Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Multiattribute evaluation of regional cotton variety trials

Summary The Australian Cotton Cultivar Trials (ACCT) are designed to investigate various cotton [Gossypium hirsutum (L.)] lines in several locations in New South Wales and Queensland each year. If these lines are to be assessed by the simultaneous use of yield and lint quality data, then a multivariate technique applicable to three-way data is desirable. Two such techniques, the mixture maximum likelihood method of clustering and three-mode principal component analysis, are described and used to analyze these data. Applied together, the methods enhance each other's usefulness in interpreting the information on the line response patterns across the locations. The methods provide a good integration of the responses across environments of the entries for the different attributes in the trials. For instance, using yield as the sole criterion, the excellence of the namcala and coker group for quality is overlooked. The analyses point to a decision in favor of either high yields of moderate to good quality lint or moderate yield but superior lint quality. The decisions indicated by the methods confirmed the selections made by the plant breeders. The procedures provide a less subjective, relatively easy to apply and interpret analytical method of describing the patterns of performance and associations in complex multiattribute and multilocation trials. This should lead to more efficient selection among lines in such trials.     

Mutagenesis of the NH2-terminal domain of elongation factor Tu

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.     

Environmental actions of agrochemicals 1. Side-effects of the herbicide 3-amino-1,2,4-triazole on a laboratory acarine/host-plant interaction (Tetranychus urticae/Phaseolus vulgaris) as revealed by electron microscopy

Foliar and soil application in concentrations below the recommended rate of the herbicide 3-amino-1,2,4-triazole to the host plantPhaseolus vulgaris L. results in structural alterations of the protein-synthesizing apparatus of midgut and salivary-gland cells of the phytophagous spider miteTetranychus urticae Koch (Acari: Tetranychidae) independent of its mode of application. With prolonged incubation times cytological defects become more intense, and spread to more cells and tissues. Resultant effects on yolk and egg formation were expressed as an inhibition of egg deposition that led to a decrease in the reproduction rate ofT. urticae.Consequences of 3-amino-1,2,4-triazole action onT. urticae are discussed with regard to its value to a host-plant/parasite model, agricultural practices and environmental impacts.     

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Breeding and post-breeding structure of the vas deferens of the long-toed salamander Ambystoma macrodactylum

The vas deferens of Ambystoma macrodactylum is composed of a peritoneal epithelium, connective tissue layer with fibroblasts, circular smooth muscle, capillaries, cells containing lipid, and a luminal epithelium composed of a single layer of cuboidal cells covered by a net of interconnected ciliated squamous cells. The cuboidal cells have abundant rough endoplasmic reticulum, mitochondria, and PAS + secretory vesicles. Squamous cells of breeding males consistently have tufts of ～100 cilia located at one end of the long axis of each cell. These cilia may help distribute secretory products. The squamous cells, absent in post-breeding males, are apparently sloughed into the lumen. Lipid vesicles are present throughout the cytoplasm of the cuboidal and squamous epithelial cells and are also in some cells of the connective tissue layer. These vesicles increase dramatically in number during the first 4 weeks after breeding and may serve as an energy pool for the next breeding season. Enzyme-histochemical tests for testosterone synthesis were negative. In addition to the accumulation of lipid and the loss of squamous cells in the vas deferens, after breeding PAS + vesicle production is terminated. These alterations appear to represent energy conservation strategies employed by the sperm-depleted vas deferens.