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.     

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.     

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)     

Germination ecology of coastal plants in relation to salt environment

Responses of seed germination to salinity were examined using 37 species collected from salt marshes, cliffs, and fore (unstable) and hind (stable) sand dunes along Japanese coasts. For comparison, seed germination of nine inland species was also examined. The soil salinities in salt marshes ranged from 150 to 300 mmol/L NaCl, whereas those in fore and hind dunes ranged from 0 to 150 mmol/L NaCl, with a few exceptions. Cliff soils showed relatively high salinities up to 300 mmol/L NaCl. Ciff and foredune soils that encountered a typhoon and storm showed high salinities >300 mmol/L NaCl. Salt tolerance in seed germination of coastal plants was ordered by comparing the responses of percentage and rate of germination to salinity conditions up to 200 mmol/L NaCl, being in the order of salt marsh>cliff>foredune≅hind dune≅inland. Thse results indicate that salt tolerance in seed germination of coastal plants is closely related to the salinity conditions of their habitats. Germination experiments under favorable conditions showed that a high percentage of the seeds of salt marsh species germinate rapidly, those of diff species germinate slowly and those of foredune species exhibit a low percentage and low rate of germination. It seems that these germination characteristics contribute to the success of germination at the ‘safe site’ and the subsequent survivorship of emerged plants in their natural habitats.     

Regulated Expression of a Gene-Fusion Product Derived from the Gene for Phytochrome I from Pisum sativum and the uidA Gene from E. coli in Transgenic Petunia hybrida

The 5'-upstream region (2.4 kb) of the gene for phytochromeI from Pisum sativum (phyl) was fused to the uidA gene fromEscherichia coli that encodes ß-glucuronidase (GUS).The resulting PHY-GUS fusion was introduced into Petunia hybridaand was used as a reporter of the expression of the phyI genewhich was recognized by GUS activity. The PHY-GUS fusion wasexpressed at a relatively high level when transgenic plantswere grown in the dark, while leaves and stems of light-grownplants showed background activity. Flowers of light-grown plantswere shown to have significant levels of GUS activity but rootsdid not have such activity. When light-grown transgenic plantswere transferred to the dark, they expressed the activity atlevels that corresponded to those of dark-grown plants. Lighttreatment prior to growth in darkness revealed red/far-red reversibilityof recovery of the activity. Thus, the 2.4-kb fragment fromthe 5' region of the phyI gene carries the information necessaryfor the light-repressible autoregulation. (Received March 30, 1991; Accepted May 20, 1991)     

Detection of Polypeptides Involved in Early Stages of Nodulation in Soybean Roots

Soluble proteins extracted from the roots of nodulating soybean[Glycine max (L.) Merr. cv. T202] and from roots of the non-nodulatingisoline rj₁ of cv. T202 (cv. T201), which had been inoculatedwith Bradyrhizobium japonicum, were analyzed by two-dimensionalpolyacrylamide gel electrophoresis and silver staining, in anattempt to identify polypeptides involved in early stages ofnodulation. Almost identical patterns of polypeptides were generatedby extracts of 3-day-old roots of uninoculated T201 and T202and of inoculated T201 and T202, but a unique spot, correspondingto a polypeptide of 38 kDa was detected in the case of inoculatedroots of T202. Western blotting analysis using "inoculated-T202-rootspecific" antiserum, prepared by titration of antiserum againstproteins from inoculated T202 roots with proteins from inoculatedT201 roots, revealed spots corresponding to polypeptides of26,27, and 33 kDa that were detectable only in the extractsof roots of inoculated T202. However, no unique polypeptidespots were detected in the case of roots of inoculated T201and T202, as compared with those from uninoculated T201 andT202 roots by Western blotting analysis using "inoculated-T201-rootspecific" antiserum prepared by titration of antiserum againstproteins from inoculated T201 roots with proteins from uninoculatedT201 roots. (Received May 27, 1991; Accepted September 30, 1991)     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda