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.     

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.     

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.     

Evaluation of the safety and efficacy of testicular biopsies in llamas

Evaluation of the reproductive function of Lama glama is generally considered to be a challenging task due to the difficulty of obtaining representative semen samples. One method that has been proposed for evaluation of testicular function in these animals is histologic examination of testicular needle biopsies. This study was undertaken to examine the safety and efficacy of using needle biopsies to assess testicular function in this species. One randomly selected testicle from each of 16 sexually mature llamas was biopsied with a 14-gauge self-firing biopsy instrument. The llamas were evaluated over a 6-week period with thermography for temperature changes of the scrotum. At the end of the 6-week trial, the llamas were castrated and sections of each testis were fixed in Bouin's solution for histologic examination. Immediately prior to castration, an additional biopsy was taken from each testis to compare the tissue obtained via biopsy with sections from the corresponding testis obtained after castration. A qualitative grading scale was used to compare the seminiferous tubules from each testis. No difference was found between the biopsied and the nonbiopsied testes (P = 0.69). The percentage of normal tubules between the biopsied and the nonbiopsied sides also did not differ (P = 0.70). Furthermore, the percentage of normal seminiferous tubules did not differ between the needle biopsy samples and the corresponding tissue samples obtained at castration (P = 0.48). The number of round seminiferous tubules counted in each biopsy section ranged from 3 to 67. There was no significant difference in the thermographic images of the scrotum between the biopsied and the nonbiopsied testes. This study supports testicular biopsies as a safe and useful procedure in the evaluation of testicular function.     

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA₃)/ indole-3-butyric acid (IBA). BA (22.19 M) was effective for induction of multiple shoots and addition of GA₃ to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 M). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 M). However, this was associated with basal callus formation. Treatment with IBA (25–100 M) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 M for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 mol m^–2s^–1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 mol m^–2s^–1 in Q. leucotrichophora and around 1500 mol m^–2s^–1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 mol m^–2s^–1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.