Isolation and Characterization of <Emphasis Type="Italic">Citrobacter</Emphasis> Strain HPC255 for Broad-Range Substrate Specificity for Chlorophenols

This study aims at development of an approach for selection of strain, which has capability for oxidation of broad-range of chloro-substitute phenols. A multiplex PCR was optimized targeting loci involved in phenol and chlorophenol degradation, which was used to select activated sludge samples and also to assess the degradative genotype of isolates. The isolated strains were screened on the basis of RAPD analysis. In parallel, physiological experiments were carried out with activated sludge samples and isolated bacteria by respirometric analysis. Based on cluster analysis of RAPD pattern and respirometric data, the isolate G20 was selected and identified by using 16S rDNA sequence analysis as Citrobacter freundii strain HPC255. The strain could oxidize different substituted chlorophenol molecules. Such strains could provide the pool of intermediates, which can further be degraded by the associated population, thus helping in maintaining the synergistic association of catabolic activity in activated sludge.     

Discrimination models using variance-stabilizing transformation of metabolomic NMR data

After the extensive work that is being done in the areas of genomics, proteomics, and metabolomics, the study of metabolites has come of interest in its own right. Metabolites in biological systems give an understanding of the state of the system and provide a powerful tool for the study of disease and other maladies. Several analytical techniques such as mass spectrometry and high-resolution NMR spectroscopy have been used to study metabolites. The data, however, from these techniques remains quite complex. Traditionally, multivariate analyses have been used for such data. These methods however have an underlying assumption that the data is multivariate normal with a constant variance. This is not necessarily the case. It has been shown that a generalized log transformation renders the variance of the data constant effectively making the data more suitable for multivariate analysis. We demonstrate the effectiveness of these transformations on NMR data taken on a set of 18 abalone that were categorized as either being healthy, stunted, or diseased. We show how the transformation makes multivariate classification of the abalone into the healthy, stunted and diseased categories much more effective and gives a tool for identifying potential metabolic biomarkers for disease.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

Novel 18beta-glycyrrhetinic acid analogues as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenases

Extensive structural modifications to the 18beta-glycyrrhetinic acid template are described and their effects on the SAR of the 11beta-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. Isoform selective inhibitors have been discovered and compound 7 N-(2-hydroxyethyl)-3beta-hydroxy-11-oxo-18beta-olean-12-en-30-oic acid amide is highlighted as a very potent selective inhibitor of 11beta-hydroxysteroid dehydrogenase 2 with an IC(50) = 4pM.     

Arylguanidine and arylbiguanide binding at 5-HT3 serotonin receptors: a QSAR study

For a series of monosubstituted arylguanidines, 5-HT3 receptor affinity was found generally related to the electron withdrawing nature of the substituent at the aryl 3-position and the lipophilicity of the 4-position substituent. A broader examination of 35 arylguanidines and arylbiguanides revealed that affinity could be described by molecular polarizability, a Chi index term (8chiP), and the sum of all (-Cl) E-State values (SsCl) in the molecule.     

Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment)

We have investigated conformational transitions in the Klenow fragment polymerase reaction by stopped-flow fluorescence using DNA substrates containing the fluorescent reporter 2-aminopurine (2-AP) on the template strand, either at the templating position opposite the incoming nucleotide (designated the 0 position) or 5' to the templating base (the +1 position). By using both deoxy- and dideoxy-terminated primers, we were able to distinguish steps that accompany ternary complex formation from those that occur during nucleotide incorporation. The fluorescence changes revealed two extremely rapid steps that occur early in the pathway for correct nucleotide incorporation. The first, detectable with the 2-AP reporter at the 0 position, occurs within the first few milliseconds and is associated with dNTP binding. This is followed by a rapid step involving relative movement of the +1 base, detectable when the 2-AP reporter is at the +1 position. Finally, when the primer had a 3'-OH, a fluorescence decrease with a rate equal to the rate of nucleotide incorporation was observed with both 0 and +1 position reporters. When the primer was dideoxy-terminated, the only change observed at the rate expected for nucleotide incorporation had a very small amplitude, suggesting that the rate-limiting conformational change does not produce a large fluorescence change, and is therefore unlikely to involve a significant change in the environment of the fluorophore. Fluorescence changes observed during misincorporation were substantially different from those observed during correct nucleotide incorporation, implying that the conformations adopted during correct and incorrect nucleotide incorporation are distinct.     

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts

The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1-DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal D-amino acid (instead of L form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1-DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner.     

Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel

The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.     

Regulation of estrogen synthesis in postmenopausal women

The decrease in ovarian estrogen production that occurs at the menopause may lead to an increase in peripheral aromatase activity. While estrogens can have beneficial effects on some body tissues, such as bone and the cardiovascular system, they also have a crucial role in supporting the growth and development of breast tumors. A number of factors, including interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and prostaglandin E(2) (PGE(2)), which can stimulate aromatase activity, have now been identified. As plasma concentrations of some cytokines increase at the menopause, this may account for the increased peripheral aromatase activity that is detected in older women. Macrophages and lymphocytes which infiltrate breast tissue are now thought to be an important source of cytokines that can stimulate aromatase activity in this tissue. Studies, we have recently carried out, have suggested that the endogenous estrogen metabolite, 2-methoxy-estradiol, may be able to modulate the ability of cytokines and PGE(2) to stimulate aromatase activity. Understanding the role of endogenous estrogen metabolites in regulating estrogen synthesis may give rise to new strategies for the prevention or treatment of breast cancer.