Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found in WS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.     

The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase

The tumour growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the K_m-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation of Walker cells with Ro07-7957 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentration ≤LD₅₀. This increase in enzyme activity does not occur in the presence of cycloheximide. These results suggest that inhibition of serine hydroxymethyltransferase in intact cells is accompanied by an increase in enzyme biosynthesis and that the growth inhibitory property of Ro07-7957 does not involve interference with the conversion of serine to glycine.     

Muscle plasticity: comparison of a 30-Hz burst with 10-Hz continuous stimulation

The changes in the contractile properties induced by a 30-Hz phasic stimulation paradigm were measured and compared with the changes induced by a 10-Hz continuous stimulation paradigm. The study was performed on the tibialis anterior muscles of cats with one paradigm applied to one hindlimb muscle and the other to the contralateral limb. Both hindlimb muscles received the same number of stimuli in a day, making the average stimulation frequency 10 Hz. Two periods of daily stimulation were studied, 8 and 24 h/day. Muscles stimulated at 30 Hz produced greater overall tetanic tension and, during a prolonged stimulation test, exerted a greater mean tension than muscles stimulated at 10 Hz (50 and 32% increase for animals stimulated for 8 and 24 h/day, respectively). Muscle mass was least reduced and fewer pathological abnormalities were observed in the muscles stimulated at 30 Hz. There were no apparent differences in the histochemistry or biochemistry between muscles stimulated at 10 and 30 Hz, which could account for these differences in muscle properties. These results indicate the 30-Hz paradigm may be better suited than 10 Hz continuous stimulation for applications requiring sustained muscle tension such as correction of scoliosis or muscle conditioning for motor prostheses.     

The vacuolar H+-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification,mitochondrial function,and iron metabolism

Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%–75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H ⁺-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H ⁺-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.     

Serotonergic Regulation of Acetylcholine Release in Rat Frontal Cortex

Abstract: The extent to which serotonin regulates the activity of cortically projecting cholinergic neurons was studied using in vivo microdialysis to monitor interstitial concentrations of acetylcholine in the frontal cortex of freely moving rats. Systemic administration of the serotonin release-inducing agent fenfluramine (3 or 10 mg/kg, i.p.) increased acetylcholine release by 110–130%. The fenfluramine-induced increase in acetylcholine release was significantly attenuated by pretreatment with the selective serotonin uptake inhibitor fluoxetine (10 mg/kg, i.p.). Pretreatment with the selective dopamine D₁ receptor antagonist SCH-23390 (0.3 mg/kg, s.c.) failed to prevent the fenfluramine-induced increase in acetylcholine release. In contrast, the serotonin 5-HT_2A receptor antagonist ketanserin (5 mg/kg, i.p.) blocked fenfluramine-induced increases in acetylcholine release. In contrast to previous studies that have concluded that serotonin has inhibitory actions on cortical acetylcholine release, the present results indicate that fenfluramine increases cortical acetylcholine release in vivo by its ability to enhance serotonin transmission and that serotonin produces these effects at least in part via actions at serotonin 5-HT_2A receptors.     

Effect of chlorambucil and indomethacin on growth and prostaglandin levels in sensitive and resistant walker carcinoma

An analysis of the effect of combinations of chlorambucil and indomethacin, or chlorambucil and prostaglandin E₂ (PGE₂) on the growth of alkylating agent sensitive and resistant Walker carcinoma in vitro has been made by the isobologram approach. Indomethacin alone acts as a growth inhibitor of the Walker carcinoma. High concentrations of indomethacin (5 μg/ml) act to inhibit the growth of the resistant line sub-additively with chlorambucil, whereas low concentrations act additively. For the sensitive line indomethacin acts either additively or supra-additively with chlorambucil at all concentrations employed. Both indomethacin and low concentrations of chlorambucil alone inhibit PGE₂ secretion into the culture medium of both cell lines and an enhanced inhibition is seen with the combination. PGE₂ itself acts as a growth inhibitor of both cell lines, although it causes greater growth inhibition of chlorambucil resistant Walker carcinoma (LD₅₀ 1.8 μg/ml) than of the sensitive line. This correlates with a greater PGE₂ secretion capacity by the resistant cell line (40 pg PGE₂/ml medium/10⁵ cells for the resistant tumour and 17 pg PGE₂/ml medium/10⁵ cells for the sensitive tumour). Combinations of PGE₂ with chlorambucil inhibit growth either additively or sub-additively. It seems unlikely that inhibition of PGE₂ secretion is responsible for the interactive effects of chlorambucil and indomethacin, since growth inhibition produced by the combination is not reversed by PGE₂ at any of the concentrations employed. Possible mechanisms of the interactive effects are discussed.     

The relationship between the growth characteristics of somatic cell hybrids and their level of camp and activities of adenylate cyclase and camp phosphodiesterase.

When avian tendon cells are transferred from an in vivo environment to an in vitro environment, they lose their ability to synthesize large amounts of collagen relative to other cell proteins and thereby forgo the hallmark of their differentiated state. This research has systematically investigated the role of various components in the standard cell culture medium in order to decipher why it is an inadequate environment for maintaining differentiated function. The results show that serum levels in excess of 0.5% can be detrimental to a high production of collagen synthesis. The magnitude of this serum effect was also found to be a function of cell density. High cell densities, apparently acting through an increase in the CO₂ concentration, reverse the inhibitory effect of serum. In addition, if the lactate ion concentration is raised to 30 mM (the highest concentration tested), the level of collagen synthesis relative to total cellular protein is restored to its initial level of 30%. Thus, the major differentiated function of avian tendon cells can be retained in cell culture. Moreover, the results appear to implicate high cell density as the normal stabilizing factor in maintaining differentiation in ovo. A high concentration of cells tend to switch the cell metabolism to one which is more anaerobic, thereby favoring high collagen synthesis. Reduction in the cell division rate, which also occurs at high cell densities, does not appear to effect the ratio of collagen to other cell proteins synthesized.