An investigation of enumeration and DNA partitioning in Bacillus subtilis L-form bacteria

R.N. WATERHOUSE, E.J. ALLAN, F. AMIJEE, V.J. UNDRILL AND L.A. GLOVER. 1994. Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.     

Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides

A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.     

Stress-Induced Cortisol Response and Fat Distribution in Women

Recent studies have shown an association between uncontrollable stress and abdominal fat distribution. It has been suggested that changes in cortisol secretion might represent one possible mechanism for this relationship. This study investigated whether body fat distribution, determined by waist-to-hip ratio (WHR), is related to salivary cortisol levels in response to laboratory stressors. Subjects were 41 overweight women with a Low or a High WHR. Multiple measures of cortisol and mood were obtained during a session of stressful tasks (eg., timed arithmetic) and during a time-matched, control rest session. Also, background life stress and psychological trait variables were assessed. Compared to Low WHR subjects, High WHR subjects secreted significantly more cortisol during the stressful session after 60 minutes of stress, and considering the total area under the curve of secretion. This difference was not seen on the rest day. In terms of background and psychological measures, High WHR subjects were characterized by poorer coping skills and differences in mood reactivity. Specifically, although all subjects became more angry in response to the stressful session, High WHR subjects showed smaller increases in anger. This could indicate that they are more likely to evidence a helpless reaction to uncontrollable stress. These findings support the hypothesis that cortisol secretion might represent a mechanism for the observed association between stress and abdominal fat distribution. Furthermore, differences in coping and appraisal may suggest that a particular psychological pattern might influence the reactivity of the adrenal-cortical system to stress, and subsequent fat distribution.     

Observations on the structure of kefir grains and the distribution of the microflora

The mixed flora of yeasts and lactobacilli of kefir is held together in non-dispersible structures which build up into large grains. The fibrillar extracellular material of the matrix in which the microflora is embedded was stained by ruthenium red and periodic acid-thiosemicarbazide silver proteinate, indicating that it was largely composed of carbohydrate. It is suggested that the carbohydrate is of bacterial origin and that this is produced by a population of lactobacilli which resides within the matrix and which separates non-carbohydrate-producing populations of lactobacilli and yeasts so that sheet-like structures are formed which show asymmetry, with yeasts predominating on one side and lactobacilli on the other.     

The biological activities of some new gibberellins (GAs) in six plant bioassays

The biological activities of GA₄₀, GA₄₃, GA₄₆, GA₄₇, GA₅₁ and GA₄ 20,4-lactone were tested over a wide range of concentrations in six plant bioassays. GA₄ 20,4-lactone showed the highest activity. Of the two 2-hydroxylated compounds GA₄₇ showed moderately high activity, and GA₄₀ was slightly active. The 2-hydroxylated compunds GA₄₃, GA₄₆ and GA₅₁ were virtually inactive.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - Me methyl - TLC thin layer chromatography - TMS trimethylsilyl The author is née Frydman     

Evaluation of tentacle regeneration as a biological assay inHydra

Summary Tentacle number in non-buddingHydra attenuata, randomly selected from mass culture varies <0.5 tentacles over a 3 month period. Replicate samples of untreated regenerates (n=50–60), however, show some variability in mean tentacle number regenerated (S _x0.13–0.15). The variability is similar whether experiments are performed using randomly selected animals or animals with identical tentacle numbers. The variability is, further, not the result of profound differences in the time of tentacle initiation in individual animals.Addition of 10^–5 M glutamate or a methanol extract to the assay medium results in both an earlier appearance of tentacles and in more tentacles being regenerated during early time periods. The mean tentacle number of methanol extract-treated animals is significantly higher than the mean tentacle number of either control or glutamate-treated animals at all time periods examined.The distribution of tentacle number classes among regenerates is normal in control and glutamate-treated animals but nonparametric in methanol extract-treated animals, making statistical analysis of the data using Student'st-test in-appropriate. The usefulness of the Mann WhitneyU and Kruskal-Wallis tests is discussed, as is the appropriateness of tentacle regeneration as an assay forhydra morphogens.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.