A dihydrofolate reductase gene from Candida albicans: molecular cloning

The dihydrofolate reductase gene from Candida albicans has been cloned and partially characterized. A genomic bank from C. albicans strain 10127/5 was constructed in Escherichia coli and screened for trimethoprim resistance. A plasmid pMF1, carrying the resistance marker was isolated and characterized by restriction mapping and Southern blotting. Cells harbouring pMF1 were as sensitive as the parental cells to a wide spectrum of antibacterial agents, except for trimethoprim; the dihydrofolate reductase activity from these cells was trimethoprim resistant.     

A combined nucleic acid and protein analysis in Friedreich ataxia: implications for diagnosis, pathogenesis and clinical trial design

Background

Friedreich''s ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls.

Methodology/Principal Findings

We enrolled 24 patients with classic FRDA phenotype (cFA), 6 late onset FRDA (LOFA), all homozygous for GAA expansion, 5 pFA cases who harbored the GAA expansion in compound heterozygosis with FXN point mutations (namely, p.I154F, c.482+3delA, p.R165P), 33 healthy expansion carriers, and 29 healthy controls. DNA was genotyped for GAA expansion, mRNA/FXN was quantified in real-time, and frataxin protein was measured using lateral-flow immunoassay in peripheral blood mononuclear cells (PBMCs). Mean residual levels of frataxin, compared to controls, were 35.8%, 65.6%, 33%, and 68.7% in cFA, LOFA, pFA and healthy carriers, respectively. Comparison of both cFA and pFA with controls resulted in 100% sensitivity and specificity, but there was overlap between LOFA, carriers and controls. Frataxin levels correlated inversely with GAA1 and GAA2 expansions, and directly with age at onset. Messenger RNA expression was reduced to 19.4% in cFA, 50.4% in LOFA, 52.7% in pFA, 53.0% in carriers, as compared to controls (p<0.0001). mRNA levels proved to be diagnostic when comparing cFA with controls resulting in 100% sensitivity and specificity. In cFA and LOFA patients mRNA levels correlated directly with protein levels and age at onset, and inversely with GAA1 and GAA2.

Conclusion/Significance

We report the first explorative study on combined frataxin and mRNA levels in PBMCs from a cohort of FRDA patients, carriers and healthy individuals. Lateral-flow immunoassay differentiated cFA and pFA patients from controls, whereas determination of mRNA in q-PCR was sensitive and specific only in cFA.     

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.     

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.     

Glycolytic enzymes in polyamine-treated bovine retina

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.     

Gene duplications in the TL region of the mouse major histocompatibility complex

We have isolated a class I gene from the TL region of the A/J mouse. The gene, T2A, is a homologue of the C57BL/10 mouse gene T2. In the process of mapping this gene we screened a number of BALB/c class I cosmid clusters with a T2A flanking probe. Several of the hybridizing clusters were found to contain identical DNA segments and could therefore be linked together into one single BALB/c TL region which appears to be identical to the TL region of the C57BL/10 mouse. However, two of the hybridizing clusters do not overlap with the C57BL/10 TL region. It appears that these two clusters represent a partial duplication of the TL region in the BALB/c mouse.     

Phylogenetic diversity and biotechnological potentials of marine bacteria from continental slope of eastern Arabian Sea

Marine environments are substantially untapped source for the isolation of bacteria with the capacity to produce various extracellular hydrolytic enzymes, which have important ecological roles and promising biotechnological applications. Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. A number of marine hydrolases have been described, including amylases, lipases and proteases, which are being used extensively for biotechnological applications. The present study was carried out to isolate marine bacteria from continental slope sediments of the eastern Arabian Sea and explore their biotechnological potential. Among the 119 isolates screened, producers of amylases (15%), caseinases (40%), cellulases (40%), gelatinases (60%), lipases (26%), ligninases (33%), phytase (11%) and Malachite Green dye degraders (16%) were detected. Phylogenetic analysis based on 16S rRNA gene sequencing showed that predominant marine sediment bacteria possessing more than four enzymatic activities belonged to the phyla Firmicutes and Proteobacteria, was assigned to the genera Bacillus, Planococcus, Staphylococcus, Chryseomicrobium, Exiguobacterium and Halomonas. Biodegradation of the dye Malachite Green using the liquid decolorization assay showed that both the individual cultures (Bacillus vietnamensis, Planococcus maritimus and Bacillus pumilus) and their consortium were able to decolorize more than 70% of dye within 24?h of incubation. This is the first report on diversity and extracellular hydrolytic enzymatic activities and bioremediation properties of bacteria from continental slope sediment of eastern Arabian Sea.     

Isolation,structure determination and biological activity of A-16686 factors A′ 1, A′ 2 and A′ 3 glycolipodepsipeptide antibiotics

Summary WhenActinoplanes strain ATCC 33076, the producer of A-16686 A1, A2 and A3 complex, is fermented in a suitable medium three additional factors, designated A1, A2 and A3 are produced. These were isolated and characterized, and were shown to differ from the parent components of the original complex by lacking one mannose unit. Bioconversion of A factors into A factors was achieved by incubation with the mycelium ofActinoplanes ATCC 33076. Factor A2 has better antibacterial activity than A2 against some bacteria.