Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs

Astrocytes can act as intermediaries between neurons and cerebral arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands. CO is a gasotransmitter that can be related to neural function and blood flow regulation in the brain. The present study addresses the hypothesis that glutamatergic stimulation promotes perivascular astrocyte CO production and pial arteriolar dilation in the newborn brain. Experiments used anesthetized newborn pigs with closed cranial windows, piglet astrocytes, and cerebrovascular endothelial cells in primary culture and immunocytochemical visualization of astrocytic markers. Pial arterioles and arteries of newborn pigs are ensheathed by astrocytes visualized by glial fibrillary acidic protein staining. Treatment (2 h) of astrocytes in culture with L-2-alpha-aminoadipic acid (L-AAA), followed by 14 h in toxin free medium, dose-dependently increased cell detachment, suggesting injury. Conversely, 16 h of continuous exposure to L-AAA caused no decrease in endothelial cell attachment. In vivo, topical L-AAA (2 mM, 5 h) disrupted the cortical glia limitans histologically. Such treatment also eliminated pial arteriolar dilation to the astrocyte-dependent dilator ADP and to glutamate but not to isoproterenol or CO. Glutamate stimulated CO production by the brain surface that also was abolished following L-AAA. In contrast, tetrodotoxin blocked dilation to N-methyl-D-aspartate but not to glutamate, isoproterenol, or CO or the glutamate-induced increase in CO. The concurrent loss of CO production and pial arteriolar dilation to glutamate following astrocyte injury suggests astrocytes may employ CO as a gasotransmitter for glutamatergic cerebrovascular dilation.     

Modulation of MthK potassium channel activity at the intracellular entrance to the pore

We used a bacterial complementation screen with the LB2003 K(+) uptake-deficient strain of Escherichia coli to analyze residues that are critical to Methanobacterium thermoautotrophicum potassium channel (MthK) function. Channel expression and relative structural integrity of mutants were analyzed by SDS-PAGE and Western blot, and mechanisms underlying altered mutant channel function were analyzed using single-channel recording. We observed that wild-type MthK expression complements K(+) uptake deficiency. Although MthK function was previously thought to require Ca(2+) in the millimolar range, we demonstrate that at elevated temperatures the requirement for Ca(2+) becomes much lower. Mutations at the cytoplasmic mouth of the MthK pore can blunt complementation, indicating that those mutant channels cannot support K(+) uptake. In contrast, substitutions at the Ca(2+)-binding site in the MthK RCK domain did not decrease complementation compared with wild-type MthK. We focused on mutations to residues Glu-92 and Glu-96, which may form the narrowest part of the pore in the channel's closed state. Mutations at these residues can yield slight changes in single-channel conductance that do not necessarily correlate with effects on bacterial complementation. However, mutations at Glu-92 could also change channel open probability, and these changes correlated with complementation effects. The most striking of these mutations was E92A, which nearly eliminated bacterial complementation by decreasing the open probability of MthK. Our results suggest that the small, hydrophobic alanine side chain at the K(+) channel bundle crossing may generate an intrinsically stable structure, which in turn shifts the closed-to-open-state equilibrium toward the closed state.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Carbon monoxide as an endogenous vascular modulator

Carbon monoxide (CO) is produced by heme oxygenase (HO)-catalyzed heme degradation to CO, iron, and biliverdin. HO has two active isoforms, HO-1 (inducible) and HO-2 (constitutive). HO-2, but not HO-1, is highly expressed in endothelial and smooth muscle cells and in adjacent astrocytes in the brain. HO-1 is expressed basally only in the spleen and liver but can be induced to a varying extent in most tissues. Elevating heme, protein phosphorylation, Ca(2+) influx, and Ca(2+)/calmodulin-dependent processes increase HO-2 activity. CO dilates cerebral arterioles and may constrict or dilate skeletal muscle and renal arterioles. Selected vasodilatory stimuli, including seizures, glutamatergic stimulation, hypoxia, hypotension, and ADP, increase CO, and the inhibition of HO attenuates the dilation to these stimuli. Astrocytic HO-2-derived CO causes glutamatergic dilation of pial arterioles. CO dilates by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. CO binds to BK(Ca) channel-bound heme, leading to an increase in Ca(2+) sparks-to-BK(Ca) channel coupling. Also, CO may bind directly to the BK(Ca) channel at several locations. Endothelial nitric oxide and prostacyclin interact with HO/CO in circulatory regulation. In cerebral arterioles in vivo, in contrast to dilation to acute CO, a prolonged exposure of cerebral arterioles to elevated CO produces progressive constriction by inhibiting nitric oxide synthase. The HO/CO system is highly protective to the vasculature. CO suppresses apoptosis and inhibits components of endogenous oxidant-generating pathways. Bilirubin is a potent reactive oxygen species scavenger. Still many questions remain about the physiology and biochemistry of HO/CO in the circulatory system and about the function and dysfunction of this gaseous mediator system.     

Study on the Lake Baikal microbial community in the areas of the natural oil seeps

We studied the composition of a natural microbial community, the distribution of different groups of microorganisms (including those able to degrade oil hydrocarbons) within the areas of natural oil seeps in the Lake Baikal. It was revealed that, in the bottom sediments, the oil-degrading microorganisms dominating the community have included the bacteria of g. Bacillus, while in the water column, dominating microbes are presented by species of genera Rhodococcus, Pseudomonas, and Micrococcus. Under the conditions of the model experiment, the potential activity of Baikal microbes towards utilization of n-alcanes has been assessed. Under such conditions it was shown that the concentration of n-alcanes decreases to 60% during 20 days of the experiment (the initial oil concentration was 0.5 mg/l, i.e., ten maximal permissible concentrations, MPC).     

Microbial community of freshwater sponges in Lake Baikal

The microbial community of Baikal sponges has been studied in five species belonging to the genera Swartschewskia, Baicalospongia, and Lubomirskia of the endemic family Lubomirskiidae. The results show that the total numbers of bacteria and bacterioplankton production have an effect on the growth of L. baicalensis body. Bacteria of the genera Pseudomonas, Bacillus, Micrococcus, Sarcina, Flavobacterium, Arthrobacter, and Acinetobacter living in the sponges are representatives of the Baikal bacterioplankton. Actinomycetes of the genera Streptomyces and Micromonospora are a permanent component of the cultivable sponge microbial community. The numbers and enzyme activities of heterotrophic, oligotrophic, and psychrophilic bacteria isolated from different sponge species and from the ambient water in autumn and in winter have been estimated.     

Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate

Endogenous carbon monoxide (CO)contributes to vasodilator responses of cerebral microvessels innewborn pigs. We investigated the expression, intracellularlocalization, and activity of heme oxygenase (HO), the key enzyme in COproduction, in quiescent cerebral microvascular endothelial cells(CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected byRT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 aremembrane-bound proteins that have a strong preference for the nuclearenvelope and perinuclear area of the cytoplasm. Betamethasone(10⁶ to 10⁴ M for 48 h) was associatedwith upregulation of HO-2 protein by ~50% and inhibition of Cox-2but did not alter HO-1 or endothelial nitric oxide synthase expressionin CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by30-60%. HO activity as ¹⁴CO production from[¹⁴C]glycine-labeled endogenous heme was inhibited bychromium mesoporphyrin (10⁶ to 10⁴ M).L-Glutamate (0.3-1.0 mM) stimulated HO activity1.5-fold. High-affinity specific binding sites forL-[³H]glutamate suggestive of the glutamatereceptors were detected in CMVEC. Altogether, these data suggest that,in cerebral circulation of newborn pigs, endothelium-derived CO maycontribute to basal vascular tone and to responses that involveglutamate receptor activation.