A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.     

Characterization of the ribosomal proteins from mosquito (Aedes albopictus) cells

Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae. albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila S6, however, Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins.     

Evidence of hydrolytic route for anaerobic cyanide degradation.

Products observed during anaerobic cyanide transformation are consistent with a hydrolytic pathway (HCN + H2O <--> HCONH2 + H2O <--> HCOOH + NH3). Formate, the most frequently observed product, was generally converted to bicarbonate. Formamide was rapidly hydrolyzed to formate upon exposure to the anaerobic consortium but was not detected as an intermediate of cyanide transformation.     

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.     

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells

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Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains

Abstract Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, β-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both Sfi I and Not I restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei .     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.