Effect of clofibrate on the adenosine triphosphatase activity of rat liver mitochondria

The antihypercholesterolemic drug clofibrate (ethyl-α-p-chlorophenoxyisobutyrate) stimulated the latent ATPase activity and “superstimulated” the uncoupler-induced ATPase activity of rat-liver mitochondria. Addition of clofibrate decreased the turbidity of mitochondrial suspensions and released considerable amount of mitochondrial protein into solution. In these properties it closely resembled detergents like Triton X-100 and deoxycholate. However, unlike the detergents, clofibrate required the presence of a permeant cation for its disruptive action. Also, it was without any such effect on sonic submitochondrial particles. The drug enhanced the uptake of both Mg2 and Cl− by mitochondria suggesting that osmotic swelling precedes lysis. Sonic submitochondrial particles prepared in the presence of clofibrate showed a greater yield and comparable ATPase activity.

Mitochondrial localization of functional ferrochelatase from Plasmodium falciparum

In the malarial parasite, enzymes of heme-biosynthetic pathway are distributed in different cellular compartments. The site of localization of ferrochelatase in the malarial parasite is crucial, since it will decide the ultimate site of heme synthesis. Earlier results have differed in terms of localization, being the mitochondrion or apicoplast and the functional enzyme has not been cloned, expressed and characterized. The present study reveals that Plasmodium falciparum ferrochelatase (PfFC) gene encodes multiple transcripts of which the one encoding the full length functional protein (PfFC) has been cloned and the recombinant protein over-expressed and purified from E. coli cells. The enzyme shows maximum activity with iron, while zinc is a poor substrate. Immunofluorescence studies with antibodies to functional ferrochelatase reveal that the native enzyme is localized to the mitochondrion of the parasite indicating that this organelle is the ultimate site of heme synthesis.

Photocytotoxic 3d-Metal Scorpionates with a 1,8-Naphthalimide Chromophore Showing Photoinduced DNA and Protein Cleavage Activity

Ternary 3d-metal complexes of formulation [M(Tp(Ph))(py-nap)](ClO4)(1-3), where M is Co(II) (1), Cu(II) (2), and Zn(II) (3); Tp(Ph) is anionic tris (3-phenylpyrazolyl)borate; and py-nap is a pyridyl ligand with a conjugated 1,8-naphthalimide moiety, have been prepared from the reaction of metal perchlorate with KTp(Ph) and py-nap in CH2Cl2. The complexes have been characterized from analytical and physicochemical data. The complexes are stable in solution as evidenced from the electrospray ionization mass spectrometry data. The complexes show good binding propensity with calf thymus (CT) DNA, giving binding constant (K-b) values of similar to 10(5) M-1 and a molecular ``light-switch'' effect that results in an enhancement of the emission intensity of the naphthalimide chromophore on binding to CT DNA. The complexes do not show any hydrolytic cleavage of DNA. They show poor chemical nuclease activity in the presence of 3-mercaptopropionic acid or hydrogen peroxide (H2O2). The Co(II) and Cu(II) complexes exhibit oxidative pUC19 DNA cleavage activity in UV-A light of 365 rim. The Zn(II) complex shows moderate DNA photocleavage activity at 365 nm. The Cu(II)complex 2 displays photoinduced DNA cleavage activity in red light of 647.1 nm and 676 rim and near-IR light of >750 rim. A mechanistic studyin UV-A and visible light suggests the involvement of the hydroxyl radical as the reactive species in the DNA photocleavage reactions. The complexes also show good bovine serum albumin (BSA) protein binding propensity, giving K-BSA values of similar to 10(5) M-1. Complexes 1 and 2 display significant photoinduced BSA cleavage activity in UV-A light. The Co(II) complex 1 shows a significant photocytotoxic effect in HeLa cervical cancer cells on exposure to UV-A light of 365 nm, giving an IC50 value of 32 mu M. The IC50 value for the py-nap ligand alone is 41.42 mu m in UV-A light. The IC50 value is >200 mu M in the dark, indicating poor dark toxicity of 1. The Cu(II) complex 2 exhibits moderate photocytotoxicity and significant dark toxicity, giving IC50 values of 18.6 mu m and 29.7 mu m in UV-A light and in the dark, respectively.

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOXI promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

Rhythmic activity of utilization of mevalonate for biogenesis of cholesterol

The utilization of mevalonate for biogenesis of cholesterol shows rhythmic activity with a peak at midnight and the step responsible is likely to be between mevalonate and isopentenyl pyrophosphate.

Synthetic polypeptide with antifreeze activity

MUCH information has been gathered in recent years on the so-called 'antifreeze' proteins which lower the freezing point of the serum of certain marine fishes living in sub-zero water temperatures1−4. The proteins from the Antarctic fish Trematomus borchgrevinki are glycoproteins with a repeating alanyl-alanyl-threonyl tripeptide sequence, the threonyl residue being linked to a disaccharide1,2. In contrast, the antifreeze protein from the winter flounder Pseudopleuronectus americanus in the North American Atlantic coastal region is made up of eight ammo acids with no apparent repeating sequence of the residues and no sugar moiety (ref. 4 and unpublished work of C. L. Hew, C. C. Yip & G. Fletcher). The antifreeze activity of these proteins is not compatible with the known colligative properties of solutes in solution and the mechanism of their action is not yet fully understood. But a common feature of both types of the antifreeze proteins is the preponderance of alanine which accounts for over 60% of the total amino residues. This fact, together with the absence of the carbohydrate in the protein from the winter flounder, prompted us to attempt the synthesis of polypeptide analogues having comparable proportions of alanine in them along with suitable other amino acids. As a first step, we made use of the lack of any obvious periodicity in the distribution of the alanyl residues in the flounder's protein and attempted the synthesis of a random copolypeptide containing about 65 mol % of alanine and 35 mol % of aspartic acid. The choice of aspartic acid was made on the basis of its being the next major amino acid in the flounder's protein3,4 and on the expectation that its polar character will help the water-solubility of the alanine-rich copolypeptide, as in other studies on alanine-containing random copolymers. In addition, Duman and DeVries4 have earlier indicated the involvement of carboxyl groups on the antifreeze activity by chemical modification studies. We report here the synthesis of this polypeptide and show that it possesses antifreeze activity.

Physicochemical, Electrochemical, and Spectroscopic Characterization of Zinc-Based Room-Temperature Molten Electrolytes and Their Application in Rechargeable Batteries

Room-temperature zinc ion-conducting molten electrolytes based on acetamide, urea, and zinc perchlorate or zinc triflate have been prepared and characterized by various physicochemical, spectroscopic, and electrochemical techniques. The ternary molten electrolytes are easy to prepare and can be handled under ambient conditions. They show excellent stability, high ionic conductivity, relatively low viscosity, and other favorable physicochemical and electrochemical properties that make them good electrolytes for rechargeable zinc batteries. Specific conductivities of 3.4 and 0.5 mS cm(-1) at 25 degrees C are obtained for zinc-perchlorate-and zinc-triflate-containing melts, respectively. Vibrational spectroscopic data reveal that the free ion concentration is high in the optimized composition. Rechargeable Zn batteries have been assembled using the molten electrolytes, with gamma-MnO2 as the positive electrode and Zn as the negative electrode. They show excellent electrochemical characteristics with high discharge capacities. This study opens up the possibility of using acetamide-based molten electrolytes as alternate electrolytes in rechargeable zinc batteries. (C) 2009 The Electrochemical Society.

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.

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.