Osmoregulation of the HtrA (DegP) protease of Escherichia coli: an Hha-H-NS complex represses HtrA expression at low osmolarity

The HtrA protein of Escherichia coli is a heat-shock inducible periplasmic protease, essential for bacterial survival at high temperatures. Expression of htrA gene depends on the alternative factor sigmaE and on the two-component regulatory system Cpx. These regulators systems respond, among others factors, to overproduction of misfolded proteins in the periplasm or to high level synthesis of various extracytoplasmic proteins. We describe in this report the osmoregulation of the expression of htrA gene. Low osmolarity conditions result in htrA repression. We report, as well, the role of the nucleoid associated proteins H-NS and Hha in the repression of htrA expression at low osmolarity.     

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.     

Environmental-dependent proline accumulation in plants living on gypsum soils

Biosynthesis of proline—or other compatible solutes—is a conserved response of all organisms to different abiotic stress conditions leading to cellular dehydration. However, the biological relevance of this reaction for plant stress tolerance mechanisms remains largely unknown, since there are very few available data on proline levels in stress-tolerant plants under natural conditions. The aim of this work was to establish the relationship between proline levels and different environmental stress factors in plants living on gypsum soils. During the 2-year study (2009–2010), soil parameters and climatic data were monitored, and proline contents were determined, in six successive samplings, in ten taxa present in selected experimental plots, three in a gypsum area and one in a semiarid zone, both located in the province of Valencia, in south-east Spain. Mean proline values varied significantly between species; however, seasonal variations within species were in many cases even wider, with the most extreme differences registered in Helianthemum syriacum (almost 30 μmol g^?1 of DW in summer 2009, as compared to ca. 0.5 in spring, in one of the plots of the gypsum zone). Higher proline contents in plants were generally observed under lower soil humidity conditions, especially in the 2009 summer sampling preceded by a severe drought period. Our results clearly show a positive correlation between the degree of environmental stress and the proline level in most of the taxa included in this study, supporting a functional role of proline in stress tolerance mechanisms of plants adapted to gypsum. However, the main trigger of proline biosynthesis in this type of habitat, as in arid or semiarid zones, is water deficit, while the component of ‘salt stress’ due to the presence of gypsum in the soil only plays a secondary role.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities “in vitro”

A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides “in vitro”. The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K_ad = 33.6 ± 0.44 mL g^?1, K_ad = 11.37 ± 0.87 mL g^?1, K_ad = 10.4 ± 0.19 mL g^?1, respectively). According to “in vitro” enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.     

Mitochondrial DNA and microsatellite loci data supporting a management plan for a critically endangered lizard from Brazil

Endemic and endangered species are highly vulnerable to habitat perturbations and may be subject to variations in their population size. Management plan for these species is crucial to avoid population decline, loss of genetic variability, inbreeding and ultimately extinction. The sand lizard, Liolaemus lutzae, is endemic to a habitat of sandy coastal plain (restinga). Its geographical distribution extends for only 200 km stretch of the coast of Rio de Janeiro state, Brazil, one of South America’s most densely populated regions. Extensive development and degradation of the beaches where the species inhabits, have led to the species becoming critically endangered. We used mitochondrial DNA sequences and microsatellite loci to resolve patterns of population connectivity and genetic variation within the species in order to provide a platform for a species management plan. Our results indicate the existence of three main populations, separated from each other by the Guanabara Bay and by the Arraial do Cabo Peninsula. The low microsatellite genetic variation and heterozygosity witnessed in each of the three populations, together with high levels of inbreeding and low effective population sizes suggest that the species is in urgent need of intensive management. Based on the results of this study we propose strong measures to protect existing restinga fragments and the implementation of programmes of captive breeding and reintroduction of individuals from the heavily threatened regions to protected refugia. Such measures may be the only way of ensuring the continuity of the species.     

Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8?hr (rice husks) and 7.3?hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5–7 (rice hulls) and 6–9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.     

Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.     

No evidence for oxidative stress as a mechanism of action of hyperhomocysteinemia in humans

Oxidative stress has been suggested as one of the physiopathologic conditions underlying the association of total plasma homocysteine (p-tHcy) with cardiovascular disease (CVD), but this hypothesis has not been validated in human epidemiological studies. We measured plasma and erythrocyte antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), along with serum lipid-soluble antioxidants alpha-tocopherol, beta-carotene, lycopene and retinol, in a sample of 123 healthy elderly subjects (54 men, 69 women). Plasma malondialdehyde (p-MDA) was determined as a marker of lipid peroxidation, and p-tHcy was quantified by HPLC. No significant differences were found for p-MDA, GPx or SOD activities or serum antioxidant concentrations, in subjects with elevated p-tHcy (≥15 μmol/l) as compared to those with lower plasma homocysteine. Hyperhomocysteinemia did not lead to increased risk of having the highest p-MDA values, in either sex. We found no evidence that p-tHcy was associated with lipid peroxidation in this elderly human sample. Our results do not support the view that hyperhomocysteinemia would induce an adaptive response of antioxidant systems, either. More epidemiologic and clinical research is needed to clarify whether homocysteine promotes atherosclerosis by means of an oxidative stress mechanism.