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.     

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.     

Detection of fimbriae and fimbrial antigens on the oral anaerobe Bacteroides gingivalis by negative staining and serological methods

We screened 63 clinical isolates of Bacteroides gingivalis from eight different laboratories for the presence of fimbriae by negative staining and by immunological methods. Techniques used were bacterial agglutination, Ouchterlony immunodiffusion and Western immunoblotting analysis using rabbit anti-fimbriae and anti-fimbrilin sera raised against fimbriae and fimbrilin (a constituent protein of B. gingivalis fimbriae) from B. gingivalis strain 381. In 49 of the 51 strains tested, fimbriae were clearly detected by negative staining, and 30 (60%) of the fimbriate strains were positive in all three of the immunological assays. A total of 37 strains (75%) were positive by immunoblotting analysis, which was the most reliable of the serological methods used in this study. The study shows that the majority of B. gingivalis strains are fimbriate, and that these fimbriae are immunologically related to the fimbriae of B. gingivalis strain 381. Molecular heterogeneity of fimbrilin was discovered by the immunoblotting analysis, when different strains were compared. With most of the strains, including strain 381, the antifimbrilin serum reacted with a protein of apparent molecular mass 43 kDa, but with 15 strains the immuno-reactive protein had an apparent molecular mass of 46 kDa.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Activity of chimeric RNAs of U6 snRNA and (-)sTRSV in the cleavage of a substrate RNA.

U6 small nuclear RNA is one of the spliceosomal RNAs essential for pre-mRNA splicing. Discovery of mRNA-type introns in the highly conserved region of the U6 snRNA genes led to the hypothesis that U6 snRNA functions as a catalytic element during pre-mRNA splicing. The highly conserved region of U6 snRNA has a structural similarity with the catalytic domain of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], suggesting that the highly conserved region of U6 snRNA forms the catalytic center. We examined whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic RNA of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and have a GU sequence. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence, which is shared by the 5' end of an intron in a pre-mRNA. We found that the highly conserved region of U6 snRNA and the catalytic domain of (-)sTRSV are strikingly similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results suggest that U6 snRNA, (-)sTRSV and the group I self-splicing intron originated from a common ancestral RNA, and support the hypothesis that U6 snRNA catalyzes pre-mRNA splicing reaction.     

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)     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.