Competition of cyclooctenes and cyclooctadienes for ethylene binding and activity in plants

trans-Cyclooctene, cis,trans-1,5-cyclooctadiene, and cis,trans-1,3-cyclooctadiene have been compared with the cis and cis,cis isomers and with 2,5-norbornadiene for competition with ethylene for binding in mung bean sprouts and tobacco and for action (induction of chlorophyll degradation) in banana. The compounds containing a trans double bond were much more effective in competition for binding and action than the cis and cis,cis compounds. trans-Cyclooctene and cis,trans-1,3-cyclooctadiene were in the general range of 50–90 times more effective than 2,5-norbornadiene.R.J. Reynolds Research Apprentice     

Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size,and for reductions in membrane surface area and immature granule volume during granule maturation

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.     

Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Exotoxin production by Aeromonas spp. in foods

A psychrotrophic toxin-producing strain of Aeromonas hydrophila grew well in a range of food slurries (scallop, prawn, fish, chicken liver paté, liverwurst, chicken luncheon slice and commercial baby food preparations) held at refrigeration temperatures. In most foods, excluding the baby food preparations, exotoxins were produced at levels comparable with production in bacteriological broth without apparent food spoilage (all but prawn and fish). Addition of ultra-heat treated (UHT) milk to toxin-containing broth culture supernatants markedly decreased or removed haemolytic and cytotoxic activities, explaining low levels of toxins found in milk in a previous study. Baby food preparations did not inactivate exotoxins under similar conditions suggesting production of toxins rather than their inactivation was inhibited in these foods.     

Seasonal dynamics of the association between sweet potato and vesicular-arbuscular mycorrhizal fungi

To better understand the behavior of selected vesicular-arbuscular mycorrhizal (VAM) isolates in the field, we documented the growth of roots, root hairs, and VAM colonization of inoculated and noninoculated sweet potato plants (Ipomea batatas (L.) Lam. cv White Star) over a growing season. We also determined the seasonal dynamics of P and Zn uptake, and shoot and storage-root growth. Shoot cuttings were inoculated with an isolate of either Glomus etunicatum Becker and Gerdemann or Acaulospora rugosa Mortan, or were not inoculated, and were harvested 2, 4, 8, 13, 20, and 27 weeks after planting (WAP). At each harvest, roots were sampled at 0 to 30, 30 to 60, and 60 to 90 cm depths and at 0, 23, 83, and 116 cm from the base of the shoot. At the end of the study, the roots of three non-inoculated plants were sampled by soil horizon. Inoculation had no affect on shoot growth or total shoot uptake of P and Zn; shoot dry mass and P and Z content increased rapidly up to 20 WAP, while shoot length continued to increase through 27 WAP. Shoot-P concentration of plants inoculated with A. rugosa at 2 and 8 WAP were higher than the noninoculated plants, while shoot-Zn concentration was not affected by inoculation. Storage-root yields of inoculated plants were higher than yields for noninoculated plants. Root length density, and percentage of root length with root hairs and VAM colonization were highest and most dynamic near the base of the plant. Percentage of root length colonization by VAM fungi was highest in the E2 horizon, intermediate in the Bh horizon, and lowest in the Ap horizon. Percentage of root length with root hairs had the opposite pattern. Intensive measurements of root characteristics close to the base of the plant, and shoot P-content and concentration during the period of rapid yield production, provided the most useful data for evaluating the activity of effective isolates.Published as Florida Agricultural Experimental Station Journal Series No. R-02576     

Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H₂, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39–42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.     

Vph6 Mutants of Saccharomyces Cerevisiae Require Calcineurin for Growth and Are Defective in Vacuolar H(+)-Atpase Assembly

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.