Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.     

Large scale in vitro experiment system for 2 GHz exposure

A beam formed radiofrequency (RF) exposure-incubator employing a horn antenna, a dielectric lens, and a culture case in an anechoic chamber is developed for large scale in vitro studies. The combination of an open type RF exposure source and a culture case through which RF is transmitted realizes a uniform electric field (+/-1.5 dB) in a 300 x 300 mm area that accommodates 49 35 mm diameter culture dishes. This large culture dish area enables simultaneous RF exposure of a large number of cells or various cell lines. The RF exposure source operates at 2142.5 MHz corresponding to the middle frequency of the downlink band of the International Mobile Telecommunication 2000 (IMT-2000) cellular system. The dielectric lens, which has a gain of 7 dB, focuses RF energy in the direction of the culture case and provides a uniform electric field. The culture case is sealed and connected to the main unit for environmental control, located outside the anechoic chamber, via ducts. The temperature at the center of the tray, which contains the culture dishes in the culture room, is maintained at 37.0 +/- 0.2 degrees C by air circulation. In addition, the appropriate CO2 density and humidity supplied to the culture case realizes stable long-term culture conditions. Specific absorption rate (SAR) dosimetry is performed using an electric field measurement technique and the Finite Difference Time Domain (FDTD) calculation method. The results indicate that the mean SAR of the culture fluid at the bottom of the 49 (7 x 7 array) culture dishes used in the in vitro experiments is 0.175 W/kg for an antenna input power of 1 W and the standard deviation of the SAR distribution is 59%. When only 25 culture dishes (5 x 5 array) are evaluated, the mean SAR is 0.139 W/kg for the same antenna input power and the standard deviation of the SAR distribution is 47%. The proliferation of the H4 cell line in 72 h in a pair of RF exposure-incubators reveals that the culture conditions are equivalent to those of a common CO2 incubator.     

Genetic divergence between and within two color morphotypes of <Emphasis Type="Italic">Parapercis sexfasciata</Emphasis> (Perciformes: Pinguipedidae) from Tosa Bay,southern Japan

The anterior half of the mitochondrial DNA control region (mtCR) sequence (ca. 400 base pairs) was compared between two color morphotypes (A, B) of Parapercis sexfasciata from Tosa Bay, southern Japan, using 16 and 21 specimens, respectively. Intramorphotypic mtCR divergences were only 0.0–0.5% and 1.0–2.5% for morphotypes A and B, respectively. In contrast, intermorphotypic mtCR divergence was much greater, 12.7–14.0%. Furthermore, phylogenetic analysis using a neighbor-joining algorithm, with P. multifasciata as an outgroup, showed that each morphotype was reciprocally monophyletic. These results and the distinct coloration and overlapping distribution indicate that the two color morphotypes of P. sexfasciata represent two distinct species. Mismatch distribution analysis suggested that both morphotypes had undergone population expansion; however, estimates of initial population sizes and mutational timescales suggested that morphotype B comprises historically larger and older populations than morphotype A.     

A new species of <Emphasis Type="Italic">Sebastes</Emphasis> (Scorpaeniformes: Scorpaenidae) from the Pacific coast of southern Japan

A new rockfish, Sebastes kiyomatsui, is described on the basis of 13 specimens from the Pacific coast of southern Japan. The new species resembles S. scythropus in having two sharp preorbital and robust preocular, supraocular, and postocular spines, but differs from the latter in having a black blotch on the opercle not extending to the subopercle, indistinct crimson marks on the body, blackish region of the oral cavity reaching to the prevomer and palatine, blackish tongue, a small ridge on the upper edge of the lachrymal, somewhat larger orbit, somewhat lower caudal peduncle, and seven lower unbranched pectoral fin rays. Sebastes kiyomatsui is a deep water (ca. 250m) inhabitant.     

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.     

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 +/- 0.7 degrees C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5 degrees C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 +/- 0.7 degrees C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).