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Abstract. Botulinum C3 exoenzyme specifically ADPribosylates a group of ms-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32p]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADPribosylated in situ in a time-and concentrationdependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-l-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzymeinduced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells. R as and ras-related genes constitute a gene family encoding a series of closely related proteins with guanine nucleotide-binding activities. To date, ~40 ras and ras-related GTP-binding proteins are known, and they are divided into four subfamilies: ms, rho, tab, and others (Hall, 1990;Bourne et al., 1991). These proteins exist in two interconvertible functional states; one in an inactive GDPbound form and the other in an active GTP-bound form. In resting cells, they are present in an inactive GDP-bound form, and upon cell stimulation, converted to the active GTP-bound form and work as molecular switches linking external stimuli to various cellular responses such as growth, differentiation, and secretion. Among the ras-related GTPbinding proteins, rho proteins are believed to be involved in organization of cytoskeleton and maintenance of cell shape. There are at least three members in this family in human, which are called rhoA, B, and C (Yeramian et al., 1987;Chardin et al., 1988). rho proteins are unique among the small GTP-binding proteins in being substrates for ADP ribosylation by the exoenzyme C3 from Clostridium botulinum (Aktories et al., 1987;Morii et al., 1988;Narumiya et al., 1988;Kikuchi et al., 1988). This enzyme ADPribosylates the proteins at an asparagine residue in the putative effector domain and inhibits their biological activities presumably by interfering with their interaction ...

A novel extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent environment (depth, 22 m) in Tachibana Bay, Nagasaki Prefecture, Japan. The cells of this organism were gramnegative rods. Growth occurred at temperatures between 50 and 85°C (optimum temperature, 80°C; doubling time at optimum temperature, 90 min), at pH 5.5 and 9.0 (optimum pH, 7.0), and in the presence of 1 and 5% NaCl (optimum NaCl concentration, 3%). The new isolate was an aerobic heterotroph which utilized the following compounds as sole energy and carbon sources: yeast extract, peptone, starch, casein, Casamino Acids, a variety of sugars, some carboxylic acids, and amino acids. As determined by a sequence analysis of the 16s rRNA, the new isolate belongs to the genus Rhodothermus and represents a modern lineage of extreme thermophiles within the domain Bacteria. On the basis of the physiological and molecular properties of the new isolate, we describe a new species, Rhodothermus obamensis. The type strain of R. obamensis is strain OKD7 (= JCM 9785).Over the last 10 years, a number of new genera and species of thermophilic organisms which are capable of growth at temperatures up to 80°C have been isolated. Most of these organisms belong to the domain Archaea (31), but there are a few genera, such as the genera Themus, Thennotoga, and Aquifex, which belong to the domain Bacteria (5, 10, 11). These thermophiles have distinct physiological differences; the members of the genus Themus are strictly aerobic heterotrophs, the members of the genus Thermotoga are strictly anaerobic heterotrophs, and the members of the genus Aquifex are microaerobic chemolithoautotrophs. On the basis of 16s rRNA analysis data, the genera Thennotoga and Aquifex represent the deepest phylogenetic branches in the domain Bacteria (7, 21). Moreover, the genus Thennus also branches deeply on the phylogenetic tree inferred from 16s rRNA sequences (21). These findings and the results of a phylogenetic study of the thermophilic genus Hydrogenobacter support the hypothesis that the ancestors of bacteria were thermophilic species (1,26).The members of the genus Rhodothemus, on the other hand, are marine thermophilic bacteria that have been isolated from sites in Iceland and the Azores (2, 20). Rhodothemus marinus is an aerobic heterotrophic bacterium that has an optimum growth temperature of 65°C and has been used in studies of genetic engineering of thermostable enzymes (28). A 16s rRNA analysis of this organism placed the genus Rhodothermus close to the root of the Flexibacter-Cytophaga-Bacteroides group with affinities to the green sulfur bacteria, fibrobacteria, and spirochetes (3). This phylogenetic position of the genus Rhodothennus differs from the phylogenetic positions of other thermophilic bacteria and suggests that there are thermophilic or extremely thermophilic bacteria which are distant from the universal root of life and that there was another origin of thermophily within the Bacteria (3).In this paper, we describe the isolation and cha...

IntroductionTraumatic brain injury (TBI) and post-traumatic stress disorder (PTSD) are risk factors for early onset of Alzheimer's disease (AD) and may accelerate the progression rate of AD pathology. As amyloid-beta (Aβ) plaques are a hallmark of AD pathology, we hypothesized that TBI and PTSD might increase Aβ accumulation in the brain.MethodsWe examined PET and neuropsychological data from Vietnam War veterans compiled by the US Department of Defense Alzheimer's Disease Neuroimaging Initiative, to examine the spatial distribution of Aβ in male veterans' who had experienced a TBI and/or developed PTSD. Subjects were classified into controls, TBI only, PTSD only, and TBI with PTSD (TBI_PTSD) groups and data were analyzed using both voxel-based and ROI-based approaches.ResultsCompared to controls, all three clinical groups showed a pattern of mainly increased referenced standard uptake values (SUVR) for the amyloid tracer [18F]-AV45 PET, with rank order PTSD > TBI_PTSD > TBI > Control, and same rank order was seen in the deficits of cognitive functions. SUVR increase was observed in widespread cortical regions of the PTSD group; in white matter of the TBI_PTSD group; and cerebellum and precuneus area of the TBI group, in contrast with controls. The [18F]-AV45 SUVR correlated negatively with cerebrospinal fluid (CSF) amyloid levels and positively with the CSF tau concentrations.ConclusionThese results suggest that both TBI and PTSD are substantial risk factors for cognition decline and increased Aβ deposition resembling that in AD. In addition, both PTSD and TBI_PTSD have a different pathways of Aβ accumulation.

A sulfotransferase (ST) specific to N‐21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N‐ST was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to N‐21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS‐PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N‐ST is a monomeric enzyme. The N‐ST was specific to only N‐21 of STX and GTX2+3, and O‐22 sulfation was not observed. Moreover, the N‐ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N‐1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N‐ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg2+ and Co2+. The Km values of the N‐ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.

ABSTRACT. Blooms of the noxious red tide phytoplankton Heterosigma akashiwo (Raphidophyceae) occurred in Hiroshima Bay. Japan. in 1994 and 1995. During these blooms we monitored rnicroorganisms which killed H. akashiwo by use of the microplate MPN (most probable number) method using an axenic culture of H. akashiwo as a susceptible host organism. At every sampling site abundance of algicidal microorganisms in seawater samples filtered through 0 8 pm nuclepore filters increased rapidly during the termination period of each bloom. However, the number of algicidal microorganisms In seawater samples filtered through 0 2 pm nuclepore filters was less abundant and correlated poorly with the extinction of H, akashiwo blooms. The latter samples were assumed to indicate viral activity. Thus, it is possible that H. akashiwo-killing bacteria (HAKB) played a more dominant role in the terrninatlon of the blooms in 1994 and 1995 in Hiroshima Bay than viruses. The number of algicidal bacteria targeting Chattonella antiqua (Raphidophyceae), which was not detected during the investigation period, was 1 or 2 orders of magnitude lower than that of HAKB. We isolated some HAKB strains capable of causing mortality in H. akashiwo. These results suggest that the population dynamics of algicidal bacteria has a close relationship to the blooms of the phytoplankton, and that, in marine ecosystems, algicidal bacteria targeting specific phytoplankton may be one of the agents which regulate the change of species structure of phytoplankton communities.

A novel hyperthermophilic, heterotrophic, rod-shaped archaeon was isolated from a terrestrial hot spring at Oguni-cho, Kumamoto Prefecture, Japan. T exhibited a close relationship to the sequences of Pyrobaculum aerophilum and Thermoproteus neutrophilus, which belong to the cluster of the genus Pyrobaculum. DNA-DNA hybridization analysis showed a low level of DNA similarity between TE7T and previously described Pyrobaculum species. As TE7 T is phenotypically and phylogenetically different from the other members of this genus, it is described as a new species named Pyrobaculum oguniense (type strain TE7 T l JCM 10595 T l DSM 13380 T ).

Motor actions can be released much sooner than normal when the go-signal is of very high intensity (> 100dBa). Although statistical evidence from individual studies has been mixed, it has been assumed that sternocleidomastoid (SCM) muscle activity could be used to distinguish between two neural circuits involved in movement triggering. We summarized meta-analytically the available evidence for this hypothesis, comparing the difference in premotor reaction time (RT) of actions where SCM activity was elicited (SCM+ trials) by loud acoustic stimuli against trials in which it was absent (SCM-trials). We found ten studies, all reporting comparisons between SCM+ and SCM-trials. Our mini meta-analysis showed that premotor RTs are faster in SCM+ than in SCM-trials, but the effect can be confounded by the variability of the foreperiods employed. We present experimental data showing that foreperiod predictability can induce differences in RT that would be of similar size to those attributed to the activation of different neurophysiological pathways to trigger prepared actions. We discuss plausible physiological mechanisms that would explain differences in premotor RTs between SCM+ and SCM-trials.