Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm. typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium , as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Experimental infection of red dwarf honeybee,Apis florea,with Nosema ceranae

This research is the first record of the infection of Apis florea by Nosema ceranae, a newly identified pathogen of honeybee in Thailand which was initially isolated from A. florea workers. Each Nosema free-bee was fed 2 μl of 50% (w/v) sucrose solution containing 0, 10,000 20,000 or 40,000 Nosema spores/bee. The survival rates of treated bees were significantly lower compared to control bees. Infectivity was not statistically different among the three spore concentrations, whereas no infection was found in control bees. Protein content of control bee hypopharyngeal glands 14 days post inoculation (p.i) was significantly higher (21.47 ± 0.17 mg/bee) compared to all treatments. The infection ratio of bees treated with 40,000 spores/bee increased with time after inoculation. These results suggest that N. ceranae has a significant negative effect on honeybee hypopharyngeal gland protein production and contributes to their shortened life span.     

The phosphorylation of human link proteins

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.     

Effect of the antioxidant dibunol on the function of the adrenal cortex, the thyroid and the adenohypophysis in adult and old rats

The effect of a single antioxidant (dibunol-D) injection (100 mg/kg body weight) on the functional activity of adrenal cortex (AC), thyroid gland (TG) and tropic hormone production by adenohypophysis (AH) has been studied in old and adult rats. For 48 hours following D administration two-phase changes in adrenocorticotropic function of AH and steroidogenesis were detected in the AC: activation during the first hours was followed by suppression 24 hours later, and recovery 48 hours later. Thyrotropic AH function and thyroidogenesis were found to be decreased during the first hours of D effect. Thyroidogenesis recovery by the end of the first day was delayed as compared to the recovery of AH thyrotropic function. It is suggested that the mechanisms of D action are based on its effects mediated by changes in the functional activity of endocrine glands and associated with resetting of endocrine regulation of body functions.     

An “orientation sphere” visualization for examining animal head movements

1. Animal behavior is elicited, in part, in response to external conditions, but understanding how animals perceive the environment and make the decisions that bring about these behavioral responses is challenging.
2. Animal heads often move during specific behaviors and, additionally, typically have sensory systems (notably vision, smell, and hearing) sampling in defined arcs (normally to the front of their heads). As such, head‐mounted electronic sensors consisting of accelerometers and magnetometers, which can be used to determine the movement and directionality of animal heads (where head “movement” is defined here as changes in heading [azimuth] and/or pitch [elevation angle]), can potentially provide information both on behaviors in general and also clarify which parts of the environment the animals might be prioritizing (“environmental framing”).
3. We propose a new approach to visualize the data of such head‐mounted tags that combines the instantaneous outputs of head heading and pitch in a single intuitive spherical plot. This sphere has magnetic heading denoted by “longitude” position and head pitch by “latitude” on this “orientation sphere” (O‐sphere).
4. We construct the O‐sphere for the head rotations of a number of vertebrates with contrasting body shape and ecology (oryx, sheep, tortoises, and turtles), illustrating various behaviors, including foraging, walking, and environmental scanning. We also propose correcting head orientations for body orientations to highlight specific heading‐independent head rotation, and propose the derivation of O‐sphere‐metrics, such as angular speed across the sphere. This should help identify the functions of various head behaviors.
5. Visualizations of the O‐sphere provide an intuitive representation of animal behavior manifest via head orientation and rotation. This has ramifications for quantifying and understanding behaviors ranging from navigation through vigilance to feeding and, when used in tandem with body movement, should provide an important link between perception of the environment and response to it in free‐ranging animals.

     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.