Electrophoretic evaluation of the taxonomic status of two species of sea anemone

Puerto Rican populations of two species of sea anemones (Bunodosoma cavernata and B. granulifera) which had previously been considered one were assayed electrophoretically for enzymes encoded by 12 loci. The two species shared no common allozymes at 6 of the 12 loci. Genetic distance and identity values based on these allozymes were computed for the Puerto Rican populations and for B. cavernata from Florida and B. granulifera from Panama. The Puerto Rican populations of both species had much higher genetic identities for their geographically distant conspecifics than for each other. These results indicate that the two species are reproductively isolated and should be considered as separate valid species. Average heterozygosities are presented which are the first published for coelenterate species.     

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFα is an apoptotic inducer in some cancer cells, it activates NFκB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFκB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFα, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTα, FasL, and TRAIL. Mechanistically, CCNs function through integrin α₆β₁ and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding α₆β₁-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an α₆β₁-HSPG-binding defective CCN1 are blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFα and related cytokines.     

Separation and Identification of the Polar Lipids of Chromatium Strain D

The polar lipids of the autotrophically grown, obligately anaerobic, photosynthetic bacterium Chromatium strain D were separated by paper chromatography. Four major phospholipids were identified: lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In addition, three glycolipids were observed and characterized, namely, monoglucosyldiglyceride, which is found in other biological systems, and (mannosyl, glucosyl)-diglyceride and (dimannosyl, glucosyl)-diglyceride, which heretofore have not been observed in nature.     

Factors affecting biomass attachment during startup and operation of anaerobic fluidized beds

Three anaerobic fluidized bed reactors at 37 degrees C were utilized to observe the effects of startup and operational procedures on biomass attachment. Using a meat-based synthetic waste and stepped-loading regime, the influences of synthetic polymer addition and maintenance of anaerobiosis during startup were investigated. Subsequently, increasing bed expansions were applied to assess shear effects. Synthetic polymer addition enhanced biomass retention but did not improve process performance. Maintenance of a reduced environment ameliorated fluctuating process parameters during start up and aided biomass retention and substrate removal. A bed expansion of 5% was detrimental to biomass attachment and COD removal but system stability was maintained at expansions between 10% and 30%. Startup was achieved in 56 days. Anaerobiosis appeared to enhance the initial evolution of a stable, well-adapted microbial population, whereas polymer addition interfered with this. Moderate bed expansions had negligible effects on attachment and performance.     

Identification of protein kinase C and its potential substrate in Entamoeba histolytica

1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.     

Intergeneric relationships ofLolium,Festuca, andVulpia (Poaceae) and their phylogeny

Morphological and seed protein analyses of 26 species of the generaLolium, Festuca andVulpia confirmed their close systematic affinities. Six inflorescence characters readily differentiatedFestuca fromLolium. Protein similarities betweenFestuca of sect.Bovinae and cross-pollinated species ofLolium, coupled with cytogenetic and crossability data, substantiate that they should be united into one genus.Vulpia had phenetic similarities with sect.Scariosae, Montanae andOvinae ofFestuca. Lolium, Festuca, andVulpia are most likely derived from a common ancestral form which was close toFestuca pratensis andLolium perenne.     

High-affinity [3H]inositol uptake by dissociated brain cells and cultured fibroblasts from fetal mice

The accumulation of [³H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent K_m values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na⁺ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain.     

Glucuronides of monohydroxylated bile acids: specificity of microsomal glucuronyltransferase for the glucuronidation site, C-3 configuration, and side chain length

The ability of rat liver microsomes to catalyze UDP-glucuronic acid-dependent glucuronidation of monohydroxy-bile acids was examined. The following bile acids were used as substrates, each as the 3 alpha and 3 beta epimer: 3-hydroxy-5 beta-cholanoic acid (C24), 3-hydroxy-5 beta-norcholanoic acid (C23), 3-hydroxy-5 beta-bisnorcholanoic acid (C22), 3-hydroxy-5 beta-pregnan-21-oic acid (C21), and 3-hydroxy-5 beta-androstane-17 beta-carboxylic acid (C20). The corresponding glucuronides were chemically synthesized to serve as standards and were characterized by thin-layer and gas-liquid chromatography as well as by nuclear magnetic resonance. Enzymatic glucuronidation reactions were optimized with respect to pH for each product formed and the kinetic parameters for each reaction were measured. Analytical techniques necessary to separate products from unreacted substrates and to identify them included thin-layer chromatography, gas-liquid chromatography, and nuclear magnetic resonance. It was found that the 3 alpha epimers of the five bile acids listed above enzymatically formed 3-O-glucuronides, C24 being the best substrate, followed by C21 and C20; C22 and C23 gave rise to only small amounts of this product. The 3 beta epimers of all bile acids tested were poorer substrates, although by a factor that varied widely. In addition to the expected hydroxyl-linked glucuronide, three of the 3 alpha-bile acids (C23, C22, and C20) and at least one 3 beta-bile acid (C20), gave rise to a novel metabolite in which the 1-OH of glucuronic acid was esterified with the steroidal carboxyl group (carboxyl-linked glucuronide).     

Excretion of cholate glucuronide

[3-3H]Cholic acid glucuronide [7 alpha,12 alpha-dihydroxy-3 alpha-O-(beta-D-glucopyranosyluronate)-5 beta- cholan-24-oate] was synthesized and administered to rats prepared with either an external biliary fistula or a ligated bile duct. When bile fistula animals were given either microgram or milligram amounts of the glucuronide, biliary secretion of label was rapid and efficient: greater than 90% of the administered label was secreted within 60 min and total recovery of label in bile was 98.6 +/- 1.2%. Studies in which [14C]taurocholate was included in the dose indicated that this bile acid was secreted into bile significantly more rapidly than was the glucuronide. In animals with ligated bile ducts, urinary excretion was the major route of elimination: after 20 hr, 83.4 +/- 9.3% of the administered dose had been excreted in urine. Urinary excretion of cholate glucuronide was significantly more rapid than that of taurocholate. Gas-liquid chromatographic analysis of the methyl ester acetate derivatives of labeled compounds isolated from bile and urine by chromatography established that the bulk (greater than 70%) of the administered material was secreted in bile or excreted in urine as the intact cholate glucuronide. From these results, we conclude that the glucuronidation of cholic acid produces a derivative which is rapidly and effectively cleared from the circulation and excreted.