I50 Concentration Research Articles

Assessment of the Phytotoxic Potential of Dregea volubilis (L.f.) Benth. ex Hook.f. and Identification of its Phytotoxic Substances for Weed Control

The phytotoxic potential of plants and the effect of their active components on another plant species is being explored as a potential alternative to synthetic herbicides for weed control. In the current study, we investigated the phytotoxic potential of the leaves of Dregea volubilis (L.f.) Benth. ex Hook.f. against four test plants [timothy (Phleum pratense L.), barnyard grass (Echinochloa crus-galli (L.) P. Beauv), lettuce (Lactuca sativa L.), and alfalfa (Medicago sativa L.)] and observed significant growth inhibition on those plants at concentrations >3 mg D.W. equivalent extract mL−1. A bioassay-governed purification of the D. volubilis extracts using different chromatography phases produced two growth inhibitory compounds, 3-hydroxy-α-ionone (compound 1) and 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one (compound 2). The compounds retarded the growth of barnyard grass and cress (Lepidium sativum L.) with I50 (concentration required for 50% growth suppression) values ranging from 0.098 to 0.450 mM for 3-hydroxy-α-ionone and 0.029 to 0.420 mM for 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one. Thus, the extracts and identified compounds may have the possibility to be utilized as bioagents for weed control.

In Vitro and In Vivo Effects of α-Amylase Inhibitor From Avena sativa Seeds on Life History and Physiological Characteristics of Sitotroga cerealella (Lepidoptera: Gelechiidae).

The inhibitory effects of Avena sativa L. seed extract were studied on life history and some physiological aspects of Sitotroga cerealella (Olivier; Lepidoptera: Gelechiidae). The inhibition of α-amylase activity in vitro by A. sativa proteinaceous extract suggested its potential antimetabolic effect on S. cerealella larvae. Although, chronic ingestion of A. sativa inhibitor (I10: 0.108 mg protein/artificial seed) did not show significant reduction of the growth and development of S. cerealella. However, a delay in the developmental time of immature stages was detected when S. cerealella larvae were continuously fed on I30 and I50 concentrations (0.429 and 1.11 mg protein/artificial seed, respectively) of the inhibitor. The highest realized fecundity was recorded for females which came from larvae fed on I10 concentration (102.46 ± 2.50 eggs/female), and the lowest fecundity was observed for females which came from larvae fed on I50 concentration (31.64 ± 3.17 eggs/female). The lightest weight of pupae of S. cerealella was observed on I50 concentration (2.76 ± 0.07 mg). The lowest glycogen and lipid contents of the pupae were detected on I50 concentration (50.00 ± 3.53 and 289.57 ± 29.00 µg/pupa, respectively). The lower survival rate of pupae at low temperature indicated that S. cerealella fed on I50 concentration of the inhibitor was less cold tolerant than control insects. The inhibitory studies indicated that A. sativa proteinaceous extract is a good candidate as an inhibitor of the α-amylase of this pest. This inhibitor can be expressed in genetically engineered plants to confer resistance to S. cerealella.

SUBSTRATE SPECIFICITY AND INHIBITION STUDIES ON AFRICAN CATFISH (<i>Clarias gariepinus</i>) LIVER GLUTATHIONE S-TRANSFERASE

The effect of some inhibitors of glutathione transferases was examined on a purified glutathione transferase from the liver of the African catfish (Clarias gariepinus). The ability of the enzyme to catalyze the conjugation of glutathione (GSH) with a variety of other compounds was also examined. The glutathione transferase (GST) from the African catfish was inhibited by all the compounds examined. Cibracron blue and triphenyltin chloride were the most potent inhibitors with I50 (concentration of inhibitors causing 50% inhibition) of 0.0875μM and 0.0525μM respectively. The enzyme displayed broad substrate specificity. The enzyme was able to catalyze the conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid , 4-nitrobenzylchloride, p-nitrophenylacetate and hydrogen peroxide. The results obtained from this work suggest that the glutathione transferase from the liver of African catfish might possibly belong to the Pi and Mu class of isoenzymes and that the enzyme is homodimeric. Key Words: Glutathione transferase; African catfish; Clarias gariepinus; substrate specificity; isoenzyme(s) Global Jnl Pure & Applied Science Vol.10(1) 2004: 179-182

Uncoupling of Rat Liver Mitochondrial Oxidative Phosphorylation by the Fasciolicide Triclabendazole and Its Sulfoxide and Sulfone Metabolites

The uncoupling activity of a narrow-spectrum benzimidazole anthelmintic triclabendazole (TCZ, 6-chloro-5-[2,3-dichlorophenoxy]-2-methylthio-benzimidazole) and its 2 principal metabolites triclabendazole sulfoxide (TCZ sulfoxide, 6-chloro-5-[2,3-dichlorophenoxy]-2-methylsulfinyl-benzimidazole) and triclabendazole sulfone (TCZ sulfone, 6-chloro-5-[2,3-dichlorophenoxyl]-2methylsulfonyl- benzimidazole) has been determined using rat liver mitochondria. With glutamate or succinate as the mitchondrial substrate, and the respiratory control index (RCI) as an indicator of uncoupling activity, we found that TCZ and its 2 main metabolites were uncouplers of oxidative phosphorylation at micromolar concentrations. The rank order of in vitro activity was TCZ sulfone > TCZ sulfoxide > TCZ. Structure-activity relationship studies revealed that the electron-withdrawing power of the substituent in the 2-position was the principal determinant of mitochondrial uncoupling activity. Correlation techniques were used to assess the strength of the relationship between the ability of TCZ and its metabolites to uncouple oxidative phosphorylation and their ability to lower the electrical resistance of planar bimolecular lipid membranes. A log-log plot of RCI I50 vs. resistance effective concentration (REC I50) gave a linear fit with a correlation coefficient (r) of 0.98; an r of 0.98 indicates a high positive relationship between the ability of these fasciolicides to uncouple oxidative phosphorylation and their ability to lower electrical resistance. These findings are consistent with the view that TCZ and its sulfoxide and sulfone metabolites are lipophilic protonophoric uncouplers of rat liver mitochondrial oxidative phosphorylation.

Altered ryanodine receptor of canine cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on ryanodine receptor in canine cardiac junctional sarcoplasmic reticulum (SR) were studied. The results show that the Bmax for [3H]ryanodine binding to cardiac junctional SR was decreased by 25% (8 +/- 0.38 vs 6 +/- 0.41 pmole/mg protein for control and endotoxic, respectively; (P less than 0.01) while the kd (13.7 +/- 1 nM for control vs 13.2 +/- 2 nM for endotoxic) was unaffected 4 hr following endotoxin administration. Ca2+ activated [3H]ryanodine binding in both groups sigmoidally but the Vmax for Ca2+ activation was decreased by 24% (P less than 0.05) while the S0.5 and the Hill coefficient values remained unchanged after endotoxin injection. Caffeine, ATP, and AMP-PCP activated while calmodulin, SKF-525A, ruthenium red, and Mg2+ inhibited [3H]ryanodine binding in both groups but the A0.5 (concentration requires for half-maximum activation) and the I50 (concentration requires for half-maximum inhibition) for the above-mentioned activators and inhibitors, respectively, were unaffected during endotoxin shock. Digestion of cardiac SR isolated from control dogs with phospholipase A2 inhibited [3H]ryanodine binding and the inhibition was reversed completely by the addition of phosphatidylserine. Addition of phosphatidylserine to cardiac SR isolated from endotoxic dogs stimulated [3H]ryanodine binding and the stimulation represents a complete reversal of the inhibition caused by endotoxin administration. Based on these findings together with previous observation that phospholipase A2 activity is activated during endotoxin shock, it is concluded that endotoxin administration decreases the number of ryanodine receptor in canine cardiac junctional SR and the decrease in ryanodine receptor is associated with a mechanism involving a modification of membrane lipid microenvironment in response to phospholipase A2 activation.

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes.

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.

Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine.

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.

Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity.

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)

Uptake and Photosynthetic Inhibition by Atrazine and its Degradation Products on Four Species of Submerged Vascular Plants

AbstractThe photosynthetic inhibitory effect of atrazine (2‐chloro‐4‐ethylamino‐6‐isopropyl amino‐1,3,5‐triazine) and three of its major metabolites (deethylated, deisopropylated, and hydroxyatrazine) were determined for four species of submerged macrophytes: Potamogeton perfoliatus L., Ruppia maritima L., Myriophyllum spicatum L., and Zannichellia palustris L. The four species showed a similar response to varied dosages of the parent atrazine compound with an average I1 (concentration at which photosynthesis is inhibited by 1%) for the four species of 20 µg/L and an average I50 (concentration at which photosynthesis is inhibited by 50%) for the four species of 95 µg/L. The three major degradation metabolites of atrazine produced varying degrees of photosynthetic inhibition in the four species, but generally the order of toxicity was deethylated > deisopropylated > hydroxyatrazine with hydroxyatrazine causing an apparent stimulation of photosynthesis in several species. Of four species tested, Myriophyllum spicatum L. was the most resistant to atrazine and its metabolites. The magnitude of the actual uptake of the compounds [µg compound/grams dry wt (gdw) plant] by the plants correlated closely with the photosynthetic inhibitory response, i.e., at the same concentration the uptake of atrazine > deethylated > deisopropylated > hydroxyatrazine. Considering that an extremely high environmental concentration (0.5 mg/L) of deethylated atrazine for an estuary only produced a photosynthetic inhibition of from 20 to 40% in four major species of submerged macrophytes, it is concluded that the degradation products of atrazine tested did not play a major role in the disappearance of the submerged vascular plants from the Chesapeake Bay.

Tolerance of Three Soybean (Glycine max) Cultivars to Metribuzin

‘Tracy′, ‘Tracy-M′, and ‘Centennial′, soybean [Glycine max(L.) Merr.] cultivars were evaluated under greenhouse and field conditions for their tolerance to metribuzin [4-amino-6-tert-butyl-3-(methylthio)-as-triazin-5 (4H)-one]. The I50(50% injury) concentration of metribuzin, as determined in nutrient culture, for Tracy-M was more than three times the I50for Tracy, but was equivalent to the I50for Centennial. Data from greenhouse and field studies on Sharkey clay and Dundee silt loam soils indicated that Tracy-M was more tolerant to metribuzin than its parent selection, Tracy, but no more tolerant than Centennial when metribuzin was applied preplant soil incorporated or surface preemergence alone or in combination with several dinitroaniline herbicides.

Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).

Fertilization in the sea urchin arbacia punctulata inhibited by fluorescein dyes: Evidence for a plasma membrane mechanism

AbstractFertilization and ionophore activation of the sea urchin Arbacia punctulata were inhibited in the presence of six analogs of the dye fluorescein. The concentration of any one dye needed for blockage of sperm or Ca‐ionophoremediated activation in 50% of the eggs (I50) was a function of the dye's lipid solubility. Substantially higher concentrations of each dye were required to block activation by Ca‐ionophore (A23187) than were needed to inhibit sperm activation. A detailed study of the action of Erythrosin B (tetraiodofluorescein) showed that its effects were readily reversible. The I50 concentration of Erythrosin B increased as temperature increased from 10 to 25°C. The kinetics of blockage indicated that Erythrosin B blocked some early step in the program of fertilization. The results suggest that these anionic dyes may inhibit fertilization by preventing successful sperm‐egg fusion.

Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.

THE SELECTIVE INHIBITION OF VIRAL DNA SYNTHESIS BY CHEMOTHERAPEUTIC AGENTS: AN INDICATOR OF CLINICAL USEFULNESS? *

A cell culture system has been utilized to measure the effects of drugs on DNA synthesis in uninfected and HSV-(herpes simplex virus)-infected KB cells. DNA from HSV-infected cells was separated into viral and cellular components by isopycnic centrifugation in CsCl gradients. The amount of [3H]thymidine incorporated into acid-insoluble material was measured in the absence and presence of drugs. Dose-response relationships were established by linearly regressing the probit value of the percent inhibition DNA synthesis against the logarithm of drug concentration. Fifty percent inhibitory (I50) concentrations were interpolated from the corresponding regression lines for inhibition of the following: (i) DNA synthesis is uninfected KB cells, (ii) total DNA synthesis in HSV-infected KB cells (iii) cellular DNA synthesis in HSV-infected cells, and (iv) viral DNA synthesis in HSV-infected cells. We have derived an index (SI, selective index) that quantifies the preferential inhibition of viral or uninfected cellular DNA synthesis. This index can be expressed as SI = log10 I50 concentration for DNA synthesis in uninfected cells divided by I50 concentration for viral DNA synthesis in HSV-infected cells. The SI is positive if viral DNA synthesis is inhibited preferentially and negative if uninfected cellular DNA synthesis is more strongly inhibited. A positive SI value of 0.5 was obtained for the clinically useful antiviral drug arabinosyladenine (ara-A) and a value of 0.4 for its metabolite, arabinosylhypoxanthine (ara-H). Although the adenosine deaminase inhibitor coformycin greatly increased the potency of ara-A, the inhibitor did not increase the selectivity of the drug (SI = 0.3). Stallimycin (distimycin A) (SI = 0.3) and phosphonoacetic acid (SI = 0.3) were similarly effective in preferentially inhibiting the synthesis of HSV DNA. In contrast, arabinosylcytosine (ara-C) and ribavirin inhibited DNA synthesis in uninfected cells to a greater degree than viral DNA synthesis (SI = -0.5 and -1.9, respectively). An analysis of the advantages and limitations of this experimental procedure is made and the suggestion is offered that the in vitro determination of a drug's selective index may be a valid predictor of clinical usefulness.

Effect of cardiac glycosides and sodium ethacrynate on transepithelial sodium transport in in vivo micropuncture experiments and on isolated plasma membrane Na-K ATPase in vitro of the rat.

In parallel micropuncture experiments employing peritubular capillary perfusion techniquesin vivo and biochemical techniques on the simultaneously isolated Na−K ATPase from kidney cortexin vitro, the effects of various cardiac glycosides and sodium ethacrynate on transepithelial sodium transport in proximal tubules of the rat were examined. Cardiac glycosides with an inhibitory potency for this ATPase varying over approximately a 100 fold range were used. Thein vitro sensitivity of this rat ATPase to Scilliroside, a glycoside from red squill, the most potent of them and used here for the first time in micropuncture experiments was comparable to that shown to Ouabain by a similar ATPase from kidneys of more glycoside sensitive animals. However, even at 250–300 × the I50 concentration, maximal glycoside inhibition of Na transportin vivo was only 40–50%. ETA at only 7 x its I50 concentration inhibited Na transport by 73%, consistent with an effect on general metabolism rather than on the Na−K ATPase. These results are discussed as compatible with the Na−K ATPase/Na pump complex being a reasonably coherent single unit using ATP as a source of energy.