A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Mechanism of function of dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter in Halobacterium halobium: pH effect

The regulatory roles of medium pH, a transmembrane pH gradient (delta pH), and an electrical potential (delta phi) on the activation of the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter were studied in the membrane vesicle of Halobacterium halobium in the dark. Neither delta pH nor delta phi independently activated the antiporter but a combination could. The initial rate of Na+ extrusion did not proportionally relate to the size of delta microH+ imposed. The delta microH+-coupled Na+ efflux in the presence of delta phi (-140 mV) increased as external pH decreased, regardless of the size of delta pH, suggesting the existence of one external H+-binding site (apparent pKa 4.6) whose protonation determines primarily the Na+/H+-exchange activity. On the other hand, the dependence of the Na+ efflux on cytoplasmic pH varied with the size of delta pH imposed and the apparent pKa for the cytoplasmic H+ increased with elevating delta pH. The resulting pKa difference across the membrane seems to be the key mechanism for the facilitation of Na+-coupled H+ influx. In other words, delta pH modulates Na+/H+-exchange activity through manipulating the H+ affinity on the cytoplasmic regulatory site. The Na+ extrusion was gated by the threshold delta phi of -100 mV regardless of the size of existing delta pH. delta phi acts on the protonated antiporter and converts it into an active state which becomes delta pH reactive.     

A toxic substance produced by Nocardia otitidiscaviarum isolated from cutaneous nocardiosis

During our studies on toxic substances from clinically isolated Nocarida, a new isolate identified as Nocardia otitidiscaviarum from cutaneous nocardiosis was found to produce a toxic substance called HS-6 that had strong in vitro as well as in vivo toxicity. The mouse intraperitoneal LD₅₀ value was 1.25 mg/kg and the ED₅₀ value for L1210 cultured cells was 0.3 ng/ml. The structure of HS-6 was determined and found to belong to the 16-membered macrocyclic group with a molecular formula of C₄₃H₆₈O₁₂. HS-6 also showed activity against pathogenic fungi such as Cryptococcus neoformans.     

Germination traits and seed-bank dynamics of a biennial plant,Oenothera glazioviana Micheli

A study was conducted on the germination traits and seed-bank dynamics ofOenothera glazioviana (=O. erythrosepala), which sets seed in August in sand-dune systems in Japan. More than 90% of freshly matured seeds germinated over a wide range of temperature in light, but less than 10% did so in continuous darkness. Stratification (chilling under moist conditions) was ineffective in diminishing the light-requirement for germination. When fresh seeds were imbibed for 24 h including a 12-h light period, followed by 7-day air-drying, 94% of them became germinable in the dark at 25°C, but remained dormant at less than 15°C. of seeds collected in March from capsules of dead plants, 58% germinated in the dark at 25°C. After four cycles of alternatc 1-day wetting followed by 2-day drying or 1.5-day wetting followed by 1.5-day drying under a 12-h photoperiod, the fraction of viable seeds declined from 76% to 40% and 22%, respectively, due to germination during the wet periods. Seed-bag experiments were conducted in the field, using seeds given and not given a light-stimulus. Forty percent of the light-stimulated seeds germinated in the soil, whereas the seeds without a light-stimulus remained dormant throughout the experiment. When seeds were placed on the soil surface or at a depth of 0.5-1 cm, the proportion of germinable seeds declined during late spring and autumn, but not during winter and early spring. The seed-bank size of a natural population just prior to current seed dispersal was 2–3% of the seed production in the previous year, suggesting a high turnover rate of the seed-bank.     

Water Potential and Mechanical Properties of the Cell Wall of Hypocotyls of Dark-Grown Squash (Cucurbita maxima Duch.) under Water-Stress Conditions

Hypocotyl growth of seedlings of dark-grown squash (Cucurbitamaxima Duch.) was greatly reduced by the addition of 60mM polyethyleneglycol (PEG) to hydroponic solution (water stress). Apoplastic solution (A) and cell sap (C) were separately collectedfrom the hypocotyl segments by a centrifugation method. Theosmotic potentials of A (_A) and C (_c), and (=_c–_A) ofstressed hypocotyls were always lower than those of unstressedhypocotyls. Suction force was measured by immersing the segments into solutionsof different concentrations of mannitol. Suction force was significantlycorrelated with _C (r= –0.99). The mechanical properties of the cell wall of hypocotyl segmentswere measured by stressrelaxation technique. Minimum stressrelaxation time (T_o), relaxation rate (R) and residual stressof unstressed hypocotyls were low during the growth period andincreased when the growth ceased. T_o and R of stressed hypocotylsdecreased one day after the stress treatment, but the residualstress was not decreased by the water stress throughout theexperiment. These results suggest that the suppressed growth of dark-grownsquash hypocotyls under water stress was due neither to thereduction of the osmotic potential difference between innerand outer space of the cell, nor to the decrease in suctionforce, but was partly due to the unchanged mechanical propertiesof the cell wall, as represented by one stress-relaxation parameter,residual stress. (Received February 5, 1988; Accepted September 8, 1988)     

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.