The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-kappaB activation of lipopolysaccharide-stimulated macrophages

It has been reported that lipopolysaccharide (LPS)-hyporesponsiveness of macrophages (Mphis) of C3H/HeJ mice with a mutated Lps gene (Lps(d)) is related to high-level expression of secretory leukocyte protease inhibitor (SLPI) in response to LPS, causing suppression of NF-kappaB activation and tumor necrosis factor-alpha (TNF-alpha) production. We thus examined the effects of SLPI on the TNF-alpha production by LPS-stimulated Mphis. Neither intact SLPI nor half-sized SLPI (1/2 SLPI) down-regulated Mphi TNF-alpha production. 1/2 SLPI weakly increased Mphi TNF-alpha production in response to LPS signaling and potentiated the LPS-induced activation of NF-kappaB, especially the binding of p65-p50 heterodimers to the DNA kappaB sites, suggesting that LPS-hyporesponsiveness of Lps(d) Mphis is not due to the overexpression of SLPI.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).     

Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector

The Genome of Bacillus subtilis 168 was used for cloning and engineering of large-sized DNAs. A mouse genomic DNA of approximately 120 kb was cloned into a locus of the B. subtilis genome by ordered assembly of 20- to 50-kb mouse DNA segments. Cloned mouse DNA, maintained stably, was engineered through B. subtilis transformation and recombination. Creation of an I-PpoI recognition sequence at both ends of the insert facilitated its isolation by pulsed field gel electrophoresis. The basic concept of genome vector technology is suited to the handling of DNAs larger than 100 kb.     

Correlation analysis between fatty acid compositions of zooplankter individuals, fed on different phytoplankton species by means of pyrolysis–gas chromatography combined with on-line methylation

Pyrolysis–gas chromatography (Py–GC) combined with on-line methylation was applied to a correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and those of ingested algae using principal component analysis (PCA). Py–GC in the presence of organic alkali, tetramethylammonium hydroxide (TMAH), was used to estimate the apparent distributions of fatty acid components contained in a single individual zooplankter weighing several tens of micrograms and a small sample size of ingested algae samples in the order of 10 μg. The observed fatty acid compositions were used as a database for the PCA in order to discriminate the zooplankton and ingested algae samples. The result obtained indicated that the fatty acid compositions of zooplankton individuals used in this work were significantly reflected in those of their ingested food in spite of some contribution from isomerization and/or elongation of fatty acid components during digestion of the ingested algae phytoplankton in living zooplankters.