Surface spreading of synaptonemal complexes in locusts

Details are given of techniques for preparing surface spreads of locust spermatocytes for light and electron microscopy. The pachytene synaptonemal complex (SC) karyotypes of Locusta migratoria and Schistocerca gregaria are analysed and compared. Up to six different SCs can be identified in Locusta migratoria based on lengths, centromere positions, and possession of nucleolar organiser regions, but only two SCs are identifiable in Schistocerca gregaria. The total SC length is significantly greater in Schistocerca gregaria than in Locusta migratoria, and this difference is almost exactly proportional to the difference in the genomic DNA contents of the two species.     

The structure of the basement membrane of human lymph node high endothelial venules: An ultrastructural, histochemical and immunocytochemical study

Summary The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase,-glucuronidase,-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.     

Kinetics of mineralization of phenols in lake water.

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semicontinuous and Continuous Production of Chloroperoxidase by Caldariomyces fumago Immobilized in k-Carrageenan

Three strains of Caldariomyces fumago were immobilized in 4% k-carrageenan and tested for semicontinuous production of chloroperoxidase (CPO). Over an 80-day period, growing in defined medium, C. fumago strains CMI 89362 and ATCC 11925 produced enzyme concentrations of 99 and 71 mg/liter, respectively, during six production periods of 12 to 14 days, while C. fumago DAOM 137632 produced only 24 mg of CPO per liter during six growth periods of 10 days. CPO production was unaffected by various regimens of washing between transfers. Mycelial growth was primarily restricted to the head surface, and bead size increased linearly with time. Attempts to restrict growth but maintain CPO production were unsuccessful. Pigment production, fructose utilization, and pH change in the immobilized cell cultures compared closely with the growth characteristics of free cell cultures. By using an airlift tower fermentor with an external loop run with continuous medium replacement of 20 ml/h (D = 0.016), strain CMI 89362 in bead form produced CPO at 40 mg/liter for 11 days.     

Method for Assessing Heterogeneity in Turnover Rates within Microbial Communities

A method is presented for determining both the average turnover rate and the standard deviation of the average turnover rate of the adenine nucleotide (AN) pool within a population of microorganisms. The method requires the calculation of the initial slope and curvature of a plot of AN specific activity versus time following the introduction of [³H]adenine. An analysis of noise-corrupted data indicated that the method is capable of detecting a lack of uniformity in the turnover rate when the coefficient of variation of the turnover rate exceeds 39%. An analysis of field data revealed a significant lack of uniformity in the turnover rates of microbial communities in a marine sediment sample and freshwater pond but no significant nonuniformity in the turnover rates of microbial communities in a seawater sample and in a second freshwater pond. Although the method has been applied only to the analysis of AN turnover rates, it is applicable to any intracellular pool for which a suitable radioactive precursor exists.     

Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants

We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus.     

Surface localization of sialic acid on Actinomyces viscosus

This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.     

Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.     

Influenza virus-specific antibody-secreting cells in the murine lung during primary influenza virus infection.

An enzyme-linked immunosorbent plaque assay is described which can reliably enumerate influenza virus-specific antibody-secreting cells and exhibits specificity similar to that of the indirect enzyme-linked immunosorbent assay. The assay was used to characterize the development of specific antibody-secreting cells, principally within lung tissue, during primary murine influenza virus infection after intranasal inoculation. Cells secreting influenza virus-specific immunoglobulin M (IgM), IgG, and IgA were detected in greatest numbers in lung tissue, and the data presented indicated that the cells may have originated from specific B-cell precursors in lung tissue which are demonstratable in vitro. At 11 months after infection, cells secreting IgG and IgA were still present in lung tissue. Influenza virus-specific antibody-secreting cells were also detected in spleen tissue and blood. Antibody-secreting cells appeared earlier in spleen than in lung tissue and declined more rapidly in spleen tissue.