Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Functional divergence of the circadian clock proteins in prokaryotes

Cyanobacteria are only prokaryotes known so far to have a circadian system. It may be based either on two (kaiB and kaiC) or three (kaiA, kaiB and kaiC) circadian genes. The homologs of two circadian proteins, KaiB and KaiC, form four major subfamilies (K1–K4) and also occur in some other prokaryotes. Using the likelihood-ratio tests, we studied a rate shift at the functional divergence of the proteins from the different subfamilies. It appears that only two of the subfamilies (K1 and K2) perform circadian functions. We identified in total 92 sites that have significantly different rates of evolution between the clades K1/K2 and K3/K4; 67 sites (15 in KaiB and 52 in KaiC) been evolving significantly slower in K1/K2 than the overall average for the entire sequence. Many critical sites are located in the identified functionally important motifs and regions, e.g. one of the Walker’s motif As, DXXG motif, and two KaiA-binding domains of KaiC. There are also 36 sites (~5%) with rate shift between K1 and K2. The rate shift at these sites may be related to the interaction with KaiA. Rate shift analyses have identified residues whose manipulation in the Kai proteins may lead to better understanding of their functions in the two different types of the cyanobacterial circadian system.     

Diversification by host switching and dispersal shaped the diversity and distribution of avian malaria parasites in Amazonia

Understanding how pathogens and parasites diversify through time and space is fundamental to predicting emerging infectious diseases. Here, we use biogeographic, coevolutionary and phylogenetic analyses to describe the origin, diversity, and distribution of avian malaria parasites in the most diverse avifauna on Earth. We first performed phylogenetic analyses using the mitochondrial cytochrome b (cyt b) gene to determine relationships among parasite lineages. Then, we estimated divergence times and reconstructed ancestral areas to uncover how landscape evolution has shaped the diversification of Parahaemoproteus and Plasmodium in Amazonia. Finally, we assessed the coevolutionary patterns of diversification in this host–parasite system to determine how coevolution may have influenced the contemporary diversity of avian malaria parasites and their distribution among Amazonian birds. Biogeographic analysis of 324 haemosporidian parasite lineages recovered from 4178 individual birds provided strong evidence that these parasites readily disperse across major Amazonian rivers and this has occurred with increasing frequency over the last five million years. We also recovered many duplication events within areas of endemism in Amazonia. Cophylogenetic analyses of these blood parasites and their avian hosts support a diversification history dominated by host switching. The ability of avian malaria parasites to disperse geographically and shift among avian hosts has played a major role in their radiation and has shaped the current distribution and diversity of these parasites across Amazonia.     

Cytokine profiles of HIV patients with pulmonary tuberculosis resulting from adjunct immunotherapy with herbal phytoconcentrates Dzherelo and Anemin

Dzherelo (Immunoxel) and Anemin when combined with standard anti-tuberculosis therapy (ATT) were shown to produce better clinical outcome than chemotherapy alone. Sixty HIV-positive patients with active pulmonary TB were equally divided into three matched groups to receive either ATT, ATT + Dzherelo, or ATT + Dzherelo + Anemin. Peripheral blood samples were measured by ELISA for plasma levels of IL-2, IL-6, TNF-α, IFN-γ, and IFN-α. After 6 months of follow-up Dzherelo and Dzherelo + Anemin combinations produced 61% (P = 0.005) and 44.4% (P = 0.06) higher levels of IL-2, whereas in ATT group they were reduced by 33.1% (P = 0.002). The levels of IL-6 increased by 17% (P = 0.15) in ATT group, but declined in both immune intervention groups by 26.2% (P = 0.007) and 21.3% (P = 0.22). TNF-α was suppressed in two immunotherapy groups by 19.1% (P = 0.06) and 76.3% (P = 0.02), respectively, but had risen by 14% (P = 0.42) in ATT patients. The pattern of production of IFN-γ was opposite to that of TNF-α, but statistical significance was stronger in patients receiving ATT and Dzherelo + Anemin than in Dzherelo group: −34% (P = 0.004), +31.9% (P = 0.008), and +17.3% (P = 0.33), respectively. Moderately decreased levels of IFN-α were observed in all treatment arms (range 0.9–16.6%) but differences were not significant. Despite considerable intra-group variation in cytokine production, the baseline inter-group averages were not statistically different indicating that the results were not biased by sample heterogeneity. Immunomodulators used in this study possibly act by enhancing natural immune response against TB. Expanded study of other cytokines and correlates relevant to control and protection from TB and HIV is needed in order to identify biomarkers of favorable treatment outcome, which may aid design of better immune interventions and vaccines.     

G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.     

Cloning and expression of the tubulin genes in barley

Tubulins are encoded by small gene families in plants. Based on the barley EST collection, cDNAs for alpha-, beta-, and gamma-tubulins were selected. Five genes for alpha-tubulin, eight newly identified beta-tubulin sequences and one gamma-tubulin gene were found to be expressed in barley. In silico analysis of relative abundance of the distinct tubulin sequences among ESTs derived from different libraries revealed that the various tubulin genes differed in their level of expression, and to some extent were tissue specific.     

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes.CONCLUSION: These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.     

Voltage-dependent gating underlies loss of ENaC function in Pseudohypoaldosteronism type 1

Here we explore the mechanism and associated structure-function implications of loss of function for epithelial Na⁺ channel (ENaC) containing a pseudohypoaldosteronism type 1 (PHA-1)-causing missense point mutation. As expected, human ENaC that contained subunits harboring PHA-1-causing substitutions within an absolutely conserved, cytosolic Gly residue (e.g., βG37S) had significantly less activity. Unexpectedly, though, such substitution also results in voltage sensitivity with greater activity at hyperpolarizing potentials. This is a consequence of voltage-dependent changes in the single-channel open probability and is not species- or subunit-dependent. Voltage sensitivity in PHA-1 mutants stems from the disruption of critical structure, rather than the development of new properties resulting from the introduction of novel side chains. Residues near the conserved His-Gly sequence of G95 in α-mENaC are particularly important for voltage sensing. Although substitution of I93 in α-mENaC results in voltage sensing, it also slows the activation and deactivation kinetics enough to enable capture of the dynamic changes in single-channel open probability that account for changes in macroscopic activity. This provides definitive proof of the mechanism that underlies loss of function. In addition, the voltage dependence of ENaC with PHA-1 substitutions is akin to that which results from substitution of a critical, interfacial Trp residue conserved at the intracellular base of TM1 (e.g., W112 in α-mENaC). Dynamic interactions between similarly positioned His and Trp residues are essential for gating and the girdle-like structure that lines the intracellular mouth of the M2 proton channel. The similar residues in ENaC may serve a shared function, suggesting the possibility of an intracellular girdle just below the mouth of the ENaC pore.