Possible involvement of queuine in regulation of cell proliferation

An increase in cell number is one of the most prominent characteristics of cancer cells. This may be caused by an increase in cell proliferation or decrease in cell death. Queuine is one of the modified base which is found at first anticodon position of specific tRNAs. It is ubiquitously present throughout the living system except mycoplasma and yeast. The tRNAs of Q-family are completely modified to Q-tRNAs in terminally differentiated somatic cells, however hypomodification of Q-tRNA is closely associated with cell proliferation and malignancy. Queuine participates at various cellular functions such as regulation of cell proliferation, cell signaling and alteration in the expression of growth associated proto-oncogenes. Like other proto-oncogenes bcl2 is known to involve in cell survival by inhibiting apoptosis. Queuine or Q-tRNA is suggested to inhibit cell proliferation but the mechanism of regulation of cell proliferation by queuine or Q-tRNA is not well understood. Therefore, in the present study regulation in cell proliferation by queuine in vivo and in vitro as well as the expression of cell death regulatory protein Bcl2 are investigated. For this DLAT cancerous mouse, U87 cell line and HepG2 cell line are treated with different concentrations of queuine and the effect of queuine on cell proliferation and apoptosis are studied. The results indicate that queuine down regulates cell proliferation and expression of Bcl2 protein, suggesting that queuine promotes cell death and participates in the regulation of cell proliferation.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2

NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1-/- with NQO2-/- mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial-associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFalpha, IL-6, and IL-1beta and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury.     

Correlation of epiphyllous bud differentiation with foliar senescence in crassulacean succulent Kalanchoe pinnata as revealed by thidiazuron and ethrel application

Leaves of Kalanchoe pinnata have crenate margins with each notch bearing a dormant bud competent to develop into a healthy plantlet. Leaf detachment is a common signal for inducing two contrastingly different leaf-based processes, i.e. epiphyllous bud development into plantlet and foliar senescence. To investigate differentiation of bud and its correlation, if any, with foliar senescence, thidiazuron (TDZ), having cytokinin activity and ethrel (ETH), an ethylene releasing compound, were employed. The experimental system was comprised of marginal leaf discs, each harbouring an epiphyllous bud. Most of the growth characteristics of plantlet developing from the epiphyllous bud were significantly inhibited by TDZ but promoted by ETH. The two regulators modulated senescence in a manner different for leaf discs and plantlet leaves. Thus, TDZ caused a complete retention whereas ETH a complete loss of chlorophyll in the leaf discs. In contrast, the former resulted in a complete depletion of chlorophyll from the plantlet leaves producing an albino effect, while the latter reduced it by 50% only. In combined dispensation of the two regulators, the effect of TDZ was expressed in majority of responses studied. The results presented in this investigation clearly show that the foliar processes of epiphyllous bud differentiation and senescence are interlinked as TDZ that delayed senescence inhibited epiphyllous bud differentiation and ETH that hastened senescence promoted it. A working hypothesis to interpret responsiveness of the disc-bud composite on lines of a source-sink duo, has been proposed.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Development of alternative support system for viable count of cyanobacteria by most probable number method

A technique was developed to evaluate alternative support systems to test tubes used in the standard most probable number technique, for simultaneous isolation and enumeration of cyanobacteria. Five different support systems were tested for their suitability in terms of accuracy, sensitivity, economics and ease of handling. PCR plates with 96 wells and carrying capacity of 300 microL per well were found to be most sensitive, besides being cost- and time-effective. This technique can also be useful for isolation of cyanobacteria, due to immobilization of colonies in the gel matrix and storage of samples at room temperature, without loss of viability for 5-6 weeks. This technique can help to process large sample size with ease--both for enumeration and isolation and can be extended for enumeration of other microorganisms from diverse sources.