Selective roles for alpha-PKC in positive signaling for O-(2) generation and calcium mobilization but not elastase release in differentiated HL60 cells

Protein kinase C (PKC) isotypes and Ca2+ mobilization have been implicated in phagocytic cell functions such as O(-)(2) generation. Ca/DG-dependent alpha-PKC and beta-PKC have similar substrate specificities and cofactor requirements in vitro. However it is not known if these isotypes play redundant or unique roles in the intact cell. In the present study, a role for alpha-PKC in positive signaling for fMet-Leu-Phe- and PMA-activated O(-)(2) generation was probed using an siRNA strategy in HL60 cells differentiated to a neutrophilic phenotype (dHL60 cells). A selective decrease in alpha-PKC in dHL60 cells attenuated O(-)(2) generation but not degranulation, and reduced ligand-induced phosphorylation of p47phox as previously shown for beta-PKC. However alpha-PKC, unlike beta-PKC, was a positive regulator of fMet-Leu-Phe-triggered Ca2+ uptake via SOCC (Store Operated Calcium Channels). The ability of a selective SOCC inhibitor, MRS1845, to decrease fMet-Leu-Phe induced Ca2+ uptake and O(-)(2) generation confirmed that Ca2+ uptake via SOCC was required for O(-)(2) generation. These results indicate that alpha-PKC and beta-PKC are required for optimal O(-)(2) generation, but play different roles in Ca2+ signaling for phagocytic responses such as O(-)(2) generation.     

Diversity of Extracellular Proteolytic Activities Among Prevotella Species from the Rumen

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B₁4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH₄Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B₁4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B₁4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult. Received: 12 January 1999 / Accepted: 19 May 1999     

Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.

The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.     

在韩国境内Potentilla fragarioides var.sprengeliana的遗传多样 …

根据２２个等位酶位点遗传变异,探讨了韩国境内委陵菜（Ｐｏｔｅｎｔｉｌｌａ ｆｒａｇａｒｉｏｉｄｅｓ Ｌ．ｖａｒ．ｓｐｒｅｎｇｅｌｉａｎａ）的遗传多样性和种群结构。酶位点的多态位点百分比为５９．１％。种和种群水平上的遗传多样性比较高,分别为Ｈｅｓ＝０．２１０,Ｈｅｐ＝０．１９９;而种群的分化水平则相对较低（ＧＳＴ＝０．０７４）。１９个种群中随机交配的偏差为ＦＩＳ＝０．３３１。每代迁移数的间接估计     

GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol,Plays a Role in Motor Coordination

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.     

Nerves in the endodermal canals of hydromedusae and their role in swimming inhibition

N eoturris breviconis (Anthomedusae) has a nerve plexus in the walls of its endodermal canals. The plexus is distinct from the ectodermal nerve plexuses supplying the radial and circular muscles in the ectoderm and no connections have been observed between them. Stimulation of the endodermal plexus evokes electrical events recorded extracellularly as “E” potentials. These propagate through all areas where the plexus has been shown by immunohistology to exist and nowhere else. When Neoturris is ingesting food, trains of “E” potentials propagate down the radial canals to the margin and cause inhibition of swimming. This response is distinct from the inhibition of swimming associated with contractions of the radial muscles but both may play a part in feeding and involve chemoreceptors. Preliminary observations suggest that the “E” system occurs in other medusae including Aglantha digitale (Trachymedusae) where the conduction pathway was previously thought to be an excitable epithelium.     

Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.