Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine?pyrimidine (Pu?Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))?(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu?Py sequences may be pertinent to gene regulation.     

Advances in Transgenic Rice Biotechnology

Rice is the most amenable crop plant for genetic manipulation amongst monocots due to its small genome size, enriched genetic map, availability of entire genome sequence, and relative ease of transformation. Improvement in agronomic traits of rice is bound to affect a sizeable population since it is a primary source of sustenance. Recent advances like use of ‘clean gene’ technology or matrix attachment regions would help improve rice transformation. Function of several novel genes and their promoters has been analyzed in transgenic rice. Significant progress has been made in introducing traits like herbicide, biotic stress and abiotic stress tolerance. Attempts also have been made to enhance nutritional characteristics of the grain and yield. Identification of genes controlling growth and development can be used to modify plant architecture and heading period. Transgenic rice can serve as a biofactory for the production of molecules of pharmaceutical and industrial utility. The drive to apply transgenic rice for public good as well as commercial gains has fueled research to an all time high. Successful field trials and biosafety of transgenic rice have been reported. This would act as a catalyst for greater acceptance of genetically modified food crops. The lessons learnt from rice can be extended to other cereals thereby opening new opportunities and possibilities.     

Possibility of an antiparallel (tetramer) quadruplex exhibited by the double repeat of the human telomere

Under physiological concentrations of Na+ and K+, human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K+, whereas Na+ facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K+ and Na+, respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands.     

Selective Insulin Resistance in Adipocytes

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.     

Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis: ROLE OF THE CD LOOP AND PRE-A MOTIF IN ELECTRON CYCLING*

Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μm/min⁻¹, which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly⁴⁸ may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10⁻⁵ eV, which is lower than the value obtained for E. coli flavoHb (8.0 10⁻⁴ eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.     

Description of two new species of ectoparasitic mites of Podapolipus and Podapolipoides (Acari: Podapolipidae) on Oxya sp. from West Bengal,India

Two new species, Podapolipus husbandi and Podapolipoides channabasavannai, collected on Oxya sp. from West Bengal, India are described and illustrated.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.