Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.     

Gene regulatory network from microarray data of colon cancer patients using TSK-type recurrent neural fuzzy network

In this work we applied a TSK-type recurrent neural fuzzy approach to extract regulatory relationship among genes and reconstruct gene regulatory network from microarray data. The identified signature has captured the regulatory relationship among 27 differentially expressed genes from microarray dataset. We applied three different methods viz., feed forward neural fuzzy, modified genetic algorithm and recurrent neural fuzzy, on the same data set for the inference of GRNs and the results obtained are almost comparable. In all tested cases, TRNFN identified more biologically meaningful relations. We found that 87.8% of the total interactions extracted by TRNFN are correct in accordance with the biological knowledge. Our analysis resulted in 2 major outcomes. First, upregulated genes are regulated by more genes than downregulated genes. Second, tumor activators activate other tumor activators and suppress tumor suppressers strongly in the disease environment. These findings will help to elucidate the common molecular mechanism of colon cancer, and provide new insights into cancer diagnostics, prognostics and therapy.     

Presence of erythromycin and tetracycline resistance genes in lactic acid bacteria from fermented foods of Indian origin

Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88 % harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.     

Inhibition of SIRT1 by HIV-1 viral protein Tat results in activation of p53 pathway

Human immunodeficiency virus-1 (HIV-1) disease is characterized by a relentless decline in CD4(+) T cells, resulting in the development of AIDS. Extracellular Tat secreted from the HIV-1 infected cells, enters non-infected T cells to induce apoptosis. A number of mechanisms, none of which is mutually exclusive, have been attributed to the cell depletion property of Tat protein. In the present communication, we provide evidence that the cell-killing effect of Tat is mediated by the activation of p53 pathway via inhibition of SIRT1, an NAD(+)-dependent deacetylase belonging to class III histone deacetylases. This evidence is based on the following experimental facts reported herein: (1) Overexpression of Tat protein decreases both the deacetylase and promoter activity of SIRT1, (2) SIRT1 inhibition by Tat involves increased levels of acetylated p53 and (3) The activation of p53 leads to subsequent increases in the expression of p53 target genes, p21 and BAX.     

Nanosphere lithography-based platform for developing rapid and high sensitivity microarray systems

A novel gold nanoarray (NA)-based platform was developed for microarray applications. This novel approach is based upon the principle of nanosphere lithography and can be used for one-step antibody immobilization. The developed platform was checked by functionalizing with cysteine followed by capturing biotinylated antibody and detecting it with dye-conjugated steptravidin. An immunoassay was performed with spiked samples containing human fetuin A antigen. The minimum limits of detection (LOD) of human fetuin A for NA-based and conventional microarray platforms were 50 pg/mL and 50 ng/mL, respectively. The developed approach was highly reproducible and unlike conventional microarray approaches the use of a spotting system was omitted because immobilization was controlled and directed on the predefined arrays. This approach could be an ideal alternative for developing microarrays. And, the ease of the strategy also allows the high throughput production of the microarrays.     

NAMPT pathway is involved in the FOXO3a-mediated regulation of GADD45A expression

Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD(+). Despite the pivotal role of NAD(+) in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD(+) level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD(+) levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD(+)-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.     

Age, photoperiod and estrogen dependent variations in the shell gland and the expression of AVT in the ovary of Japanese quail

Present work was undertaken to describe (i) age dependent (prepuberal-3, 4, 5 and 6 weeks old, puberal and actively laying 8 and 12 weeks old and aged 78 weeks old) (ii) photoperiodic response dependent (photosensitive and photorefractory) and sex steroid dependent (estradiol benzoate and its antagonist tamoxifen treated) variation in the ovary and shell gland activity of Japanese quail (Coturnix coturnix japonica). Further, in view of the role of neurohypophysial peptide arginine vasotocin (AVT) in many physiological processes including age/reproduction related oviposition, expression of ir-AVT was also monitored in the ovary of quail. All the parameters associated with histodifferentiation increased rapidly during the developing stages followed by a decrease in old age, which also increased in reproductively quiescent photorefractory birds following estradiol treatment and decreased in reproductively active photosensitive quail following tamoxifen treatment. Using AVT-specific antibody, expression of immunoreactive AVT (ir-AVT) observed in the ovary of photosensitive quail was not detected in the photorefractory quail. However, administration of estrogen in the photorefractory quail stimulated the growth and activity of ovary and shell gland also resulted in the expression of ovarian ir-AVT. On the other hand, tamoxifen eliminated the localization of ir-AVT in the ovary of photosensitive quail in addition to a decrease in the shell gland protein and alkaline phosphatase activity. It is concluded that estrogen not only affects the growth and differentiation of ovary and oviduct including shell gland but also regulates the expression of ovarian AVT. It is also suggested that in addition to reported paracrine effect of AVT in the shell gland of Japanese quail for oviposition, ovarian AVT may also affect ovarian function (ovulation), and in part, this regulation is estrogen dependent.     

Anti-neoplastic action of aspirin against a T-cell lymphoma involves an alteration in the tumour microenvironment and regulation of tumour cell survival

The present study explores the potential of the anti-neoplastic action of aspirin in a transplantable murine tumour model of a spontaneously originated T-cell lymphoma designated as Dalton's lymphoma. The antitumour action of aspirin administered to tumour-bearing mice through oral and/or intraperitoneal (intratumoral) routes was measured via estimation of survival of tumour-bearing mice, tumour cell viability, tumour progression and changes in the tumour microenvironment. Intratumour administration of aspirin examined to assess its therapeutic potential resulted in retardation of tumour progression in tumour-bearing mice. Oral administration of aspirin to mice as a prophylactic measure prior to tumour transplantation further primed the anti-neoplastic action of aspirin administered at the tumour site. The anti-neoplastic action of aspirin was associated with a decline in tumour cell survival, augmented induction of apoptosis and nuclear shrinkage. Tumour cells of aspirin-treated mice were found arrested in G0/G1 phase of the cell cycle and showed nuclear localization of cyclin B1. Intratumoral administration of aspirin was accompanied by alterations in the biophysical, biochemical and immunological composition of the tumour microenvironment with respect to pH, level of dissolved O2, glucose, lactate, nitric oxide, IFNγ (interferon γ), IL-4 (interleukin-4), IL-6 and IL-10, whereas the TGF-β (tumour growth factor-β) level was unaltered. Tumour cells obtained from aspirin-treated tumour-bearing mice demonstrated an altered expression of pH regulators monocarboxylate transporter-1 and V-ATPase along with alteration in the level of cell survival regulatory molecules such as survivin, vascular endothelial growth factor, heat-shock protein 70, glucose transporter-1, SOCS-5 (suppressor of cytokine signalling-5), HIF-1α (hypoxia-inducible factor-1α) and PUMA (p53 up-regulated modulator of apoptosis). The study demonstrates a possible indirect involvement of the tumour microenvironment in addition to a direct but limited anti-neoplastic action of aspirin in the retardation of tumour growth.     

Evaluation of Interferon Response Induced by Anti-Myostatin shRNA Constructs in Goat (Capra hircus) Fetal Fibroblasts by Quantitative Real TIME-Polymerase Chain Reaction

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p?相似文献