessels in the substantia nigra of monkeys rendered parkinsonian by MPTP injection and compared the results with those obtained in control monkeys. Using stereological methods, we observed an increase in the number of VEGF-expressing neurons and an increase of the number of blood vessels and their volume occupying the substantia nigra pars compacta of monkeys rendered parkinsonian by chronic MPTP intoxication. These changes in vascularization may therefore modify the neuronal availability of blood nutrients, blood cells or toxic substances and neuronal susceptibility to parkinsonism.

Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei.

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Ultradian and circadian body temperature and activity rhythms in chronic MPTP treated monkeys.

The body temperature and locomotor activity rhythms of seven 1-Methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgous monkeys were registered over a week on two separate occasions over an interval of 2 months. Motor disability was absent in two animals and present in five: it was mild in one, moderate in two and severe in two. Both temperature and motor activity were recorded every minute using a radio telemetry system. Analysis of circadian rhythms revealed less robustness of the 24-hour circadian components of body temperature and locomotor activity with increasing motor impairment, and a fragmentation of the body temperature rhythm into 8 hour-period components. Both total activity and daytime activity correlated inversely with the degree of motor impairment. On the contrary, the monkeys did not show differences in night time activity. The proportions of variance accounted for by the body temperature and locomotor activity of 24 h + 12 h + 8 h components were correlated. Also, the average levels at which the circadian rhythm varies between body temperature and locomotor activity were correlated. The results were almost identical in the two 1-week recording sessions. The present study confirms individual differences in the vulnerability to MPTP of the nigrostriatal system of monkeys, suggesting that if a cumulative dose does not provoke stable motor alterations, this cumulative dose will not produce circadian body temperature and locomotor activity rhythm alterations either. Similarly, if a dose is able to produce motor impairment, this dose will also be able to produce circadian rhythm alterations.

Status report on primary health care (1987-1993).

PIP: This article describes the legislative mandate for the development of primary health care (PHC) in the Philippines and provides a discussion of the attempts made to realize the goals of that mandate. Two major Department of Health thrusts are delineated: 1) from 1986 to 1991 Under-Secretary Alfredo Bengzon sought to deemphasize the implementation of primary health care in favor of an approach which sought to forge partnerships with nongovernmental organizations (NGOs) to realize a concept of "Community Health Development" and 2) from 1992 to 1995, Secretary Juan Flavier restored attention to the implementation of PHC which would be operational under the guidance of local government units. The difference in the two approaches is primarily a difference in their manner of execution. Despite some problems, the first initiative achieved important goals such as 1) implementing an information system to allow targeting of areas for social development, 2) organizing the First National Convention of NGOs for Health, 3) encouraging collaborative activities with NGOs, and 4) support of the activities of Barangay Health Workers (BHWs). The second initiative involved recognition of additional volunteer health workers; following community organizing as a basic approach for empowerment; expanding the prior initiatives; and making plans to identify model PHC barangays, monitor levels of PHC implementation, and prepare a BHW operational manual. This review ends by considering various issues and offering recommendations which include: 1) spelling out the role of local chief executives, 2) adopting a single terminology to describe the current approach, 3) defining the role of the BHW (multipurpose worker or health worker), 4) adopting a convergence of efforts strategy, 5) monitoring levels of PHC, 6) documenting the PHC implementation process, 7) dealing with program sustainability issues, and 8) improving the management of local health facilities.^ieng