CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.     

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

Highly sensitive second antibody format enzymeimmunoassay for determination of 13,14-dihydro-15-keto-PGF2alpha in blood plasma of mithun (Bos frontalis)

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in blood plasma of mithun (Bos frontalis; bovine) on microtitreplates using second antibody coating technique and PGFM-horseradish peroxidase as a label has been developed. The wells of the microtitreplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20microl plasma. The PGFM standard curve, with doses ranging from 0.1 to 50pg/well was linear. The sensitivity of the assay was 5pg/ml. PGFM standard curve in buffer showed parallelism with serially diluted mithun plasma containing high endogenous PGFM. Plasma PGFM concentrations estimated by using the developed EIA and commercially available PGFM EIA kit in the same samples were significantly correlated (r=0.98) and showed linearity. Intra- and inter-assay coefficients of variation were below 7%. Recovery of known concentrations of added PGFM in charcoal stripped plasma was linear (r=0.99). The developed EIA was further validated biologically by estimating PGFM in cyclic cows for the entire estrous cycle and in peri-parturient cows beginning day 7 prior to calving till day 30 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of PGFM directly in bovine plasma.     

Biomarkers in cancer screening, research and detection: present and future: a review

Biomarkers provide a powerful and dynamic approach to understanding the spectrum of malignancies with applications in observational and analytic epidemiology, randomized clinical trials, screening, diagnosis and prognosis. Defined as alterations in the constituents of tissues or body fluids, these markers offer a means for homogeneous classification of a disease and risk factor, and they can extend one's basic information about the underlying pathogenesis of disease. The goals in cancer research include finding biomarkers that can be used for the early detection of cancers, design individual therapies, and to identify underlying processes involved in the disease. Because so many myriad processes are involved in the diseased states, the goal is similar to 'finding a needle in a haystack'. However, the development of many -omic technologies, such as genomics and proteomics, has allowed us to monitor a large number of key cellular pathways simultaneously. This has enabled the identification of biomarkers and signalling molecules associated with cell growth, cell death and cellular metabolism. These are also facilitating in monitoring the functional disturbance, molecular and cellular damage, and damage response. This brief review describes the development of biomarkers in cancer research and detection with emphasis on different proteomic tools for the identification and discovery of new biomarkers, different clinical assays to detect various biomarkers in different specimens, role of biomarkers in cancer screening and last but not the least, the challenges in this direction of cancer research.     

Synthesis and DNA-binding ability of pyrrolo[2,1-c][1,4]benzodiazepine-azepane conjugates

A series of pyrrolobenzodiazepine-azepane conjugates linked through different alkane spacers have been prepared and their DNA thermal denaturation studies have been carried out. One of the compound (4b), elevates the DNA helix melting temperature of the CT-DNA by 2.0 degrees C after incubation for 36 h at 37 degrees C.     

DNA binding potential and cytotoxicity of newly designed pyrrolobenzodiazepine dimers linked through a piperazine side-armed-alkane spacer

New pyrrolobenzodiazepine (PBD) dimers have been developed that are composed of two DC-81 subunits tethered to their C8 positions through piperazine moiety side-armed with alkaneoxy linkers (composed of 2-5 carbons). DNA thermal denaturation studies show that after 18h of incubation with calf thymus DNA at a 1:5 ligand/DNA ratio, one of them, 6a, increases the DeltaT(m) value by 24.0 degrees C. Thus, incorporation of a piperazine moiety instead of an inert alkanedioxy linker alone significantly enhances the DNA binding ability, and the analogous dimer 4 that lacks a piperazine moiety in the linker spacer elevates melting by only 15.1 degrees C under identical experimental conditions. This illustrates the effect of introducing a piperazine ring in the middle of such an alkanedioxy linker which produces several hydrophobic interactions and could also achieve a superior isohelical fit within the DNA minor groove. Interestingly, these dimers 6a-d are significantly more cytotoxic than 4 in a number of human cancer cell lines, in particular, compound 6c is highly potent for almost all the nine human cancer cell lines.     

Synthesis and evaluation of alpha-hydroxymethylated conjugated nitroalkenes for their anticancer activity: inhibition of cell proliferation by targeting microtubules

The Morita-Baylis-Hillman (MBH) type reaction of a variety of aromatic and heteroaromatic conjugated nitroalkenes with formaldehyde in the presence of stoichiometric amounts of imidazole and catalytic amounts (10 mol %) of anthranilic acid at room temperature provided the corresponding hydroxymethylated derivatives in moderate to good yield. The parent nitroalkenes and their MBH adducts were subsequently screened for their anticancer activity. Some of the MBH adducts were found to inhibit cervical cancer (HeLa) cell proliferation at low micromolar concentrations with half-maximal inhibitory concentrations in the range of 1-2 microM. The antiproliferative activity of 3-((E)-2-nitrovinyl)furan and three potent MBH adducts, namely, hydroxymethylated derivatives of 3-((E)-2-nitrovinyl)thiophene, 1-methoxy-4-((E)-2-nitrovinyl)benzene, and 1,2-dimethoxy-4-((E)-2-nitrovinyl)benzene was correlated well with their antimicrotubule activity. At their effective concentration range, the tested compounds perturbed the organization of mitotic spindle microtubules and chromosomes. In the presence of hydroxymethylated nitroalkenes, abnormal bipolar or multipolar mitotic spindles were apparent. Interphase microtubules were found to be significantly depolymerized at relatively higher concentrations of the tested compounds. These compounds inhibited tubulin assembly into microtubules in vitro by binding to tubulin at a site distinct from the vinblastine and colchicine binding sites. The compounds reduced the intrinsic tryptophan fluorescence of tubulin and the fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid (ANS) complex indicating that they induced conformational changes in the tubulin. The results suggest that hydroxymethylated nitroalkenes exert their antiproliferative activity at least in part by depolymerizing cellular microtubules through tubulin binding and indicate that hydroxymethylated nitroalkenes are promising lead compounds for cancer therapy.     

Solvent free microwave synthesis and evaluation of antimicrobial activity of pyrimido[4,5-b]- and pyrazolo[3,4-b]quinolines

A series of 2-oxopyrimido[4,5-b]-, 2-thio[4,5-b]-, 1-(p-tosyl)pyrazolo[3,4-b]- and 1-(2',4'-dinitrophenyl)pyrazolo[3,4-b]-quinolines have been synthesized in good to excellent yields by environmentally benign solvent free microwave-induced techniques involving the condensation of 2-chloro-3-formylquinolines with urea, thiourea, p-toluenesulfonylhydrazide and 2,4-dinitrophenylhydrazine, respectively, using PTSA as a catalyst. All the synthesized compounds were evaluated for their antibacterial and antifungal activities. Most of the compounds showed the best activity against Escherichia coli and Pseudomonas aeruginosa.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.