Decrease of cholesterol in mouse melanoma causes secretion of lysosomal enzymes

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Inhibitory effect of fermented milk on delayed-onset muscle damage after exercise

Milk fermented with a starter containing Lactobacillus helveticus and Saccharomyces cerevisiae is drunk on a daily basis by many people in Japan and has several beneficial effects. We studied the influence of this fermented milk product on muscle damage after prolonged exercise in rats. Wistar rats were divided into four groups: rested controls, rested rats given fermented milk diet, exercised rats and exercised rats given fermented milk diet. After 3 weeks of acclimatization, both exercise groups were made to run on a treadmill at 26 m/min for 60 min. Exercise increased the serum creatine kinase level, as well as myeloperoxidase activity and the level of thiobarbituric-acid-reactive substances in the gastrocnemius muscle after 24 h. These changes were ameliorated by intake of fermented milk. An increase of CINC-1 was also ameliorated by fermented milk. Furthermore, milk diet increased the mRNA and protein levels of protective proteins such as antioxidants and chaperone proteins. These results indicate that fermented milk can ameliorate delayed-onset muscle damage after prolonged exercise, which is associated with an increased antioxidant capacity of muscles.     

Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature

A feasibility test of a 17 m3-pilot-scale sewage treatment system was carried out by continuous feeding of raw municipal sewage under ambient temperature conditions. The system consisted of a UASB and an aerated fixed bed reactor. Some of the effluent from the fixed bed reactor was returned to the UASB influent in order to provide a sulfate source. The total BOD of 148-162 mg l(-1) in the influent was reduced to a more desirable 11-25 mg l(-1) in the final effluent. The levels of methane-producing activity from acetate and H2/CO2 gas at 10 degrees C were only 2% and 0% of those at 35 degrees C, respectively. On the other hand, the sulfate-reducing activity levels of the UASB sludge were relatively high at 10 degrees C, for example, 18% for acetate and 9% for H2/CO2 gas, compared to the activity levels at 35 degrees C. Therefore, BOD oxidization by sulfate reduction in the UASB was greater than that by methane production under low temperature conditions. This sulfate-reducing activity tended to be proportional to the copy number of adenosine-5'-phosphosulfate (APS) reductase genes in DNA extracted from the sludge.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Genotype matrix mapping: searching for quantitative trait loci interactions in genetic variation in complex traits.

In order to reveal quantitative trait loci (QTL) interactions and the relationship between various interactions in complex traits, we have developed a new QTL mapping approach, named genotype matrix mapping (GMM), which searches for QTL interactions in genetic variation. The central approach in GMM is the following. (1) Each tested marker is given a virtual matrix, named a genotype matrix (GM), containing intersecting lines and rows equal to the total allele number for that marker in the population analyzed. (2) QTL interactions are then estimated and compared through virtual networks among the GMs. To evaluate the contribution of marker combinations to a quantitative phenotype, the GMM method divides the samples into two non-overlapping subclasses, S(0) and S(1); the former contains the samples that have a specific genotype pattern to be evaluated, and the latter contains samples that do not. Based on this division, the F-measure is calculated as an index of significance. With the GMM method, we extracted significant marker combinations consisting of one to three interacting markers. The results indicated there were multiple QTL interactions affecting the phenotype (flowering date). GMM will be a valuable approach to identify QTL interactions in genetic variation of a complex trait within a variety of organisms.