Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998     

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.     

Isolation of Orientia tsutsugamushi from patients in Shikoku and finding of a strain which grows preferentially at low temperatures

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.     

Physiological and subjective responses in the elderly when using floor heating and air conditioning systems

The purpose of this study was to investigate the effects of a floor heating and air conditioning system on thermal responses of the elderly. Eight elderly men and eight university students sat for 90 minutes in a chair under the following 3 conditions: air conditioning system (A), floor heating system (F) and no heating system (C). The air temperature of sitting head height for condition A was 25 degrees C, and the maximum difference in vertical air temperature was 4 degrees C. The air and floor temperature for condition F were 21 and 29 degrees C, respectively. The air temperature for condition C was 15 degrees C. There were no significant differences in rectal temperature and mean skin temperature between condition A and F. Systolic blood pressure of the elderly men in condition C significantly increased compared to those in condition A and F. No significant differences in systolic blood pressure between condition A and F were found. The percentage of subjects who felt comfortable under condition F was higher than that of those under condition A in both age groups, though the differences between condition F and A was not significant. Relationships between thermal comfort and peripheral (e.g., instep, calf, hand) skin temperature, and the relationship between thermal comfort and leg thermal sensation were significant for both age groups. However, the back and chest skin temperature and back thermal sensation for the elderly, in contrast to that for the young, was not significantly related to thermal comfort. These findings suggested that thermal responses and physiological strain using the floor heating system did not significantly differ from that using the air conditioning system, regardless of the subject age and despite the fact that the air temperature with the floor heating system was lower. An increase in BP for elderly was observed under the condition in which the air temperature was 15 degrees C, and it was suggested that it was necessary for the elderly people to heat the room somehow in winter. Moreover, it is particularly important for elderly people to avoid a decrease in peripheral skin temperature, and maintain awareness of the warmth of peripheral areas, such as the leg, in order to ensure thermal comfort.     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in humans

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, suppresses allergic immunoglobulin responses and inflammation caused by polymorphonuclear leukocytes (PMNL) in mice. However, few placebo-controlled clinical trials have examined the efficacy and safety of polyphenolic phytochemicals for treatment of allergic inflammatory diseases in humans. The present study determined whether oral supplementation with rosmarinic acid is an effective intervention for patients with seasonal allergic rhinoconjunctivitis (SAR). In this 21-day, randomized, double-blind, age-matched, placebo-controlled parallel group study, patients with mild SAR were treated daily with extract of Perilla frutescens enriched for rosmarinic acid (200 mg [n=10] or 50 mg [n=9]) or placebo (n=10). Patients recorded symptoms daily in a diary. Profiles of infiltrating cells and concentrations of eotaxin, IL-1beta, IL-8, and histamine were measured in nasal lavage fluid. Serum IgE concentrations and routine blood tests were also examined. As compared with placebo supplementation, supplementation with extract of Perilla frutescens enriched for rosmarinic acid resulted in a significant increase in responder rates for itchy nose, watery eyes, itchy eyes, and total symptoms (P<0.05). Active treatment significantly decreased the numbers of neutrophils and eosinophils in nasal lavage fluid (P<0.05 vs. placebo). Patients reported no adverse events, and no significant abnormalities were detected in routine blood tests. In conclusion, extract of Perilla frutescens enriched for rosmarinic acid can be an effective intervention for mild SAR at least partly through inhibition of PMNL infiltration into the nostrils. Use of this alternative treatment for SAR might reduce treatment costs for allergic diseases.     

Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase,at Ser-446

We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.