Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2

The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, we identify 11 kinases and phosphatases controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. We focus on casein kinase 2 (CK2) and demonstrate that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β, mediated cooperatively by two N-terminal residues of CK2α, is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches we identify Ankyrin2 as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. Our study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.     

PASSAGE OF 5-HYDROXYTRYPTAMINE THROUGH THE BLOOD-BRAIN BARRIER, ITS METABOLISM IN THE BRAIN AND ELIMINATION OF 5-HYDROXYINDO-LEACETIC ACID FROM THE BRAIN TISSUE

Abstract— 5-HT was injected intravenously in rats (10 mg/kg) and a marked increase in brain 5-HT and 5-HIAA was observed. For the first 10 min after injection the penetration of 5-HT into the brain and formation of 5-HIAA is evident. After 10 min degradation of exogenous 5-HT and elimination of 5-HIAA are prominent. Metabolism of exogenous 5-HT in the brain is very fast (half-life between 5 and 10 min; completely metabolized in approximately 80 min). The importance of these results in explaining the permeability of blood-brain barrier to 5-HT is discussed. Experiments on brain slices show that 5-HT is more readily metabolized in brain tissue than eliminated into incubation medium. In contrast, 5-HIAA very easily leaves brain tissue.     

Increased Summer Temperatures Reduce the Growth and Regeneration of Larix sibirica in Southern Boreal Forests of Eastern Kazakhstan

The larch forests at the southern limit of the Siberian boreal forest in Central Asia have repeatedly experienced strong recent growth declines attributed to decreasing summer precipitation in the course of climate warming. Here, we present evidence from the southernmost Larix sibirica forests in eastern Kazakhstan that these declines are primarily caused by a decrease in effective moisture due to increasing summer temperatures, despite constant annual, and summer precipitation. Tree-ring chronologies (>800 trees) showed a reduction by 50–80% in mean ring width and an increase in the frequency of missing rings since the 1970s. Climate-response analysis revealed a stronger (negative) effect of summer temperature (in particular of the previous year’s June and July temperature) on radial growth than summer precipitation (positive effect). It is assumed that a rise in the atmospheric vapor pressure deficit, which typically increases with temperature, is negatively affecting tree water status and radial growth, either directly or indirectly through reduced soil moisture. Larch rejuvenation ceased in the 1950s, which is partly explained by increasing topsoil desiccation in a warmer climate and a high drought susceptibility of larch germination, as was demonstrated by a germination experiment with variable soil moisture levels. The lack of regeneration and the reduced annual stem increment suggest that sustainable forest management aiming at timber harvesting is no longer feasible in these southern boreal forests. Progressive climate warming is likely to cause a future northward shift of the southern limit of the boreal forest.     

Physiological,biochemical, and cytological characteristics of a halotolerant and alkalitolerant methanotroph grown on methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h-1) was twice as high as on methane (0.1 h-1). The maximum growth rate increased with increasing medium salinity and was lower at neutral than at alkaline pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

New thermophilic methanotrophs of the genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and 0-12, growing in the temperature range from 30 to 61 degrees C with an optimum at 55 degrees C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes, membrane-bound methane monooxygenase, and phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulose phosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55 degrees C that in cells grown at 37 degrees C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH4+ assimilation occurred through reductive amination of alpha-ketoglutarate and via the glutamate cycle. The relationship between the physiological-biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Taxonomic characterization of new alkaliphilic and alkalitolerant methanotrophs from soda lakes of the Southeastern Transbaikal region and description of Methylomicrobium buryatense sp.nov

Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5-10.5) are taxonomically described. These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes. All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO). They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP). The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5-11.0 and optimally at pH 8.5-9.5. These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9-1.4 M NaCl or 1 M NaHCO3. Although being mesophilic, all the isolates are resistant to heating (80 degrees C, 20 min), freezing and drying. Their cellular fatty acids profiles primarily consist of C(16:1). The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The main quinone is Q-8. The DNA G+C content ranges from 49.2-51.5 mol %. Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus. However, they differ from the known members of this genus by DNA-DNA relatedness. Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium Methylomicrobium buryatense sp. nov.     

2′-OH of mRNA are critical for the binding of its codons at the 40S ribosomal P site but not at the mRNA entry site

The roles of 2′-OH groups in the binding of mRNA to human ribosomes were studied using site-directed cross-linking. We found that both mRNA and mDNA analogues bearing a cross-linker can modify ribosomal proteins (rps) S3e and S2e at the mRNA entry site independently on tRNA presence, but only mRNA analogues were capable of a tRNA^Phe-dependent binding to human ribosomes and cross-linking to rpS26e in the mRNA binding centre. Thus, 2′-OH groups of mRNA are unimportant for binding at the entry site but they are crucial for codon-anticodon interactions at the P site, implying the existence of mRNA-ribosome contacts that do not occur in bacteria.     

Transventricular and transpial absorption of cerebrospinal fluid into cerebral microvessels

It is generally accepted that volume of cerebrospinal fluid (CSF) is secreted in brain ventricles and flows to subarachnoid space to be absorbed into dural venous sinuses or/and into lymphatics via perineural sheats of cranial nerves. Since 99% of CSF volume is water, in experiments on cats 3H-water was slowly infused into lateral ventricle and found that it does not flow to subarachnoid space but that it is rapidly absorbed transventricularly into periventricular capillaries. When 3H-water was infused in cortical subarachnoid space, it was absorbed locally into cerebral capillaries via pia mater. On the contrary, when macromolecule 3H-inulin is applied in CSF it is very slowly eliminated in bloodstream, and, with time, is carried by systolic-diastolic pulsations and mixing of CSF bidirectionally along CSF system. Thus, CSF volume (water) is absorbed rapidly into adjacent cerebral capillaries while inulin is distributed bidirectionally due to its long residence time in CSF Previously, the macromolecules have been used to study CSF volume hydrodynamics and with this misconception of CSF physiology arose.     

UP-PCR Genotyping and rDNA Analysis of Ascochyta pisi Lib

Isolates of Ascochyta pisi, previously identified as separate pathotypes, were genotyped by rDNA-RFLP and UP-PCR using 8 UP-primers and 2 arbitrary primers individually or in pair-wise combinations. The appearance of polymorphic UP-PCR products for all studied isolates facilitated their differentiation. The markers were suitable in identifying isolates, and may be useful for developing isolate- or pathotype-specific PCR-based diagnostic assays. In studying genetic relatedness by UP-PCR among A. pisi isolates, two distinct clusters were revealed. rDNA analysis of the isolates based on endo-ntjclease digestion of amplified ITSl and 1TS2-25S nuclear rDNA regions did not separate the isolates, whereas the variabihty of the isolates using UP-PCR was more than 40%.     

Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy

Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)?hydroperoxide (9(S)?HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60?s at 0?°C, pH?4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9?hydroxy?9?[(1′E,3′Z)?nonadienyloxy]?nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [¹³C₁₈]9(S)?HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS).