The role of glutathione in cancer

Glutathione is an abundant natural tripeptide found within almost all cells. Glutathione is highly reactive and is often found conjugated to other molecules via its sulfhydryl moiety. It instils several vital roles within a cell including antioxidation, maintenance of the redox state, modulation of the immune response and detoxification of xenobiotics. With respect to cancer, glutathione metabolism is able to play both protective and pathogenic roles. It is crucial in the removal and detoxification of carcinogens, and alterations in this pathway, can have a profound effect on cell survival. However, by conferring resistance to a number of chemotherapeutic drugs, elevated levels of glutathione in tumour cells are able to protect such cells in bone marrow, breast, colon, larynx and lung cancers. Here we present a number of studies investigating the role of glutathione in promoting cancer, impeding chemotherapy, and the use of glutathione modulation to enhance anti-neoplastic therapy.     

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)_n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)_n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.     

Fractal dimension in endometrial carcinoma

OBJECTIVE: To mathematically assess in a pilot study, endometrial glandular margin irregularity in simple hyperplasia, complex atypical hyperplasia and well-differentiated endometrial carcinoma with the help of box counting of fractal dimension and to discriminate these lesions on the basis of box counting of fractal dimension of the gland. STUDY DESIGN: Ten cases each of endometrial simple hyperplasia (without atypia), complex hyperplasia with atypia and endometrial carcinoma (well-differentiated, endometrioid) were assessed in the study. Five fields at 20 x magnification from each case were randomly selected, and the glands were outlined with the help of a pointer. Using the box counting method, the fractal dimension of each case was measured. RESULTS: Mean fractal dimension in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma was, 0.899 +/- 0.13, 0.932 +/- 0.042 and 0.939 +/- 0.02, respectively. Statistical analysis showed that the fractal dimension of glands of simple hyperplasia were significantly different from that of complex atypical hyperplasia and endometrial carcinoma (P = .041 and .013, respectively, ANOVA). However, there was no significant difference in fractal dimension between glands of complex hyperplasia and of endometrial carcinoma (P = .659, ANOVA). CONCLUSION: This study provides mathematical (objective) assessment of the measurement of glandular margin irregularities in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma. Fractal dimension of gland margin may have diagnostic potential in the future.     

Genetic admixture of European FRDA genes is the cause of Friedreich ataxia in the Mexican population

Friedreich ataxia accounts for approximately 75% of European recessive ataxia patients. Approximately 98% of pathogenic chromosomes have large expansions of a GAA triplet repeat in the FRDA gene (E alleles), and strong linkage disequilibrium among polymorphisms spanning the FRDA locus indicates a common origin for all European E alleles. In contrast, we found that only 14 of 151 (9.3%) Mexican Mestizo patients with recessive ataxia were homozygous for E alleles. Analysis of polymorphisms spanning the FRDA locus revealed that all Mestizo E alleles had the common European haplotype, indicating that they share a single origin. Genetic admixture levels were determined, which revealed that the relative contributions to the Mestizo FRDA gene pool by Native American and European genes were 76-87% and 13-24%, respectively, commensurate with the observed low prevalence of Friedreich ataxia in Mestizos. This indicates that Friedreich ataxia in Mexican Mestizos is due to genetic admixture of European mutant FRDA genes in the Native American gene pool that existed prior to contact with Europeans.     

Molecular activity of 1,25-dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] exerts anti-proliferative, differentiating and apoptotic effects on prostatic cells. These activities, in addition to epidemiologic findings that link Vitamin D to prostate cancer risk, support the use of 1,25(OH)(2)D(3) for prevention or therapy of prostate cancer. The molecular mechanisms by which 1,25(OH)(2)D(3) exerts antitumor effects on prostatic cells are not well-defined. In addition, there is heterogeneity among the responses of various prostate cell lines and primary cultures to 1,25(OH)(2)D(3) with regard to growth inhibition, differentiation and apoptosis. To understand the basis of these differential responses and to develop a better model of Vitamin D action in the prostate, we performed cDNA microarray analyses of primary cultures of normal and malignant human prostatic epithelial cells, treated with 50 nM of 1,25(OH)(2)D(3) for 6 and 24 h. CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) was the most highly upregulated gene. Significant and early upregulation of dual specificity phosphatase 10 (DUSP10), validated in five additional primary cultures, points to inhibition of members of the mitogen-activated protein kinase (MAPK) superfamily as a key event mediating activity of 1,25(OH)(2)D(3) in prostatic epithelial cells. The functions of other regulated genes suggest protection by 1,25(OH)(2)D(3) from oxidative stress. Overall, these results provide new insights into the molecular basis of antitumor activities of Vitamin D in prostate cells.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.     

Two-stage optimization process for formulation of chitosan microspheres

The objective of the present study was to optimize the concentration of a chitosan solution, stirring speed, and concentration of drugs having different aqueous solubility for the formulation of chitosan microspheres. Chitosan microspheres (unloaded and drug loaded) were prepared by the chemical denaturation method and were subjected to measurement of morphology, mean particle size, particle size distribution, percentage drug entrapment (PDE), drug loading, and drug release (in vitro). Morphology of the microspheres was dependent on the level of independent process parameters. While mean particle size of unloaded microspheres was found to undergo significant change with each increase in concentration of chitosan solution, the stirring rate was found to have a significant effect only at the lower level (ie, 2000 to 3000 rpm). Of importance, spherical unloaded microspheres were also obtained with a chitosan solution of concentration less than 1 mg/mL. Segregated unloaded microspheres with particle size in the range of 7 to 15 microm and mean particle size of 12.68 microm were obtained in the batch prepared by using a chitosan solution of 2 mg/mL concentration and stirring speed of 3000 rpm. The highest drug load ( microg drug/mg microspheres) was 50.63 and 13.84 for microspheres containing 5-fluorouracil and methotrexate, respectively. While the release of 5-fluorouracil followed Higuchi's square-root model, methotrexate released more slowly with a combination of first-order kinetics and Higuchi's square-root model. The formation of chitosan microspheres is helped by the use of differential stirring. While an increase in the concentration of water-soluble drug may help to increase PDE and drug load over a large concentration range, the effect is limited in case of water-insoluble drugs.     

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

LOC3D: annotate sub-cellular localization for protein structures

LOC3D (http://cubic.bioc.columbia.edu/db/LOC3d/) is both a weekly-updated database and a web server for predictions of sub-cellular localization for eukaryotic proteins of known three-dimensional (3D) structure. Localization is predicted using four different methods: (i) PredictNLS, prediction of nuclear proteins through nuclear localization signals; (ii) LOChom, inferring localization through sequence homology; (iii) LOCkey, inferring localization through automatic text analysis of SWISS-PROT keywords; and (iv) LOC3Dini, ab initio prediction through a system of neural networks and vector support machines. The final prediction is based on the method that predicts localization with the highest confidence. The LOC3D database currently contains predictions for >8700 eukaryotic protein chains taken from the Protein Data Bank (PDB). The web server can be used to predict sub-cellular localization for proteins for which only a predicted structure is available from threading servers. This makes the resource of particular interest to structural genomics initiatives.