Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

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.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

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 molecular orbital study of the metabolic pathway with hydroquinone intermediate from benzene as carcinogen

A possible metabolic activation pathway of benzenein vivo is the nonenzymatic oxidation of hydroquinone producedvia the cytochrome P-450-mediate two-step oxidation of benzene. The mechanism of the further oxidation of hydroquinone and the nature of the most reactive intermediate have not yet been clarified, although it is speculated that the intermediate isp-benzoquinone and/orp-benzosemiquinone. The theoretical result of using molecular orbital calculations (ab initio and CNDO/2 methods) indicates that although the mechanism of the nonenzymatic oxidation of hydroquinone cannot yet be determined, the intermediate is thep-benzosemiquinone anion radical. It is also suggested that active-oxygen species such as hydroxyl radical, which accelerates the nonenzymatic oxidation, play an important role in the metabolic pathway in question.     

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.