Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium.

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.     

Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor.

Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'.     

A new 'paralyzed' flagella mutant, OC-10, in Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) that can be reactivated with adenosine triphosphate

A new ‘paralyzed’ mutant. OC–10, was isolated in Chlamydomonas reinhardtii Dangeard. OC-10 cannot swim and generally shows very little flagellar movement. However, when OC-10 was demembranated, axonemal motility was reactivated in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The beating form of the reactivated axonemes was almost the same as that of the wild-type axonemes. Flagellar regeneration of OC-10 was slower than that of the wild-type. Electron microscopic examination showed no abnormality in OC-10 flagella, but SDS/PAGE revealed that mobility of a flagellar membrane protein was changed and a few bands disappeared in OC-10 flagella, When the mutant was crossed to wild-type to form temporary dikaryon cells with 4 flagella, OC-10 flagella did not regain motility. Tetrad analysis of crosses between OC–10 and wild-type demonstrated a 1:1 segregation on the basis of flagellar motility. From these results, we suppose that OC-10 may be limited in ATP availability inside the flagella, or altered in flagellar membrane proteins important for motility.     

Carbon source nutrition of rapamycin biosynthesis inStreptomyces hygroscopicus

Summary Chemically-defined media were developed for rapamycin production byStreptomyces hygroscopicus. Thirty-five carbon sources were tested for their effect on production. Eight failed to support growth and seven appeared to repress or inhibit rapamycin formation. The best combination of two carbon sources were 2% fructose and 0.5% mannose. Acetate and propionate, which are known to contribute most of the carbon atoms of the lactone ring, were unsatisfactory for growth and/or rapamycin production.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Parabolic flight experiments on physiological data acquisition and processing technologies using small jet aircraft (MU300)

The parabolic aircraft flight provides a short low gravity environment for approximately 20 seconds, which may not be sufficient for a research on the physiological phenomenon induced by actual weightlessness in space. However, the method is still useful to reveal essential and characteristic feature of physiological signs, and is available for testing hardware and also training of crew member during altered gravity. This paper reports the summary of parabolic flight experiments recently conducted as a NASDA program (1990-1992). The program is providing opportunities in low gravity research with small jet aircraft for researchers and agencies. The flight experiments in the life science area have been conducted mostly focused on a physiological changes and basic methodology which may be effective under the altered gravity condition. In this study, the following research team, NASDA, Research Institute of Environmental Medicine, Nagoya University, Toyohashi University of Technology, Tokyo Metropolitan Hospital, Torey Research Center and JSUP were involved and coordinated for the research.     

The main neutrophil and neutrophil-related functions may compensate for each other following exercise-a finding from training in university judoists.

In order to clarify the relationship between exercise and neutrophil function, we measured three major neutrophil and neutrophil-related functions, viz. the reactive oxygen species (ROS) production capability and phagocytic activity (PA) of neutrophils and serum opsonic activity (SOA), simultaneously before and after a unified loading exercise under three different sets of conditions. Thirteen female collegiate judoists were examined with a unified exercise loading (2 h) immediately before and after a 64 day training period. Immediately thereafter, the athletes took part in a 6 day intensified training camp, following which the same exercise loading was repeated. Responses from circulating neutrophils were estimated by comparing the two sets of values obtained before and after the two instances of exercise loading. The parameters assessed included neutrophil count, SOA, PA and ROS production capability. ROS production increased after the exercise loading performed immediately before and after the 64 day training period just before the camp, (p < 0.01) but decreased following the exercise loading performed after the camp (p < 0.05). This suggested depressed bacteriocidal capability of the circulating neutrophils. PA decreased after the exercise loading sessions imposed prior to and after the 64 day training period (p < 0.01) but did not change in the loading session after the camp. No changes were seen in SOA produced with the loading exercise either before the 64 day exercise period or before the camp, but increased significantly following the post-camp session (p < 0.05). In conclusion, athletic training-induced changes in immune functional activities of neutrophils, such as ROS production and PA, and neutrophil-related factors, such as SOA, may compensate for each other to maintain the overall integrity of the neutrophil immune function.     

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

A new class of corticotropin releasing factor 1 (CRF₁) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF₁ receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF₁ receptor antagonist. The synthesized tricyclic derivatives 2a–e showed CRF₁ receptor binding activity with IC₅₀ values of less than 400?nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF₁ receptor binding activity (IC₅₀?=?7.1?nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF₁ receptor binding activity (IC₅₀?=?58?nM) with good oral bioavailability (F?=?68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [¹²⁵I]-CRF binding in the frontal cortex (5 and 10?mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10?mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF₁ receptors in the brain and exhibits the potential to be further examined for clinical studies.     

Heterogeneity in response to interleukin 2 and interleukin 2-producing ability of adult T cell leukemic cells

To examine the possibility of heterogeneous mechanisms in the proliferation of adult T cell leukemia (ATL) cells, leukemic cells from 13 patients, nine acute-type and four chronic-type ATL, were examined for the production of interleukin 2 (IL 2) with or without mitogenic stimulation and their response to recombinant IL 2 when exogeneously added. The leukemic cells were classified into four groups, as follows. Group 1 (two patients): Cells of this group produced IL 2 messenger RNA, secreted IL 2, and proliferated when cultured in mitogen-free medium. The spontaneous proliferation of the cells in mitogen-free medium was inhibited by anti-Tac/IL 2 receptor and anti-IL 2 monoclonal antibodies. Moreover, the thymidine incorporation by the cells was enhanced in response to exogeneously added recombinant IL 2 and IL 2 produced by themselves. These results indicate that the ATL cells of this group proliferate with autostimulation by IL 2. Group 2 (seven patients): Cells of this group did not secrete IL 2 when cultured in mitogen-free medium, but the cells showed response to exogeneously added recombinant IL 2 and proliferated in culture. These results indicate that the ATL cells of this group proliferate by a paracrine mechanism. Group 3 (one patient): Cells of this group secreted IL 2 in mitogen-free medium. However, the spontaneous proliferation of these cells in vitro was very low, and the response to recombinant IL 2 was also very low. Group 4 (three patients): Cells of this group did not secrete IL 2 in mitogen-free medium. Spontaneous proliferation and the response to recombinant IL 2 were also very low. The clinical feature of all patients of Groups 1 and 2 was acute-type, and that of Groups 3 and 4 was chronic-type. Thus, we conclude that heterogeneous mechanisms exist in the proliferation of leukemic cells, and that growth rate in mitogen-free medium and response to IL 2 of the cells may have a significant relationship to the clinical feature, acute- or chronic-type.