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.     

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

One-stage repair of blepharophimosis

Congenital blepharophimosis is a congenital anomaly characterized by abnormalities in the area of the eyes, including bilateral ptosis, shortening of the horizontal fissure of the lid, expansion of the intercanthal distance, and epicanthus inversus. The condition is subject to autosomal-dominant heredity and is said to occur more frequently in Orientals than in Occidentals. Over the past 9 years, we have surgically treated 11 cases of congenital blepharophimosis using a procedure in which levator resection and medial canthoplasty are performed in one stage. It has been commonly believed that when levator resection and medial canthoplasty are performed at the same time, tension in the eyelid becomes too strong to achieve favorable results; therefore, the standard procedure has been to divide the operation into two stages. In all 11 cases we experienced, however, it was possible to obtain good results with a single-stage operation.     

Secretory pathways and processing of human proopiomelanocortin (POMC) using transformed mouse cultured fibroblasts (L cells) and AtT20 cells by human POMC gene--preembedding immunoelectron microscopic studies.

In order to elucidate the relationship between secretory pathway and processing for precursor molecule of peptide hormones, we performed immunoelectron microscopic studies to localize POMC-derived peptides in mouse cultured L cells (fibroblasts without secretory granules) and in mouse AtT20 cells (ACTH secreting pituitary tumor cells with secretory granules) which had been transformed with human POMC gene. From the electron microscopic localization patterns, L42 cells were considered to serve as a model of constitutive pathway without processing of POMC, and A53 cells were considered to serve as a model of transgranular (regulated) pathway with processing of POMC. Immunoblotting supported these interpretations.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional analysis of a polyoma-related mammalian plasmid (L factor): the enhancer activity and plasmid establishment.

L factor is a unique plasmid DNA which was originally discovered in a subclone (B822) of mouse L cells at a high copy number (more than 5,000 copies/cell). The presence of L factor caused no detectable abnormalities to the plasmid-bearing cells. We determined the total DNA sequence of the L factor I (and a part of L factor II) and compared it with that of polyoma DNA. Both DNA are common to the general construction of DNA frames such as early, late and noncoding regions, suggesting the two to be closely related. On the other hand, the L factor DNA sequences differ substantially from that of polyoma in the DNA sequences corresponding to the polyoma large T antigen, capsid proteins and a portion of the enhancer region. In order to investigate the mechanism of plasmid establishment of L factor, we compared the enhancer activity, capacity of DNA replication and efficiency of plasmid establishment of L factor with those of polyoma. The results indicate that L factor enhancer activity and DNA replication capacity were considerably lower than those of polyoma, suggesting that these altered (lowered) activities associated with L factor contribute to the plasmidal establishment and stable maintenance of L factor.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

The effect of ovarian steroidogenesis on ovulation and fertilizability in the in vitro perfused rabbit ovary

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.