The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Synthesis, crystal structure and DNA cleavage activity of (aqua)bis(dipyridophenazine)copper(II) complex

A copper(II) complex of dipyridophenazine, viz., [Cu(dppz)(2)(H2O)](ClO4)(2) (I), has been prepared and structurally characterized by X-ray crystallography. The crystal structure of the complex shows a five-coordinate structure in which two N,N-donor dipyridophenazine (dppz) and one aqua ligand bind to the copper(II) center giving Cu-O and Cu-N bond distances in the range of 1.981(6) to 2.043(6) angstrom. The ESI-MS spectrum of 1 in MeCN shows a peak at m/z value of 313 (100%) indicating the dissociation of the aqua ligand in the solution phase. The complex is one-electron paramagnetic (mu(eff), 1.86 mu(B)). It displays a quasi-reversible Cu(II)/Cu(I) redox process at 0.096 V. The complex is an avid binder to CT DNA giving a binding constant value of 3.5 x 10(5) M-1. It shows significant hydrolytic cleavage of supercoiled pUC19 DNA in dark ill the absence of any external agents. The complex exhibits chemical nuclease activity oil treatment with 3-mercaptopropionic acid as a reducing agent forming hydroxyl radicals. Complex 1 is a model synthetic nuclease and hydrolase showing both modes of DNA cleavage under different reaction conditions. The DNA cleavage activity of 1 is significantly better than its phen analogue but similar to that of the bis-dpq complex.

Variations in the levels of aminoacylation and modified nucleotide content between total tRNAs from chloroplasts and etioplasts in cucumber cotyledons

Total tRNAs isolated from chloroplasts and etioplasts of cucumber cotyledons were compared with respect toamino acid acceptance, isoacceptor distribution and extent of modification. Aminoacylation of the tRNAs with nine different amino acids studied indicated that the relative acceptor activities of chloroplast total tRNAs for four amino acids are significantly higher than etioplast total tRNAs. Two dimensional polyacrylamide gel electrophoresis(2D-PAGE) of chloroplast total tRNAs separated at least 32 spots, while approximately 41 spots were resolved from etioplast total tRNAs. Comparison of the reversed-phase chromatography (RPC-5) profiles of chloroplast and etioplast leucyl-, lysyl-, phenylalanyl-, and valyl-tRNA species showed no qualitative differences in the elution profiles. However, leucyl-, lysyl- and valyl-tRNA species showed quantitative differences in the relative amounts of the isoaccepting species present in chloroplasts and etioplasts. The analysis of modified nucleotides of total tRNAs from the two plastid types indicated that total tRNA from etioplasts was undermodified with respect to ribothymidine, isopentenyladenosine/hydroxy-isopentenyladenosine, 1 -methylguanosine and 2-o-methylguanosine. This indicates that illumination may cause de novo synthesis of chloroplast tRNAmodifying enzymes encoded for by nuclear genes leading to the formation of highly modified tRNAs in chloroplasts. Based on these results, we speculate that the observed decrease in levels of aminoacylation, variations in the relative amounts of certain isoacceptors, and differences in the electrophoretic mobilities of some extra tRNA spots in the etioplast total tRNAs as compared to chloroplast total tRNAs could be due to some partially undermodified etioplast tRNAs. Taken together, the data suggested that the light-induced transformation of etioplasts into chloroplasts is accompanied by increases in the relative levels of some functional chloroplast tRNAs by post transcriptional nucleotide modifications.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Conjugated Ternary Copper(II) Complexes

Ferrocene-conjugated ternary copper(II) complexes [Cu(L)(B)](ClO4)(2), where L is FcCH(2)N(CH2Py)(2) (Fc = (eta(5)-C5H4)Fe-II(eta(5)-C5H5)) and B is a phenanthroline base, viz., 2,2'-bipyridine (bpy, 1), 1, 10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), have been synthesized and characterized by various spectroscopic and analytical techniques. The bpy complex 1, as its hexafluorophosphate salt, has been structurally characterized by X-ray crystallography. The molecular structure shows the copper(II) center having an essentially square-pyramidal coordination geometry in which L with a pendant ferrocenyl (Fc) moiety and bpy show respective tridentate and bidentate modes of binding to the metal center. The complexes are redox active, showing a reversible cyclic voltammetric response of the Fc(+)-Fc couple near 0.5 V vs SCE and a quasi-reversible Cu(II)-Cu(I) couple near 0.0 V. Complexes 2-4 show binding affinity to calf thymus (CT) DNA, giving binding constant (K-b) values in the range of 4.2 x 10(4) to 2.5 x 10(5) M-1. Thermal denaturation and viscometric titration data suggest groove binding and/or a partial intercalative mode of binding of the complexes to CT DNA. The complexes show good binding propensity to the bovine serum albumin (BSA) protein, giving K-BSA values of similar to 10(4) M-1 for the bpy and phen complexes and similar to 10(5) M-1 for the dpq and dppz complexes. Complexes 2-4 exhibit efficient chemical nuclease activity in the presence of 3-mercapto-propionic acid (MPA) as a reducing agent or hydrogen peroxide (H2O2) as an oxidizing agent. Mechanistic studies reveal formation of hydroxyl radicals as the reactive species. The dpq and dppz complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to visible light of different wavelengths including red light using an argon-krypton mixed gas ion laser. Mechanistic investigations using various inhibitors reveal the fort-nation of hydroxyl radicals in the DNA photocleavage reactions. The dppz complex 4, which shows efficient photoioduced BSA cleavage activity, is a potent multifunctional model nuclease and protease in the chemistry of photodynamic therapy (PDT) of cancer.

Synthesis, crystal structures, DNA binding and cleavage activity of L-glutamine copper(II) complexes of heterocyclic bases

Ternary L-glutamine (L-gln) copper(II) complexes [Cu(L-gln)(B)(H2O)](X) (B = 2,2'-bipyridine (bpy), X = 0.5SO(4)(2-), 1; B = 1,10-phenanthroline (phen), X = ClO4-, 2) and [Cu(L-gln)(dpq)(ClO4)] (3) (dpq, dipyridoquinoxaline) are prepared and characterized by physicochemical methods. The DNA binding and cleavage activity of the complexes have been studied. Complexes 1-3 are structurally characterized by X-ray crystallography. The complexes show distorted square pyramidal (4+1) CuN3O2 coordination geometry in which the N,O-donor amino acid and the N, N-donor heterocyclic base bind at the basal plane with a H2O or perchlorate as the axial ligand. The crystal structures of the complexes exhibit chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The complexes display a d-d electronic band in the range of 610-630 nm in aqueous-dimethylformamide (DMF) solution (9:1 v/v). The quasireversible cyclic voltammetric response observed near -0.1 V versus SCE in DMF-TBAP is assignable to the Cu(II)/Cu(I) couple. The binding affinity of the complexes to calf thymus (CT) DNA follows the order: 3 (dpq) > 2 (phen) >> 1 (bpy). Complexes 2 and 3 show DNA cleavage activity in dark in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent via a mechanistic pathway forming hydroxyl radical as the reactive species. The dpq complex 3 shows efficient photoinduced DNA cleavage activity on irradiation with a monochromatic UV light of 365 nm in absence of any external reagent. The cleavage efficiency of the DNA minor groove binding complexes follows the order:3 > 2 >> 1. The dpq complex exhibits photocleavage of DNA on irradiation with visible light of 647.1 nm. Mechanistic data on the photo-induced DNA cleavage reactions reveal the involvement of singlet oxygen (O-1(2)) as the reactive species in a type-II pathway. (C) 2008 Elsevier B.V. All rights reserved.

A role for nocturnal serum testosterone surge in regulating spermatogenesis in the adult non-human primate

Adult male bonnet monkeys exhibit nychthemeral rhythms in testosterone (T) secretion but the precise role of this heightened level of T secretion in regulating spermatogenesis is not known. Intranasal administration of microdoses (500 mu g or 250 mu g/day) of Norethisterone (IN-NET) to adult monkeys (n = 6) at 1600 h each day selectively and completely suppressed the nocturnal surge levels of serum T. Concomitant with this was a significant reduction (P<0.01) in serum LH but not FSH levels. DNA flow cytometric analysis of testicular biopsy tissue showed by week 10 of IN-NET treatment an arrest in meiotic transformation of primary spermatocytes (4C) to round/elongate (1C/HC) spermatids and by week 20 there was a complete absence of 4C, 1C and HC cells (with a relative accumulation in 2C cells). The accumulated meiotic (4C) cells at week 10 showed an increase (>80%, P<0.01) in coefficient of variation and a decrease in intensity of DNA-bound ethidium bromide fluorescence, parameters characteristic of degenerating 'apoptotic' subpopulation of germ cells. While two monkeys exhibited acute oligozoospermia 4 became azoospermic by 20 weeks of IN-NET treatment. A complete, qualitative reversal in the regressive changes in spermatogenesis and near-normal sperm output were apparent at the end of a 20-week recovery phase. These data demonstrate that prolonged, selective suppression of nocturnal surge levels of serum T secretion exerts a primary effect on meiosis in spermatogenesis leading to oligo/azoospermic status in adult bonnet monkeys.

Analysis of a Genomic DNA Expression Library of Mycobacterium tuberculosis Using Tuberculosis Patient Sera: Evidence for Modulation of Host Immune Response

DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments, The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins, Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, others whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients, This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression.

Metal-Ion-Dependent Oxidative DNA Cleavage by Transition Metal Complexes of a New Water-Soluble Salen Derivative

A new water-soluble, salen [salen = bis(salicylidene) ethylenediamine]-based ligand, 3 was developed. Two of the metal complexes of this ligand, i.e., 3a, [Mn(III)] and 3b, [Ni(II)], in the presence of cooxidant magnesium monoperoxyphthalate (MMPP) cleaved plasmid DNA pTZ19R efficiently and rapidly at a concentration similar to 1 mu M. In contrast, under comparable conditions, other metal complexes 3c, [Cu(II)] or 3d, [Cr(III)] could not induce any significant DNA nicking. The findings with Ni(II) complex suggest that the DNA cleavage processes can be modulated by the disposition of charges around the ligand.

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a process 5'-3' single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ and inhibited by Cd2+ suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays, Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interactio between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.

Dynamical Transition of Water in the Grooves of DNA Duplex at Low Temperature

At low temperature (below its freezing/melting temperature), liquid water under confinement is known to exhibit anomalous dynamical features. Here we study structure and dynamics of water in the grooves of a long DNA duplex using molecular dynamics simulations with TIP5P potential at low temperature. We find signatures of a dynamical transition in both translational and orientational dynamics of water molecules in both the major and the minor grooves of a DNA duplex. The transition occurs at a slightly higher temperature (TGL ≈ 255 K) than the temperature at which the bulk water is found to undergo a dynamical transition, which for the TIP5P potential is at 247 K. Groove water, however, exhibits markedly different temperature dependence of its properties from the bulk. Entropy calculations reveal that the minor groove water is ordered even at room temperature, and the transition at T ≈ 255 K can be characterized as a strong-to-strong dynamical transition. Confinement of water in the grooves of DNA favors the formation of a low density four-coordinated state (as a consequence of enthalpy−entropy balance) that makes the liquid−liquid transition stronger. The low temperature water is characterized by pronounced tetrahedral order, as manifested in the sharp rise near 109° in the O−O−O angle distribution. We find that the Adams−Gibbs relation between configurational entropy and translational diffusion holds quite well when the two quantities are plotted together in a master plot for different region of aqueous DNA duplex (bulk, major, and minor grooves) at different temperatures. The activation energy for the transfer of water molecules between different regions of DNA is found to be weakly dependent on temperature.

Copper(II) Complexes of l-Arginine as Netropsin Mimics Showing DNA Cleavage Activity in Red Light

Copper(II) complexes [Cu(L-arg)(2)](NO3)(2) (1) and [Cu(L-arg)(B)Cl]Cl (2-5), where B is a heterocyclic base, namely, 2,2'-bipyridine (bpy, 2), 1,10-phenanthroline (phen, 3), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 4), and dipyrido[3,2-a:2',3'-c)phenazine (dppz, 5), are prepared and their DNA binding and photoinduced DNA cleavage activity studied. Ternary complex 3, structurally characterized using X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor L-arginine and N,N-donor 1,10-phenanthroline form the basal plane with one chloride at the elongated axial site. The complex has a pendant cationic guanidinium moiety. The one-electron paramagnetic complexes display a metal-centered d-d band in the range of 590-690 nm in aqueous DMF They show quasireversible cyclic voltammetric response due to the Cu(II)/Cu(I) couple in the range of -0.1 to -0.3 V versus a saturated calomel electrode in a DMF-Tris HCl buffer (pH 7.2). The DNA binding propensity of the complexes is studied using various techniques. Copper(II) bis-arginate 1 mimics the minor groove binder netropsin by showing preferential binding to the AT-rich sequence of double-strand (ds) DNA. DNA binding study using calf thymus DNA gives an order: 5 (L-arg-dppz) >= 1 (biS-L-arg) > 4 (L-arg-dpq) > 3 (L-arg-phen) >> 2 (L-arg-bpy). Molecular docking calculations reveal that the complexes bind through extensive hydrogen bonding and electrostatic interactions with ds-DNA. The complexes cleave supercoiled pUC19 DNA in the presence of 3-mercaptopropionic acid as a reducing agent forming hydroxyl ((OH)-O-center dot) radicals. The complexes show oxidative photoinduced DNA cleavage activity in UV-A light of 365 nm and red light of 647.1 nm (Ar-Kr mixed-gas-ion laser) in a metal-assisted photoexcitation process forming singlet oxygen (O-1(2)) species in a type-II pathway. All of the complexes, barring complex 2, show efficient DNA photocleavage activity. Complexes 4 and 5 exhibit significant double-strand breaks of DNA in red light of 647.1 nm due to the presence of two photosensitizers, namely, L-arginine and dpq or dppz in the molecules.

Influence of Martensite Content and Morphology on Tensile and Impact Properties of High-Martensite Dual-Phase Steels

A series of dual-phase (DP) steels containing finely dispersed martensite with different volume fractions of martensite (V-m) were produced by intermediate quenching of a boron- and vanadium-containing microalloyed steel. The volume fraction of martensite was varied from 0.3 to 0.8 by changing the intercritical annealing temperature. The tensile and impact properties of these steels were studied and compared to those of step-quenched steels, which showed banded microstructures. The experimental results show that DP steels with finely dispersed microstructures have excellent mechanical properties, including high impact toughness values, with an optimum in properties obtained at similar to 0.55 V-m. A further increase in V-m was found to decrease the yield and tensile strengths as well as the impact properties. It was shown that models developed on the basis of a rule of mixtures are inadequate in capturing the tensile properties of DP steels with V-m > 0.55. Jaoul-Crussard analyses of the work-hardening behavior of the high-martensite volume fraction DP steels show three distinct stages of plastic deformation.

Interaction of Co, Mn, Mg and AI with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T-m values. Mn enhanced T-m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T-m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Appended L-Methionine Reduced Schiff Base Copper(II) Complexes of Phenanthroline Bases

Ferrocene-appended ternary copper(H) complexes of phenanthroline bases having CuN3OS coordination with an axial Cu-S bond derived from L-methionine reduced Schiff base shows red light induced oxidative DNA cleavage activity following a hydroxyl radical pathway. The dipyridophenazine complex, in addition, displays photoinduced oxidative cleavage of bovine serum albumin protein in UV-A light.