Ecological significance of bacterial polyphosphate metabolism in sediments

The purpose of this study was to find theoretical evidence that bacteria, in particular those capable of polyphosphate (polyP) metabolism, are directly implicated in sediment phosphorus (P) dynamics and control P metabolism of freshwater ecosystems. The specific attributes and functional role of such bacteria were investigated on successive levels of ecological organization: individual microorganism, microbial community, freshwater ecosystem. The results of this systematic approach have been formulated as a number of hypotheses.

1. PolyP metabolism is the mechanism which enables individual polyP bacteria to survive and grow under the fluctuating redox conditions characteristic of their habitat at the sediment-water interface.
2. PolyP metabolism together with anaerobic Mn and/or Fe respiration is the mechanism that confers upon polyP bacteria the advantage required to fill a unique ecological niche within the microbial community to which they belong.
3. To the freshwater ecosystem as a whole bacterial polyP metabolism is a homeostatic mechanism which limits P availability and makes ecosystem productivity self-correcting as a function of oxygen availability. Bacterial polyP pools in the sediment are vital components of the P cycle. It was suggested that the impact of this bacterial mechanism should be tested with regard to the eutrophication issue.

     

Targeting of superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Phenotypic effects of overexpression of Agrobacterium rhizogenes T-DNA ORF13 in transgenic tobacco plants are mediated by diffusible factor(s)

Nicotiana tabacum cv. Xanthi transgenic plants expressing ORF13 of Agrobacterium rhizogenes 8196 T-DNA under the 35S RNA promoter from the cauliflower mosaic virus displayed developmental abnormalities. They were small, with short and variable internodal lengths, their root systems were poorly developed; leaves were small, asymmetric, rounded, wrinkled and dark green; flowers were short, and irregularly shaped. They exhibited reduced apical dominance and regularly produced offshoots at the base of the plant. This phenotype was also exhibited by offshoots of normal N. tabacum cv. Xanthi stock grafted with a transgenic scion indicating that expression of ORF13 influences plant development via diffusible factor(s).     

Sucrose consumption in mice: Major influence of two genetic Loci affecting peripheral sensory responses

Individual variability in sucrose consumption is prominent in humans and other species. To investigate the genetic contribution to this complex behavior, we conducted behavioral, electrophysiological, and genetic studies, using male progeny of two inbred mouse strains (C57BL/6ByJ [B6] and 129/J [129]) and their F₂ hybrids. Two loci on Chromosome (Chr) 4 were responsible for over 50% of the genetic variability in sucrose intake. These loci apparently modulated intake by altering peripheral neural responses to sucrose. One locus affected the response threshold, whereas the other affected the response magnitude. These findings suggest that the majority of difference in sucrose intake between male B6 and 129 mice is due to polymorphisms of two genes that influence receptor or peripheral nervous system activity. Received: 27 January 1997 / Accepted: 17 March 1997     

Propylthiouracil Tasting: Determination of Underlying Threshold Distributions using Maximum Likelihood

The ability to taste low concentrations of propylthiouracil(PROP) and related bitter compounds is heritable. The currentanalysis determines whether the distribution of PROP taste thresholdsis consistent with an additive or a dominant mode of Mendeliantransmission. To that end, the lowest concentration of PROPdetectable was determined for 1015 subjects and models of bi-or tri-modal distributions of PROP taste thresholds were tested.The model with the greatest likelihood had three distributionsand followed an additive model of PROP taste sensitivity ifthe variances associated with the distributions were assumedto be equal. However, if the taste thresholds were transformedto remove skewness, or if the variances were unequal, then three-or two-distribution models were equally likely. Resolution ofthe mode of inheritance for bitter taste perception awaits additionalfamily studies and the characterization of the molecular basisof taste perception for these bitter compounds. Chem. Senses20: 529–533, 1995.     

Immunotherapy of human colon cancer by antibody-targeted superantigens

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.     

Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K12

Summary Periplasmic-leaky (lky) Hfr mutant strains of Escherichia coli K12, grown in low-phosphate Tris medium, excreted alkaline phosphatase (AP) into the extracellular fluid. The lky207 mutation, which proved to induce the highest AP excretion rate, was transferred to an F^- host, carrying a phoS, T mutation allowing constitutive AP biosynthesis. Use of high-phosphate LB-rich medium for growing this F^- lky strain improved cell biomass, extracellular AP activity and excretion specificity in favour of the enzyme. Physiological studies helped us to develop a new culture medium (LB 8.3) giving higher enzyme and excretion yields. LB 8.3 medium also increased cell viability of lky mutants stored at 4° C.Using optimized culture conditions, the highest extracellular enzyme activity produced by lky mutant 706 was reached in the late stationary growth phase and was equal to 1,400 U/ml of culture medium (i.e., 6 times the intracellular AP content of wild-type strain, Ga15, developed in derepressed conditions); AP released into the extracellular fluid corresponded to 34% of total excreted proteins and was equivalent to a purified enzyme preparation.