Anaerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture: involvement of the pentose phosphate pathway.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.     

Virtual screening to identify <Emphasis Type="Italic">Leishmania braziliensis N</Emphasis>-myristoyltransferase inhibitors: pharmacophore models,docking, and molecular dynamics

Leishmaniasis is caused by several protozoa species belonging to genus Leishmania that are hosted by humans and other mammals. Millions of new cases are recorded every year and the drugs available on the market do not show satisfactory efficacy and safety. A hierarchical virtual screening approach based on the pharmacophore model, molecular docking, and molecular dynamics was conducted to identify possible Leishmania braziliensis N-misristoyltransferase (LbNMT) inhibitors. The adopted pharmacophore model had three main features: four hydrophobic centers, four hydrogen-bond acceptor atoms, and one positive nitrogen center. The molecules (n=15,000) were submitted to alignment with the pharmacophore model and only 27 molecules aligned to model. Six molecules were submitted to molecular docking, using receptor PDB ID 5A27. After docking, the ZINC35426134 was a top-ranked molecule (? 64.61 kcal/mol). The molecule ZINC35426134 shows hydrophobic interactions with Phe82, Tyr209, Val370, and Leu391 and hydrogen bonds with Asn159, Tyr318, and Val370. Molecular dynamics simulations were performed with the protein in its APO and HOLO forms for 37 ns in order to assess the stability of the protein–ligand complex. Results showed that the HOLO form was more stable than the APO one, and it suggests that the ZINC35426134 binding stabilizes the enzyme. Therefore, the selected molecule has the potential to meet the herein proposed target.     

Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior)

The use of diagnostic methods that prevent irreplaceable samples (from museum collections, archaeological and paleontological samples) of being consumed or that increase their yield is relevant. For museum collections, archaeological and paleontological samples it is essential to conserve samples, subsamples or portions for future research. We are addressing methods for conservation of irreplaceable samples that could be fully consumed. Innovations in methodologies that are used in studies of Paleoparasitology and Paleomicrobiology will contribute to the preservation of collections. Therefore, to the development of archaeology and paleontology in the future, we evaluated whether the discarded material of the immunochromatography test could be used for molecular diagnosis and vice versa. We used a genotyped experimental coprolite positive for Giardia duodenalis. The diagnosis was positive for giardiasis in both cases. This methodology can be corroborated with the coprolite of a Paleolama maior (extinct llama) previously diagnosed for G. duodenalis with an immunoenzymatic test. The residue of the pre-digestion step of the DNA extraction before adding Proteinase K was confirmed positive with the immunochromatographic test. Also, the DNA extraction residue from a coprolite of Nothrotherium maquinense (ground sloth) was tested positive with immunochromatographic test for G. duodenalis. These are the oldest findings for G. duodenalis confirming that this intestinal parasite occurred among Northeastern Brazilian Megafauna animals from the late Pleistocene period, correlated to human occupation. The relevance of these results will allow the study by different methodological approaches from a small amount of material, reusing discarded materials.     

Production of actinorhodin by Streptomyces coelicolor A3(2) grown in chemostat culture

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.     

Diversity and seasonal dynamic of a lizard assemblage in a Neotropical semiarid habitat

We assessed the lizard assemblage from a priority conservation Caatinga area from northeastern Brazil, through a pluriannual ecological approach, to expand the understanding on biodiversity patterns of Neotropical semiarid habitats. The studied area presented one of the richest lizard faunas among Caatinga sites, being composed primarily by species typical from open landscapes. The local species composition was more similar to assemblages from adjacent Caatinga ecoregions than to those from other areas within the same ecoregion. The inventoried lizard assemblage consisted of a few common species and a majority of low abundance ones, and its overall richness and abundance did not differ between rainy and dry months. Our findings demonstrated that the composition of lizard assemblages did not match with the current proposed Caatinga ecoregions, and revealed that the studied assemblage followed a lognormal species-abundance distribution, showing no significant seasonal fluctuation in richness and abundance.     

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice

Ascaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host’s homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris.     

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 M CaCl₂ in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl₂ was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg²⁺ and Ca²⁺ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca²⁺ ions could replace Mg²⁺ ions. The high GDH activity which has been found in cells grown under Mg^2–-limited conditions in the presence of higher concentrations of Ca²⁺ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.Abbreviations PQQ pyrroloquinoline quinone - GDH glucose dehydrogenase (EC 1.1.99.17) - GaDH gluconate dehydrogenase (EC 1.1.99.3) - CAP chloramphenicol - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

Establishment of the steady state in glucose-limited chemostat cultures of Klebsiella pneumoniae

To investigate the relationship between growth rate and concentration of the nutrient that limits growth, 'Klebsiella aerogenes' NCTC 418 (K. pneumoniae) was grown in a glucose-limited chemostat. The actual time required to establish a steady-state glucose concentration exceeded that expected theoretically. Apparently, there is a long-term adaptation of the cells to nutrient limitation. As yet, it is not clear whether this has a phenotypic or genetic origin. In the final steady state, the dependence of the growth rate on glucose concentration could be mathematically described equally well by a hyperbolic and by a logarithmic function.     

Diffusible Fraction of Serum Cholesterol and Atherogenesis

CHOLESTEROL is found in the blood as a structural component of lipoproteins concerned with the transport of other lipids¹. The high resolution nuclear magnetic resonance spectra of high density serum lipoproteins are similar to that observed when lipids are dissolved in organic solvents, or dispersed in water by bile salts or detergents, or in sonicated form. The lipid component in lipoproteins is therefore probably in an extremely fluid condition². If human serum is mixed with paraffin oil, some of the cholesterol diffuses into the oil without affecting the ultraviolet absorption spectrum of serum proteins. This procedure avoids any protein denaturing action used for cholesterol extraction^3–5. It therefore seems that serum cholesterol has two fractions, one strongly bound by lipoprotein structures and the other loosely bound and diffusible in an oil phase. In this article I designate the loosely bound fraction “diffusible”.