Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.     

Immunochemical detection of precursor proteins of yolk and vitelline envelope, and their annual changes in the blood of Diodon holocanthus

Two distinct groups of female-specific proteins, vitellogenin (VTG) and vitelline envelope proteins (VEP), were detected in the blood of the porcupine fish Diodon holocanthus , and annual changes in concentration were measured immunochemically. Using antisera against yolk proteins (ab.a-E) and VEP (ab. a-VEP), VTG and VEP could be detected in the blood of maturing female fish and oestradiol-17β (E₂)-treated fish. Neither protein was detected in the blood of male fish. Immunohistochemistry showed that yolk globules and the vitelline envelope enclosing developing oocytes stained with ab.a-E. The vitelline envelope was stained specifically with ab.a-VEP. Hepatocytes from the E2-treated fish had immunoreactivity with both antisera. Thus, VTG and VEP appear to be synthesized in the liver by direct stimulation of E₂, released into the circulation, and incorporated into respective target sites. VTG and VEP in female serum maintained high levels from April until June, suggesting that yolk accumulation, as well as vitelline envelope formation, are occurring actively during these months. Unlike VTG, small amounts of VEP were detected between December and March, suggesting that vitelline envelope formation precedes yolk accumulation and that a slightly different hormonal regulation exists in the synthesis of both proteins in the liver during the early phase of oogenesis.     

Microhabitat size distribution affects local difference in community structure: Metazoan communities in treeholes

I studied the effect of treehole (microhabitat) size distribution in local habitats on geographic difference in aquatic metazoan community structure by comparing differences between two sites on Iriomote Island, and between Iriomote Island (subtropical) and Tsushima Island (temperate), in southwestern Japan. In treeholes at each local site, the amount of litter, the species richness and total biomass of metazoa were positively correlated with treehole capacity. Between the two sites on Iriomote Island (Shirahama and Komi), the amount of litter, biomass and species number per treehole was greater at Komi where the mean and variance of treehole size were greater, while the dependencies of these parameters on treehole capacity were common to both sites. Total species number was larger at Komi (2 predators and 20 saprophages) than at Shirahama (1 predator and 19 saprophages). Most of the dominant taxa colonized larger treeholes with higher probabilities, although one taxa showed the opposite trend. Treeholes on Tsushima were smaller than those on Iriomote. The metazoan fauna in treeholes consisted of 15 saprophages on Tsushima, being less richer than that on Iriomote Island which had 2 predators and 21 saprophages. However, the dependencies of litter amount and biomass on treehole capacity did not differ significantly between the islands, although treeholes on Iriomote harbored a greater number of species per treehole than those on Tsushima. This study indicated that there are general correlates between community structure within individual treeholes (infracommunity structure) and treehole capacity (microhabitat size). Therefore, microhabitat-size distribution is potentially a significant constraint of local community structure, and its variation may contribute to the variation in local and regional species richness.     

Phylogeography of the Coastal Mosquito Aedes togoi across Climatic Zones: Testing an Anthropogenic Dispersal Hypothesis

The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1—L3; although individuals with L1—L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.     

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.     

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.     

Synthesis and pharmacological profile of serofendic acids A and B

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.     

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).