Glutamate-induced calcium signals stimulate CO production in piglet astrocytes

Glutamate-stimulated, astrocyte-derived carbon monoxide (CO) causes cerebral arteriole dilation by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) channels. Here, we examined the hypothesis that glutamate activates heme oxygenase (HO)-2 and CO production via the intracellular Ca(2+) concentration ([Ca(2+)](i))/Ca(2+)-calmodulin signaling pathway in newborn pig astrocytes. The major findings are: 1) glutamate stimulated Ca(2+) transients and increased steady-state [Ca(2+)](i) in cerebral cortical astrocytes in primary culture, 2) in astrocytes permeabilized with ionomycin, elevation of [Ca(2+)](i) concentration-dependently increased CO production, 3) glutamate did not affect CO production at any [Ca(2+)](i) when the [Ca(2+)](i) was held constant, 4) thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase blocker, decreased basal CO production and blocked glutamate-induced increases in CO, and 5) calmidazolium, a calmodulin inhibitor, blocked CO production induced by glutamate and by [Ca(2+)](i) elevation. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca(2+)](i) in astrocytes, leading to Ca(2+)- and calmodulin-dependent HO-2 activation, and CO production.     

Oxygen binding by marine fish blood under hypothermic experimental conditions

Influence of temperature in the range of 1-15 degrees C on oxygen binding properties of blood of thermophilic--golden mullet (Liza aurata), anchovy (Engraulis encrasicolus), and cold-tolerant--sardelle (Clupeonella cultriventris) fishes has been investigated under experimental conditions. Heat dependence of oxygenation reaction in thermophilic fish blood at temperature below 10 degrees C considerably increases, which is evidenced by high deltaH values. That is accompanied by a substantial increase of blood oxygen affinity and complicates blood deoxygenation at the tissue level. This reaction is apparently determined by the change of hemoglobin interaction with intraerythrocyte medium. The concentration of NTP in erythrocytes increases, that partially compensates negative changes of blood oxygen affinity (parameter P50 is raised) under long-term maintenance of fishes at 5 degrees C. However this reaction is not observed at low temperatures (1-2 degrees C).     

Stratified distribution of nutrients and extremophile biota within freshwater ice covering the surface of Lake Baikal

Biological entities and gradients of selected chemicals within the seemingly barren ice layers covering Lake Baikal were investigated. Ice cores 40-68 cm long were obtained from in shore and offshore sites of Southern Lake Baikal during the cold period of a year (March-April) in 2007 and 2008. In microscopic observations of the melted ice, both algae and bacteria were found in considerable numbers (>10(3) cells/L and >10(4) cells/ml, respectively). Among all organisms found, diatom was generally the most predominant taxon in the ice. Interestingly, both planktonic and benthic algae were present in considerable numbers (2-4×10(4) cells/L). Dominant phototrophic picoplankton were comprised of small green algae of various taxa and cyanobacteria of Synechococcus and Cyanobium. The bacterial community consisted mostly of short rod and cocci cells, either free-living or aggregated. Large numbers of yeast-like cells and actinomycete mycelium were also observed. Concentrations of silica, phosphorus, and nitrate were low by an order of magnitude where biota was abundant. The profile of the ice could be interpreted as vertical stratification of nutrients and biomass due to biological activities. Therefore, the organisms in the ice were regarded to maintain high activity while thriving under freezing conditions. Based on the results, it was concluded that the freshwater ice covering the surface of Lake Baikal is considerably populated by extremophilic microorganisms that actively metabolize and form a detritus food chain in the unique large freshwater ecosystem of Lake Baikal.     

Diversity of cultivable bacteria isolated from the water column and bottom sediments of the Kara Sea shelf

In this work, the results of microbiological and molecular genetic investigation of the microorganisms inhabiting the Kara Sea and the adjacent Yenisei and Gydanskii Bays are presented. The microorganisms isolated from the samples collected in the studied area belonged to 4 phyla and 11 genera. Bacteria of two phyla, Firmicutes and Actinobacteria, prevailed; representatives of the Gammaproteobacteria and Bacteroidetes were isolated as well. According to their phenotypic properties, the obtained pure cultures were classified with the genera Streptomyces, Rhodococcus, Micrococcus, Bacillus, Pseudomonas, Acinetobacter, Flavobacterium, and Marinococcus. Analysis of the obtained nucleotide sequences of the 16S rRNA genes confirmed that the isolates belonged to the genus Bacillus. One strain was reidentified as Brevibacillus laterosporus, and two strains were identified Aeromonas piscicola and Plantibacter sp. The results of the study of the enzymatic activity of the obtained pure psychrotolerant cultures suggest that the microbial community is actively involved in the destruction processes occurring in the studied area.     

Structural-functional transformation of the malate dehydrogenase system of the bacterium <Emphasis Type="Italic">Sphaerotilus</Emphasis> sp. strain D-507 depending on nutritional mode

High-purity preparations of malate dehydrogenase (EC 1.1.1.37) were obtained by multistage purification from the bacterium Sphaerotilus sp. strain D-507 growing under different conditions. Under organotrophic conditions, the enzyme was dimeric; under mixotrophic conditions, dimeric and trimeric. On the basis of studied properties of the enzyme preparations, data on the activity of enzymes of the glyoxylate and tricarboxylic-acid cycles, and analysis of published data, it can be concluded that malate dehydrogenase isoforms are implicated in the adaptive response of bacteria to changing culturing conditions.     

Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.     

Mechanism of glutamate stimulation of CO production in cerebral microvessels

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.     

Endogenous heme oxygenase prevents impairment of cerebral vascular functions caused by seizures

In newborn pigs, the mechanism of seizure-induced cerebral hyperemia involves carbon monoxide (CO), the vasodilator product of heme catabolism by heme oxygenase (HO). We hypothesized that seizures cause cerebral vascular dysfunction when HO activity is inhibited. With the use of cranial window techniques, we examined cerebral vascular responses to endothelium-dependent (hypercapnia and bradykinin) and endothelium-independent (isoproterenol and sodium nitroprusside) dilators during the recovery from bicuculline-induced seizures in saline controls and in animals pretreated with a HO inhibitor, tin protoporphyrin (SnPP). SnPP (3 mg/kg iv) blocked dilation to heme and reduced the CO level in cortical periarachnoid cerebrospinal fluid, indicating HO inhibition in the cerebral microcirculation. In saline control piglets, seizures increased the CO level, which correlated with the time-dependent cerebral vasodilation; during the recovery (2 h after seizure induction), responses to all vasodilators were preserved. In SnPP-treated animals, cerebral vasodilation and the CO responses to seizures were greatly reduced, and cerebral vascular reactivity was severely impaired during the recovery. These findings suggest that HO in the cerebral microcirculation is rapidly activated during seizures and provides endogenous protection against seizure-induced vascular injury.     

Expression of urokinase plasminogen activator, its receptor and plasminogen activator inhibitor type 1 in different types of atherosclerotic lesion in human aorta

The role of plasminogen activators in the regulation of key processes of atherosclerosis progression stays unclear. The aim of this study was to evaluate the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and the plasminogen activator inhibitor type 1 (PAI-1) in human aorta, and to balance them with the stage of atherosclerotic lesion. We have shown that uPA and uPAR in normal aorta are mostly expressed by intimal smooth muscle cells. The expression of these proteins was up-regulated in diseased aorta compared to normal artery. The most part of cells in both fatty streak and fibro-fatty lesion were monocytes/macrophages, and about 60% of these cells expressed uPA and its receptor. PAI-1 was mostly localized on the lumonal part of the aorta and in the extracellular matrix of the intima. We observed a moderate increase of PAI-1 expression in atherosclerotic lesion. Thus, our data indicate participation of plasminogen system in atherogenesis.     

Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression

The extended postictal state is characterized by neurological problems in patients. Inadequate blood supply to the brain and impaired cerebral autoregulation may contribute to seizure-induced neuronal damage. Recent evidence in newborn pigs indicates that activation of the antioxidative enzyme heme oxygenase (HO) at the onset of seizures is necessary for increased cerebral blood flow during the ictal episode and for normal cerebral vascular functioning during the immediate postictal period. We hypothesized that seizures cause prolonged postictal cerebral vascular dysfunction that can be accentuated by HO inhibition and rescued by HO overexpression. Cerebral vascular responses to endothelium-dependent (hypercapnia, bradykinin) and -independent (isoproterenol, sodium nitroprusside) stimuli were assessed 48 h after bicuculline-induced seizures in: 1) saline-control newborn piglets, 2) HO-inhibited animals (HO was inhibited by tin protoporphyrin, SnPP, 3 mg/kg iv), and 3) HO-overexpressing piglets (HO-1 was upregulated by cobalt protoporphyrin, CoPP, 50 mg/kg ip). Extended alterations of HO expression in cerebral microvessels were confirmed by measuring CO production and inducible HO (HO-1) and constitutive HO (HO-2) proteins. Our data provide evidence that seizures cause a severe, sustained, postictal cerebral vascular dysfunction as reflected by impaired vascular reactivity to physiologically relevant dilators. During the delayed postictal state, vascular reactivity to all dilator stimuli was reduced in saline control and, to a greater extent, in HO-inhibited animals. In CoPP-treated piglets, no reduction in postictal cerebral vascular reactivity was observed. These findings may indicate that CoPP prevents postictal cerebral vascular dysfunction by upregulating HO-1, a finding that might have implications for preventing postictal neurological complications.