Structure-activity relationships for the inhibition of DNA polymerase alpha by aphidicolin derivatives.

Aphidicolin and 17 derivatives that have been structurally modified in the A- and D-rings were assessed for their ability to inhibit DNA polymerase alpha. No derivative surpassed the activity of aphidicolin; derivatives with structural alterations in the A-ring exhibited significantly greater loss of activity relative to derivatives with structural alterations in the D-ring. The conclusions of these studies indicate a critical role for the C-18 function in the interaction of aphidicolin with polymerase alpha. Molecular modelling studies could not identify structural features of the aphidicolin-dCTP "overlap" that is unique to dCTP, relative to the remaining dNTPs, and that is consistent with the extant structure-activity data.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

In vitro acetylation of the liver HMG non-histone proteins and its modulation by spermine and dexamethasone during aging of rats

The in vitro acetylation of HMG proteins was studied using liver slices of young (18-week) and old (138-week) male rats. Acetylation of total HMG proteins is lower in old age. The incorporation of (¹⁴C) acetate into individual HMG proteins varies remarkably with advancing age. Whereas acetylation of high mol. wt. proteins (HMG 1 and 2) is higher, that of low mol. wt. proteins (HMG 14 and 17) is lower in the liver of young rats as compared to the old ones. Spermine stimulates the acetylation of HMG 1 and 14 in young and HMG 1, 2 and 14 in old age. It inhibits the acetylation of HMG 17 in both ages. Dexamethasone decreases the level of incorporation of (¹⁴C) into HMG 1 and 17 in young and HMG 14 and 17 in old rats. On the other hand, it stimulates the acetylation of HMG 14 by two-fold in young and that of HMG 1 and 2 by more than three-fold in old rats. Such alteration in the acetylation of HMG proteins may account for age-related changes in the structure and function of chromatin.     

Efficacy of medicinal plant (Andrographis peniculata) extract on aflatoxin production and growth of Aspergillus flavus

The efficacies of four different concentrations (3, 5, 8 and 10 mg/ml) of an aqueous extract of the Andrographis peniculata were tested on growth and aflatoxin production by Aspergillus flavus in liquid SMKY medium. The maximum inhibition of aflatoxin production and growth of A. flavus were marked at 10 mg/ml (i.e. 78.6% aft. B₁ and 75.1% growth). Growth and aflatoxin production were co-related processes.     

Oxygen free radicals in volume overload heart failure

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.     

Effect of atrazine on some aspects of lipid metabolism in fresh water fish

The impact of atrazine, a triazine herbicide, on the lipid metabolism of fish, Sarotherodon mossambica was studied. Significant changes were reported in the lipid profiles of liver and muscle as a function of exposure period.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.     

Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.