New ternary copper(II) complexes of L-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of alpha-amino acid having polypyridyl bases of general formulation [Cu(L-ala)(B)(H2O)](X)(1-4), where L-ala is L-alanine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2',3'-f] quinoxaline (dpq, 4), and X = ClO4-/NO3- are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN3O2 coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near - 100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 >>>1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.

Synthesis and Structure-Activity Correlation Studies of Secondary- and Tertiary-Amine-Based Glutathione Peroxidase Mimics

In this study, a series of seeondary- and tertiary-amino-substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H2O2, cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert-amino groups in benzylamine-based diselenides by sec-amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N-propyl- and N-isopropylamino-substituted diselenides are 8-18 times more active than the corresponding N,N-dipropyl- and N,N-diisopropylamine-based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec-amino-substituted disclenides is similar to that of the tert-amine-based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx-like activities. it is observed that the sec-amino groups are better than the tert-amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol-exchange reactions in the selenenyl sulfides derived from sec-amine-based diselenides. Furthermore, the seleninic acids (RSeO2H) derived from the sec-amine-based compounds are more stable toward further reactions with peroxides than their tert-amine-based analogues.

Feedback-type inhibition of activity of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ubiquinone

Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.

The influence of esterification of carboxyl groups of ribonuclease-a on its structure and immunological activity

The effect of modification of carboxyl groups of Ribonuclease-Aa on the enzymatic activity and the antigenic structure of the protein has been studied. Modification of four of the eleven free carboxyl groups of the protein by esterification in anhydrous methanol/0.1 M hydrochloric acid resulted in nearly 80% loss in enzymatic activity but had very little influence on the antigenic structure of the protein. Further increases in the modification of the carboxyl groups caused a progressive loss in immunological activity, and the fully methylated RNase-A exhibited nearly 30% immunological activity. Concomitant with this change in the antigenic structure of the protein, the ability of the molecule to complement with RNase-S-protein increased, clearly indicating the unfolding of the peptide "tail" from the remainder of the molecule. The susceptibility to proteolysis, accessibility of methionine residues for orthobenzoquinone reaction and the loss in immunological activity of the more extensively esterified derivatives of RNase-A are suggestive of the more flexible conformation of these derivatives as compared with the compact native conformation. The fact that even the fully methylated RNase-A retains nearly 30% of its immunological activity suggested that the modified protein contained antibody recognizable residual native structure, which presumably accommodates some antigenic determinants.

The plant Banjauri (Vicoa indica) exhibits antifertility activity in adult female bonnet monkeys (Macaca radiata)

The antifertility activity of the plant Vicoa indica was tested in proven fertile bonnet monkeys. The dry powder of the whole plant was fed to the cycling monkeys on day 1 to 14 of menstrual cycle or day 9 to 14 of cycle or on day 2 to 5 after delivery and the fertility was evaluated in the following cycle in cycle fed monkey or after weaning the young one in the post-partum fed monkeys. Results indicated that while feeding in the post-partum monkeys did not confer any protection against pregnancy feeding during day 1 to 14 of cycle, protected from pregnancy. The monkeys did not become pregnant even after exposure to the proven fertile male monkeys for 13 ovulatory cycles while all the vehicle fed monkeys became pregnant within 3 cycles.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Responses of the Circadian Locomotor Activity Rhythm of Mus Booduga to Shifts in LD Schedules

The responses of the field mouse Mus booduga to shifts in schedules of LD cycles were monitored and the results were interpreted with the help of a PRC constructed for the same species. The results reveal that, M. booduga reentrained faster with a lesser number of transients after delay shifts than advance shifts, thus exhibiting “asymmetry effect.” A positive correlation was observed between the number of transients and the number of hours of shift. In most of the shifts, the sign of the transients (negative for delaying transients and positive for advancing transients) coincided with the direction of the shift. Interestingly, 11 and 12 h of advance shifting resulted in delaying transients. An 11-h advance shift can also be interpreted as a 13-h delay. Reentrainment through delaying transients is faster as compared to reentrainment through advancing transients. Thus, this animal might have taken a “shorter route,” as proved by the fact that an 11-h advance shift has evoked delaying transients. But a 13-h advance shift evoked only advancing transients. This prompts us to speculate that there may be a “phase jump” in M. booduga. Further, irrespective of whether L or D has been doubled in a 12-h shift, both evoked only delaying transients.

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity.

Glycodelin A triggers T cell apoptosis through a novel calcium-independent galactose-binding lectin activity

Glycodelin A (GdA) is one of the progesterone inducible endometrial factors that protect the fetal semiallograft from maternal immune rejection. The immumoregulatory effects of GdA are varied, with diverse effects on the fate and function of most immune cell types. Its effects on T cells are particularly relevant as it is capable of regulating T cell activation, differentiation, as well as apoptosis. We have previously reported that GdA triggers mitochondrial stress and apoptosis in activated T cells by a mechanism that is distinct and independent of its effects on T cell activation. In this study we describe the characterization of a cell surface receptor for GdA on T cells. Our results reveal a novel calcium-independent galactose-binding lectin activity of GdA, which is responsible for its apoptogenic function. This discovery adds GdA to a select group of soluble immunoregulatory lectins that operate within the feto-placental compartment, the only other members being the galectin family proteins. We also report for the first time that both CD4(+) and CD8(+) T cell subsets are equally susceptible to inhibition with GdA, mediated by its novel lectin activity. We demonstrate that GdA selectively recognizes complex-type N-linked glycans on T cell surface glycoproteins. and propose that the galectin-1 glycoprotein receptor CD7 maybe a novel target for GdA on T cells. This study, for the first time, links the lectin activity of GdA to its biological function.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutL alpha which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX(2)EX(4)E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and it mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX(2)EX(4)E motif.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Development and characterization of lysine based tripeptide analogues as inhibitors of Sir2 activity

Sirtuins are NAD(+) dependent deacetylases that modulate various essential cellular functions. Development of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effectors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen's 1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein, we report the synthesis of peptide analogues prepared using click reactions on N epsilon-propargyloxycarbonyl protected lysine residues and their characterization as inhibitors of Plasmodium falciparum Sir2 activity. The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-peptide substrate and parabolic non-competitive inhibition with NAD(+) supporting the formation of EI2 and E.NAD(+).I-2 complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotinamide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site, suggesting the presence of a unique alternate pocket commodating the inhibitor. One of these compounds was also found to be a potent inhibitor of the intraerythrocytic growth of P. falciparum with 50% inhibitory concentration in the micromolar range.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.