Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the     

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.     

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1_Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2_Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.