The effect of restricted feeding on the wheel-running activity rhythms of the predatory marsupial Dasyurus viverrinus

Summary The effects of restricted feeding schedules on the circadian rhythms of wheel-running of Dasyurus viverrinus were examined under a light/dark cycle and in constant darkness (experiment 1) and in constant light (experiment 2). The results of the 2 experiments showed that: (1) in contrast to the light/dark cycle, restricted feeding is only a weak zeitgeber for the wheel-running activity rhythms of D. viverrinus; (2) restricted feeding elicits meal anticipatory activity in D. viverrinus comparable to that elicited by restricted feeding in the rat; (3) transient cycles of the anticipatory activity free-run with a period different to that of the main component of activity for several cycles after the termination of restricted feeding; and (4) activity suggestive of beating between 2 oscillators occurs during restricted feeding and after the termination of restricted feeding. Taken together the latter 3 observations suggest that the activity rhythms of D. viverrinus are controlled by at least 2 separate circadian oscillators.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis.

An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate (N-Ac-Asp) and N-acetylaspartylglutamate (N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5-2 mM HCl. At concentrations of electrolytes exceeding 10 mM the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 micrograms wet weight brain tissue extracted with a mixture of methanol and 4 mM HCl (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.     

Molecular Fate of Heterologous Bacterial DNA in Competent BACILLUS SUBTILIS. I. Processing of B. PUMILUS and B. LICHENIFORMIS DNA in B. SUBTILIS

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donorrecipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.