Analysis of Grassland Vegetation of the Southwest Heilongjiang Steppe (China) Using the Power Law

In 1997, we conducted a vegetation survey in three semi-arid natural grasslands (steppes) withdifferent livestock grazing intensities in Southwest Heilongjiang Province, China. The dominant grasslandspecies was the grass Stipa baicalensis Roshev. Grasslands with light, intermediate, and heavy grazingintensities were located 10, 5, and 2 km from a village, respectively. Villagers use the steppe to raise cattle,horses, sheep, and goats. Each of the three grasslands was surveyed by placing 100 quadrats (50 cm×50 cm)along a 50 m line transect. Each quadrat was divided into four equal areas (25 cm×25 cm; S-quadrats) and allplant species occurring in each of these smaller areas were identified and recorded. These data were summa-rized into frequency distributions and the percentage of S-quadrats containing a given species and thevariance of each species were estimated. The power law was applied to these estimates. The power law wasused to evaluate the spatial heterogeneity and frequency of occurrence for each species in the grasslandcommunity. The lightly grazed grassland exhibited high spatial heterogeneity (caused by large plant size),the highest species diversity, and a high occurrence of S. baicalensis. In contrast, the heavily grazedgrassland exhibited high spatial heterogeneity (caused by patchy populations of small plant size), lowspecies diversity, and a low occurrence of S. baicalensis. We judged that the heavily grazed grassland wasovergrazed and exclusion of livestock from the degraded areas is necessary for recovery.     

Spherical Parasporal Inclusions of the Lepidoptera-Specific and Coleoptera-Specific Bacillus thuringiensis Strains: A Comparative Electron Microscopic Study

Four Lepidoptera-specific Bacillus thuringiensis strains that belong to the four H serogroups (serovars sumiyoshiensis, fukuokaensis, darmstadiensis, and japonensis) and a Coleoptera (Scarabaeidae)-specific strain belonging to serovar japonensis were examined for comparative ultrastructure of spherical parasporal inclusions. The prominent feature of the inclusions of the Lepidoptera-specific strains was the existence of thick, highly electron-dense envelopes surrounding a homogeneous protein matrix. The envelopes were 15.0–66.7 nm thick and consisted of 5–12 layers of membrane. This is also the case with inclusions of a Coleoptera-specific strain. The ultrastructure of inclusions from the five strains was in marked contrast to that of the bipyramidal parasporal inclusions produced by a Lepidoptera-specific serovar sotto strain. Received: 26 July 1999 / Accepted: 30 August 1999     

Occurrence of Bacillus thuringiensis in Fresh Waters of Japan

Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions. Received: 4 September 1999 / Accepted: 8 October 1999     

Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus

The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na(+)-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na(+) than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na(+) conduction to flagellar rotation.     

Concerted effects of amino acid substitutions in conserved charged residues and other residues in the cytoplasmic domain of PomA, a stator component of Na+-driven flagella

PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42 degrees C and was restored to the original level approximately 10 min after the temperature was returned to 20 degrees C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.     

Activation of m-calpain is required for chromosome alignment on the metaphase plate during mitosis

Calpains form a superfamily of Ca(2+)-dependent intracellular cysteine proteases with various isoforms. Two isoforms, micro- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca(2+) concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not mu-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.     

A role of anti-verotoxin antibody immunoreactive peptide, Virp5, from rat spinal cord

An anti-verotoxin 2 (VT2) antibody immunoreactive 5-kDa polypeptide (Virp5), has been obtained through screening of the rat spinal cord cDNA library with the aid of anti-VT2 antibody. Virp5 was mainly expressed in the central nervous system, liver and kidney, and localized at glia-like cells and nerve fibers in the central nervous system, vascular endothelial cells and hepatic cells in the liver, as well as epithelial cells of distal tubules in the kidney. Intravenous administration of purified Virp5 elicited a dose-dependent increase in blood pressure. These results suggest that Virp5 commonly exists in the body, being partly playing a role in regulating the blood pressure.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O₂ and low CO₂ concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased . At 30 °C, increases in endo--N-acetylglucosaminidase (EndoS) and -amylase were also detected, while at 41 °C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.Abbreviations SpeB Streptococcal pyrogenic exotoxin B - SpeF Streptococcal pyrogenic exotoxin F - Sic Streptococcal inhibitor of complement - EndoS Endo--N-acetylglucosaminidase