Primary sequence of the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.     

The use of electrophoresis of the water-soluble (sarcoplasmic) proteins of fish muscle to differentiate the closely related species of hake (Merluccius sp.).

1. Electrophoretic separation patterns of water-soluble proteins of five species of hake, Merluccius merluccius; M. australis; M. productus; M. bilinearis and M. angustimanus, have been examined. 2. The patterns were reproducible within each species and distinguishable from one another. 3. The frequency distribution of numbers of vertebrae and of gillrakers were obtained for the same fish. 4. Only the electrophoretic patterns gave an unequivocal identification of the species.     

Tetracycline Residues and Tetracycline Resistance Genes in Groundwater Impacted by Swine Production Facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.

To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators—including inorganic ions, antibiotics, and antibiotic resistance genes—were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 μg/L.

Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes—tet(M), tet(O), tet(Q), and tet(W)—were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Coordination in physonectid siphonophores

Intracellular and extracellular recordings from the stem, gastrozooids, palpons, tentacles and nectophores of physonectid siphonophores are presented. The stem organization previously described for Nanomia applies with only minor differences to Forskalia and Agalma. The endodermal epithelium of the stem is shown to be the pathway for slow potentials.

Pumping cycles and feeding activities are organized locally in gastrozooids and palpons. Protective retractions are coordinated, probably through a direct nervous link with the stem. This is also true of tentacles. The ectoderm of bracts is a conducting epithelium; excitation in it can induce nervous activity in the stem, but the mechanism is unknown.

Impulse traffic between stem and zooids is erratic and breaks down rapidly with repeated stimulation. The motor centres of the nectophores are connected to the stem by a labile nervous link, but an alternative epithelial pathway exists.     

Unconventional signalling in tunicates

The zooids in colonial tunicates do not appear to be directly interconnected by nerves, but this has not prevented the evolution of coordinated behaviour in several groups. In Botryllus and other colonial styelid asci‐dians the endothelium lining the blood vessels is excitable and transmits action potentials from cell to cell via gap junctions. These signals mediate protective contractions of the zooids and synchronize contractions of the vascular ampullae. In didemnid ascidians such as Diplosoma a network of myocytes in the tunic serves to transmit excitation and to cause contractions of the cloacal apertures. Individual zooids of Pyrosoma protect themselves by closing their siphons and arresting their branchial cilia when stimulated. At the same time a flash of light is emitted. Neighbouring zooids sense the flash with their photoreceptors and respond in turn with protective responses and light emission. Protective responses thus spread by photic signalling and propagate from zooid to zooid through the colony in a saltatory manner. In chains of Salpafusifortnis, changes in the direction and/or speed of swimming are transmitted from zooid to zooid via adhesion plaques. When a zooid is stimulated, its body‐wall epithelium conducts action potentials to the plaque connecting it to the next zooid, exciting receptor neurons in that zooid. These receptors have sensory processes that bridge the gap between the two zooids. The sensory neurons so excited in the second zooid conduct impulses to the brain where they alter the motor output pattern, and at the same time generate epithelial action potentials that travel to the next zooid in line, where the same thing happens.

It is not clear why these unconventional signalling methods have evolved but the tunic may be an inhospitable environment for nerves, making conventional nervous links impossible.     

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

Food intake regulates oleoylethanolamide formation and degradation in the proximal small intestine

Oleoylethanolamide (OEA) is a lipid mediator that inhibits food intake by activating the nuclear receptor peroxisome proliferator-activated receptor-alpha. In the rodent small intestine OEA levels decrease during food deprivation and increase upon refeeding, suggesting that endogenous OEA may participate in the regulation of satiety. Here we show that feeding stimulates OEA mobilization in the mucosal layer of rat duodenum and jejunum but not in the serosal layer from the same intestinal segments in other sections of the gastrointestinal tract (stomach, ileum, colon) or in a broad series of internal organs and tissues (e.g. liver, brain, heart, plasma). Feeding also increases the levels of other unsaturated fatty acid ethanolamides (FAEs) (e.g. linoleoylethanolamide) without affecting those of saturated FAEs (e.g. palmitoylethanolamide). Feeding-induced OEA mobilization is accompanied by enhanced accumulation of OEA-generating N-acylphosphatidylethanolamines (NAPEs) increased activity and expression of the OEA-synthesizing enzyme NAPE-phospholipase D, and decreased activity and expression of the OEAdegrading enzyme fatty acid amide hydrolase. Immunostaining studies revealed that NAPE-phospholipase D and fatty acid amide hydrolase are expressed in intestinal enterocytes and lamina propria cells. Collectively, these results indicate that nutrient availability controls OEA mobilization in the mucosa of the proximal intestine through a concerted regulation of OEA biosynthesis and degradation.     

Preferential cleavage of degradative intermediates of rpsT mRNA by the Escherichia coli RNA degradosome

RNase E, the principal RNase capable of initiating mRNA decay, preferentially attacks 5'-monophosphorylated over 5'-triphosphorylated substrates. Site-specific cleavage in vitro of the rpsT mRNA by RNase H directed by chimeric 2'-O-methyl oligonucleotides was employed to create truncated RNAs which are identical to authentic degradative intermediates. The rates of cleavage of two such intermediates by RNase E in the RNA degradosome are significantly faster (2.5- to 8-fold) than that of intact RNA. This verifies the preference of RNase E for degradative intermediates and can explain the frequent "all-or-none" behavior of mRNAs during the decay process.