A study of the carboxyl groups of sheep brain tubulin.

Tubulin was isolated from whole sheep brain by ammonium sulphate fractionation followed by ion exchange chromatography using DEAE-Sephadex in 10 mM sodium phosphate buffer pH 7.0. It was essentially free from contaminating proteins as shown by SDS-PAGE electrophoresis and was active by the criterion of colchicine binding. Hydrogen ion titration of the sheep brain tubulin was performed under native conditions. In the present study, only that pH range has been selected for analysis where carboxyl groups (most of which are located in the C-terminal regions of both the alpha- and beta- subunits) deprotonate. The results indicated the presence of 75 carboxyl groups per dimer upto pH 5.5. Of these, 46 titrated with a pK of 3.7 and the remaining 29 with a pK of 3.5.


Biochemical, cytogenetic, and morphological characteristics of human primary and metastatic prostate cancer cell lines.

Metastatic properties of human prostatic cancer cell lines (ND-1 and DU-145) were examined using various biochemical techniques. DU-145 cells had a higher metastatic potential than ND-1 cells. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with considerable structural rearrangement. ND-1 cells had a modal chromosome number range lower than DU-145 cells (45-66, compared to 54-62). Ploidy analysis revealed that DU-145 cells showed hyperdiploidy with a greater amount of proliferation than the majority of ND-1 cells. Electron microscopic studies revealed little change in the cell morphology of either line. DU-145 cells had lower phosphatidyl choline levels and higher sphingomyelin levels than ND-1. DU-145 cells had much lower arachidonic acid levels than ND-1 cells. SDS-polyacrylamide gel electrophoresis revealed protein differences between the two cell lines. This study demonstrates for the first time that lipids, proteins and cytogenetic parameters differ in human primary and secondary prostate cancer cell lines.


Immunospecificity and affinity studies on anti-luteinizing hormone releasing hormone antibodies developed by azo-LHRH-TT and azo-LHRH-BSA conjugates.

Monoclonal and conventional antibodies against the 'self' decapeptide LHRH produced by azo-LHRH-TT and azo-LHRH-BSA conjugates were studied for immunoreactivity with native LHRH, azo-LHRH, LHRH (OH) and its fragments of sequence 1-10, 7-10, 4-10 and 4-6. The CoAb reacted 8-20% to azo-LHRH, 2-6% to LHRH-Lys, 1-3% LHRH-LYS-MDP and 0.5-1.68% to LHRH-Ala-Ala-Tuftsin but failed to react to LHRH free acid and its fragments. On the other hand MoAb reacted 4-6% to azo-LHRH and 10-18% to LHRH-Lys, 3-5% to LHRH-Ala-Ala-Tuftsin. These results indicate that the azotization drastically inhibited immunoreactivity but that the azo-LHRH-carrier conjugate preserved the immunogenic epitope responsible for the induction of an effective anti-LHRH antibody response.


OmpT proteolysis of E. coli initiation factor IF2. Elimination of a cleavage site by site-directed mutagenesis.

A serious problem during purification of the E. coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation. We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290. In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant. By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT. However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.


Changes in proteolytic activity during the growth of Trypanosoma cruzi epimastigotes.

The proteolytic pattern of Trypanosoma cruzi epimastigotes was studied using electrophoresis in gelatin containing SDS polyacrylamide gels. Under conditions that represent a nutritional stress such as the depletion of glucose in an axenic medium the results obtained showed a significant increase in the activity of two proteases of M(r) = 50,000 and M(r) = 60,000 as well as the appearance of higher molecular weight bands during the transition from log to stationary phase parasites. The use of different protease inhibitors strongly suggests that all these proteases are of the cysteine type, and their acid pH optimal would be in agreement with the possible localization in the parasite lysosomes. The reincubation of stationary phase parasites in fresh LIT medium or the addition of 0.4% glucose partially reverted the proteolytic pattern to that observed in log phase parasites. The regulation in the activity of these enzymes could be related to the well established use of amino acids for catabolic purpose after depletion of the medium glucose.


Evaluation of sialic acid in lipoprotein fractions in fibrosarcoma bearing rats by 14C-glucose chase.

The changes in sialoglycoconjugates synthesis after administration of 14C-glucose to fibrosarcoma bearing rats were studied. After 1 hr treatment 14C-glucose tumor bearing rat synthesized hexosamine which contained more radioactivity when compared to sialic acid. On fractionation of lipoproteins, low density lipoproteins (LDL) contained more sialic acid. This was further confirmed by separating the lipoproteins, as more radioactivity incorporation was observed in the LDL fraction. It is noteworthy that inspite of the tumor, high density lipoproteins (HDL) did not show any change.


Comparison of 2,5,5-trimethyl-1-pyrroline-N-oxide (M3PO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps.

2,5,5-Trimethyl-1-pyrroline-N-oxide (M3PO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) were examined for their potential as spin traps used in biological samples, and the results obtained for M3PO and M4PO were compared with those of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) reported previously. The rate constants for the reactions of M3PO and M4PO with hydroxyl radicals were in the same order of that constant of DMPO. The hyperfine splitting constants of M4PO spin adducts were as informative as those of DMPO adducts. In the presence of ferric ions, M3PO and M4PO formed 2-hydroxy-2,5,5-dimethyl-1-pyrrolidinyloxy (M3PO-OH) and 2-hydroxy-3,3,5,5-tetramethyl-1-pyrrolidinyloxyl (M4PO-OH), respectively, by the nucleophilic addition of a water molecule, as we have already reported for DMPO. M4PO-OH was subjected to oxidation by ferric ions with a rate much slower than that for DMPO-OH, while M3PO-OH was not. The results indicate that M4PO is a spin trap suitable for biological applications and that nitrones having a methyl group at C2 represent improved spin traps.


Changes in cholinesterase system in different brain areas during the development of behavioral tolerance to monocrotophos toxicity in male albino rats.

During the development of behavioral tolerance to the organophosphate monocrotophos, the activities of AChE and BuChE and the content of ACh were affected in different brain areas of male albino rats. The inhibition of AChE and BuChE activities and the elevation of ACh content were progressive till 7 days and were followed by a recovery trend towards normalcy. The changes correlated with the appearance and disappearance of signs and symptoms of toxicity, which were mainly cholinergic. The results suggest the possibility of de novo synthesis of cholinesterases resulting in the development of behavioral tolerance, as was evident by the recovery trend. The shift of toxicity from greater AChE inhibition during initial periods to greater BuChE inhibition during later periods indicates the possible involvement of pseudocholinesterases in the development of tolerance and in the reduction of toxicity. The brain areas were differentially affected. Cerebral cortex was more affected initially but it was the striatum that was affected more during later dosings. The results show that behavioral tolerance to monocrotophos toxicity develops despite changes in AChE and BuChE activities and ACh content.


Identification of the number of genes encoding rat liver glutathione S-transferase Yc, and their close location on the genome.

We have tried to determine the number of genes for the rat liver glutathione S-transferase Yc, using specific probes prepared by polymerase chain reaction. DNA blot hybridization with specific probes and analysis in isolated genomic clones indicated that two copies of the Yc gene exist on the rat genome. Furthermore, DNA blot hybridization after clamped homogenous electric field gel electrophoresis indicated that the two copies of Yc gene family are closely located on the genome.


New trypsin inhibitors are present in the coelomic fluid of the earthworm Lumbricus terrestris.

Proteinase-inhibiting components of the coelomic fluid of the earthworm Lumbricus terrestris were examined. Inhibition of proteinases of serine, aspartate and thiol families was tested. Very strong inhibition was observed only in the case of trypsin. Additional data suggest that the inhibition is related to proteins of molar mass of 42 kDa and 20 kDa, respectively. These two proteins are present in the coelomic fluid in several forms which differ in their isoelectric points.


Effect of Helicobacter pylori lipopolysaccharide on the synthesis of sulfated gastric mucin.

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucus glycoprotein in gastric mucosa was investigated. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis, exerted a profound inhibitory effect on the process of mucus glycoprotein glycosylation and sulfation, and evoked a rapid (within 15 min) inhibition (65%) in both mucin glycosylation and sulfation at its optimal concentration of 100 micrograms/ml. The data on mucin secretory responses indicated that the added lipopolysaccharide caused a 57% stimulation in sulfated mucin secretion within 15 min followed thereafter by inhibition, which reached a maximum of 32% by 45 min. The high molecular weight mucin form predominated in the initial secretion, while prolonged exposure to the lipopolysaccharide caused a significant increase in the low molecular weight mucin form. The results suggest that H. pylori lipopolysaccharide exerts detrimental effect on mucus glycoprotein sulfation and assembly.


Actin binding to cross-linked 10 S smooth muscle myosin and 9 S heavy meromyosin.

Unphosphorylated gizzard myosin and heavy meromyosin were cross linked in the 10 S and 9 S states, respectively, by the cleavable cross linker, 3,3'-dithiobis (sulfosuccinimidyl-propionate) (DTSSP). The 10 S to 6 S transition for cross-linked 10 S myosin appeared to cease; myosin appeared to remain in the 10 S state from measurements of viscosity and Mg(2+)-ATPase activity. The loss of the transition for cross-linked 9 S heavy meromyosin (HMM) was also indicated by Mg(2+)-ATPase activity. The cross links were cleaved by incubation with 50 mM dithiothreitol. From direct binding measurements, the estimated Kd's of actin to cross-linked and control heavy meromyosin were 167 and 16 microM, respectively. The binding affinity of cross-linked HMM to actin was restored to the control level by dithiothreitol.


Adenosine metabolism in small coronary arteries of pig.

Adenosine preferentially relaxes small coronary arteries over large ones, and small bovine coronary arteries are also known to have a higher density of adenosine receptors. Here we report a possible role of adenosine metabolism in this process. Subcellular fractions, from right coronary artery (lumen diameter of 2-3 mm) of pig designated as large coronary artery and its subsequent branches (lumen diameter of 0.5-1 mm) as small coronary arteries, were prepared and characterized. In comparison to the various large artery subcellular fractions, the corresponding small artery fractions were richer in 5'-nucleotidase but poorer in adenosine deaminase. Thus a cascade of events may promote adenosine relaxation in small coronary arteries: higher activity of 5'-nucleotidase leads to production of more adenosine, larger number of receptors allows greater reactivity to adenosine, and lower adenosine deaminase level promotes prolonged action of adenosine.


Ponceau S as a dye for quantitative protein assay. Its use in the presence of Triton X-100.

A Ponceau S reagent with high stability and slight response to pH changes, presence of organic solvents and detergents was prepared. Under these conditions, the reagent was tested to be suitable to quantify, in one-step procedure, different standard proteins. Moreover, this method offers the possibility to measure the protein content in the presence of the detergent Triton X-100.


Crosslinking analysis of an endothelin receptor protein from human placenta.

Crosslinking of [125I]ET-1 to human placental membrane preparations in the absence of divalent cations revealed a [125I]ET-1.ET-1-receptor complex of 49 kDa and of 205 kDa on SDS-PAGE. In the presence of 20 mM MnCl2, however, only a complex of 205 kDa was observed. About 64% of the radioactively labeled receptor.ligand complex could be solubilised by digitonin and CHAPS. Size exclusion chromatography of the solubilised placental membranes, prelabeled with [125I]ET-1 in the presence of MnCl2, revealed two binding proteins of 200 and 33 kDa. In the absence of MnCl2, however, only a binding protein of 50 kDa was observed. Binding of [125I]ET-1 to the 200 kDa and 50 kDa but not to the 33 kDa protein was suppressed by unlabeled ET-1. Moreover the ET-1-receptor density in placental membranes (Bmax) increased significantly in the presence of MnCl2, whereas the dissociation constant (Kd) decreased. Thus, divalent cations exhibit marked and possibly physiologically important effects on molecular mass, Bmax and Kd of the [125I]ET-1.ET-1-receptor complex. The potential mechanism of action of Mn2+ ions on the ET-1-receptor is discussed.


Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).

Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.


Concentrations and ratios of immunoreactive big-endothelin-1 and endothelin-1 in human, rat and rabbit plasma.

We have developed a radioimmunoassay for big-endothelin-1, based on a new antiserum recognizing human big-endothelin(1-38). On a molar base the antibody showed virtually no cross-reactivity with endothelin-1, -2, -3 (less than 0.001%), 14% cross-reactivity with the C-terminal big-endothelin(22-38) and only 0.85% cross-reactivity with porcine big-endothelin(1-39). However, immunoreactive big-endothelin-1 could be detected in human, rat and rabbit plasma. Thus, big-endothelin-1 in rats and rabbits seem to be structurally similar to human big-endothelin-1. Immunoreactive endothelin-1 was measured using a commercially available anti-endothelin-1 antibody. The measured concentrations of immunoreactive endothelin-1 and big-endothelin-1 and their molar ratios differed significantly in the three species.


Evaluation of the predicted secondary structure of bacteriorhodopsin. Prediction of the bovine rhodopsin secondary structure and its sequence similarity with bacteriorhodopsin.

The secondary structure of bacteriorhodopsin (bacR) is predicted using the Chou and Fasman method in conjunction with the hydropathic index of Kyte and Doolittle. The predicted bacR structure was compared with the structure determined by Henderson et al. (1990) using electron diffraction and was found to correlate extremely well. The secondary structure of bovine rhodopsin (bovR) was then predicted using the same techniques. The proposed transmembrane regions of bovR were then examined and found to have sequence similarity with those transmembrane regions of bacR.


Lifetime determination of low sample concentrations: a new cuvette for highly accurate and sensitive fluorescence measurements.

A new cuvette was developed for spectrofluorometric measurements which increases the efficiency of recorded fluorescence emission by at least two-fold, decreasing, at the same time, the noise component of the record. It is suited for both steady-state and dynamic fluorescence measurements. Comparative data for both the classical and the novel cuvettes are presented here.


Effects of insulin and a tumour promoter, TPA, on glucose transport and metabolism in retinal pigmented epithelium in vitro.

We have studied the effects of insulin, adenosine and 12-O-tetradecanoylphorbol-13-acetate (TPA) on glucose metabolism of the retinal pigmented epithelial (RPE) cells in vitro. Insulin stimulates glucose transport, glucose oxidation and lipogenesis in RPE cells. TPA at low concentrations of insulin increases the rates of glucose transport and glucose oxidation. Depletion of adenosine in RPE cells by adenosine deaminase increases the rate of both glucose transport and 14CO2 formation and improves insulin-sensitivity of both processes. The effects of TPA on RPE cells cannot be explained by the activation of protein kinase C. An alternative possibility is that the effects of TPA on insulin-stimulated glucose disposal in RPE cells is mediated by a change in adenosine concentration and/or the affinity/number of its receptors.


Calcium ionophore A-23187 inhibits the secretion of beta-hexosaminidase from the GG2EE mouse macrophage cell line.

Secretion of the lysosomal enzyme beta-N-acetylhexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two hexosaminidase isoenzymes corresponding to hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to hexosaminidase S.


Effect of insulin and thyroxine on catalase, glutathione-s-transferase, GSH and GSSG in alloxan diabetic rat red cells.

The levels of catalase (CAT), glutathione-s-transferase (GST), reduced glutathione (GSH) and oxidised glutathione (GSSG) were measured in red blood cells from control (C) and diabetic rats (D). Diabetes was induced by alloxan administration and diabetic rats were treated with insulin (D+I) and thyroxine (D+T4). On the third day of insulin withdrawal the CAT activity increased significantly. The GST activity showed an increase in D and D+I for one week, thyroxine treatment to D rats resulted in maintaining the GST activity at control levels. The levels of GSH and GSSG increased in D red cells after one week of insulin withdrawal but later, the GSH level was below the control level while the GSSG was at its control level. Insulin treatment to D rats did not reverse GSH level to control initially but controlled it at a later stage. Thyroxine, though, reversed GSH levels but enhanced GSSG in D rat red cells.


Effect of lead on lipid peroxidation of the hepatic subcellular organelles of developing chick embryos.

The effect of lead on hepatic mitochondria and microsomes of chick embryo was studied with special attention to the role of lipid peroxidation in the manifestation of lead toxicity. Mitochondrial enzymes such as succinate dehydrogenase, cytochrome C oxidase and ATPase were found to decrease in a dose dependent manner upon lead administration. Further, mitochondrial cytochromes, microsomal cytochrome P-450 and heme levels were reduced considerably with concomitant increase of mitochondrial and microsomal lipid peroxides. In the present investigation an attempt has been made to correlate the lead induced lipid peroxidation, the loss of mitochondrial and microsomal hemoproteins and the inhibition of mitochondrial enzymes in chick embryo.


Catecholamines are required for testosterone induction of ornithine decarboxylase in the mouse kidney.

This report presents a study on the influence of the adrenergic system on androgen-regulated ornithine decarboxylase activity (ODC) in the mouse kidney. We have found that the existing levels of renal ODC in male mice and the increase induced by testosterone in male, female, castrated male and hypophysectomized mice were dramatically impaired by catecholamine depletion produced by treatment with either alpha-methyl-p-tyrosine or reserpine. Blockade of beta-receptors by treatment of the animals with the antagonists propranolol or bisoprolol was unable to prevent the effect of testosterone on the renal enzyme. However, alpha i-blockade obtained by treatment with prazosin or phenoxybenzamine was partially effective to produce the decrease of basal ODC in control male or to diminish ODC induction produced by androgens. All these findings demonstrate that catecholamines and alpha 1-adrenergic receptors are implicated in androgen-regulated ODC activity in the mouse kidney and suggest a plausible role of sympathetic renal innervation in enzyme induction by steroid hormones in this organ.


D-xylose fermentation and catabolism in Fusarium oxysporum.

Fusarium oxysporum exhibits different fermentative capacities when grown under different aeration conditions. Highest ethanol and acetic acid yield coefficients were obtained under semi-aerobic culture conditions. Effect of aerobiosis on the levels of key enzymes and intracellular intermediary metabolites of pentose phosphate pathway, tricarboxylic acid cycle and glycolysis exhibited striking correlation with the fermentative character of metabolism. The results are discussed in relation to the energy levels in the cell, the redox balance and mitochondrial function.


Androgenic status in cyclic and postmenopausal women: a comparison between smokers and nonsmokers.

Serum total testosterone (T), free testosterone (fT) sex hormone binding globulin (SHBG), androstenedione (A) and 3 alpha androstanediol glucuronide (3 alpha-diol G) levels as well as serum free androgen index (FAI), fT/T and 3 alpha-diol G/T ratios were measured in premenopausal and cyclic women, grouped according to sexual status and smoking status. Our results showed that serum T, fT, SHBG, A and 3 alpha-diol G levels were lower in cyclic women in the follicular than in the luteal phase of the ovarian cycle, although the differences between these values were not significant. Postmenopausal women had significantly lower values of T, fT, A 3 alpha-diol G, FAI and 3 alpha diolG/T but not SHBG and fT/T than cyclic women. When we compared women smokers and nonsmokers, women smokers had serum levels of T, fT, SHBG, 3 alpha-diol G, and values of FAI, fT/T and 3 alpha-diol G/T, similar to those in nonsmokers. Serum A levels were higher in women smokers than in nonsmokers, although the difference was significant only in postmenopausal women.


The transfer of phosphatidylserine from rat brain microsomes to mitochondria is regulated by microsomal lipid pattern.

We label the phosphatidylserine of rat brain microsomes through the base-exchange reaction and study the export of this lipid to mitochondria in a reconstituted system. We fuse microsomes to liposomes to vary the lipid composition of donor membranes and investigate the effect of membrane lipid pattern on phosphatidylserine movement. The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmented in microsomes and that the radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid. The transfer of phosphatidylserine from microsomes, where it forms through the base-exchange reaction, to mitochondria, where it decarboxylates to phosphatidylethanolamine, is enhanced by phosphatidylserine itself, and by other lipid classes. This is proposed as a part of a possible mechanism for regulating phosphatidylserine metabolism in the brain.


Chemical modification of the monkey brain cytosolic aminopeptidase that cleaves enkephalin to its constituent amino acids. Evidence for essential tyrosine residue (residues).

A cytosolic monkey brain aminopeptidase is known to cleave amino acids sequentially from the amino termini of peptides like enkephalins, angiotensins, and tuftsin. This enzyme is stimulated by ATP-Mg2+ and inactivated by cyclic AMP-dependent phosphorylation. Chemical modification of this aminopeptidase by several amino acid-modifying agents indicated essential lysine, histidine, arginine, cysteine, and tyrosine residues. The involvement of tyrosine residues in the catalytic activity was confirmed by modification by N-acetylimidazole and acetic anhydride, hydroxylamine treatment, and absorbance studies. Moreover, acetic-1-14C anhydride could label the enzyme that was inhibited by ATP or by bestatin, a competitive inhibitor of the enzyme.


Acidic tripeptidyl aminopeptidase in rat liver tritosomes: partial purification and determination of its primary substrate specificity.

We have found the enrichment in rat liver tritosomes of an enzyme which hydrolyzed the synthetic substrate, Ala-Ala-Phe-pNA, under acidic conditions. The enzyme cleaved the substrate in an exopeptidolytic manner, releasing Ala-Ala-Phe. The enzyme was solubilized from tritosomes and partially purified. Beside Ala-Ala-Phe-pNA, the obtained enzyme was shown to cleave angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) and des-1Tyr-dynorphin A 1-8 (Gly-Gly-Phe-Leu-Arg-Arg-Ile) with tripeptide units. The results obtained suggested that the enzyme preferred an aromatic amino acid at the third position of a substrate peptide.


Oxidative modification of lactate dehydrogenase by a non-enzymatic metal ion-catalyzed oxidation system.

Exposure of lactate dehydrogenase from rabbit muscle to the Fe(III)/EDTA/ascorbate oxidation system leads to a time-dependent enzymatic inactivation (rate of inactivation of 7.35 x 10(-3) min-1), as well as to a spontaneous fragmentation of the protein. Fe(III) is the most important compound in this system, having the highest inactivating effects at concentrations above 10 microM. The substrate pyruvate and the products of the enzymatic reaction, when added at high concentration to the full mixture of the system, have a partial protective effect on the catalytic activity.


Different interactions of spermine with a "curved" and a "normal" DNA duplex: (CA4T4G)n and (CT4A4G)n. Gel electrophoresis and circular dichroism studies.

Two nucleotide sequences, (CA4T4G)n and (CT4A4G)n, are generally considered as prototypes of a "curved" and a "normal" DNA duplex on the basis of their different behaviour with respect to physico-chemical and/or biochemical properties. We have studied their gel electrophoretic mobilities in the presence of the biologically significant tetracation spermine and have found that spermine causes a decrease of the gel electrophoretic mobility of "curved" DNA, but does not influence that of "normal" DNA. Circular dichroism spectra of the complexes between spermine and the two examined DNAs show dramatically different features. These findings suggest that different DNA superstructures are relevant in interactions not only with proteins, but also with polycations, such as spermine.


Effects of low-iron status and deficiency of essential fatty acids on some biochemical constituents of rat brain.

The effects of iron and essential fatty acids (EFA) on the development of the brain were studied in young rats. Male weanling rats were maintained on diets deficient in EFA, low in iron (9 ppm) or deficient in both EFA and iron. The low-iron status aggravated signs of EFA deficiency indicating decreases in both growth and brain weight. While the protein content of the brain was considerably reduced, the alkaline phosphatase (ALP) activity increased in all dietary regimes. The lipids and fatty acid components of the brain were affected both qualitatively and quantitatively by the diets. Myelin lipids and docosahexaenoic acid (DHA, 22:6) were reduced by the iron deficient diets and the effect was found to be greater in the deficiency of both EFA and iron. It appears that dietary iron has a significant role to play in the biosynthesis of prostaglandins and in the proper configurational development and functioning of the brain.


Stimulation of prostaglandin production in rat thymic epithelial cells by protein kinase C mediated activation of phospholipase A2.

Tumor promoter (phorbol-12-myristate-13-acetate:PMA) stimulates the production of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cloned rat thymic epithelial cells, TEA3A1. This stimulation of PGE2 production by PMA was blocked by pretreatment of cells with protein kinase C (PKC) inhibitor staurosporin and was abolished in PKC down modulated cells. PMA treatment significantly stimulated the release of arachidonic acid from the cells, but had no effect on arachidonic acid incorporation and on cyclooxygenase enzymatic activity of the cells. These results indicate that PMA stimulates PGE2 production through PKC mediated activation of phospholipase A2 (PLA2) in thymic epithelial cells.


The sea bream liver contains receptors for growth hormone and prolactin.

Different tissues of the black sea bream Mylio macrocephalus including the liver, gills, intestine, muscle, gonad, swim bladder, spleen, heart and kidney were examined for the presence of prolactin and growth hormone receptors. Membranes were prepared from the tissues and 125I-labeled ovine prolactin and bovine growth hormone were used as ligands. It was found that the liver contained the highest level of specific 125I-labeled ovine prolactin and bovine growth hormone binding, suggesting the existence of hepatic prolactin and growth hormone receptors. The protein nature of the hepatic growth hormone receptor was revealed by the reduction of specific 125I-labeled growth hormone binding after treatment of hepatic membranes with trypsin and chymotrypsin.


Anti-oxidant status in an in vitro model for hyperglycaemic lens cataract formation: competition for available nicotinamide adenine dinucleotide phosphate between glutathione reduction and the polyol pathway.

The level of NADPH, total glutathione and sorbitol have been measured in a normal (5mM) and hyperglycaemic (35mM glucose) in vitro rat lens model. In hyperglycaemic conditions, these intermediates are 50%, 84% and 3628% of the normal level. When oxidatively stressed with H2O2 (0.1mM-1.0mM) a gradation in the NADPH and total glutathione decrease is seen, at both glucose levels. This effect is most pronounced in lenses incubated in 35mM glucose, with levels already decreased, the NADPH falls to 15% of the normal lens. Sorbitol levels are correspondingly lower when the lens is oxidatively stressed. The inclusion of the ethyl ester of glutathione alleviates the disruption in anti-oxidant status caused by H2O2 but is unable to restore the NADPH level depleted by hyperglycaemia. These results are discussed in relation to the competitive requirements for NADPH between anti-oxidant preservation and sorbitol formation, as a mechanism for lens opacification in diabetes.


Zidovudine-induced blockade of the expression and function of the erythropoietin receptor.

Treatment of bone marrow progenitor cells (BMPC) with zidovudine (AZT) at various concentrations (0.5 to 20 microM) in vitro for 24 hr caused a concentration-dependent decrease in erythropoietin (Epo) receptor expression. The decrease in Epo receptors correlated with a decline in mRNA levels of the receptor. These results suggest that AZT-induced down-regulation of Epo receptor expression followed by loss of Epo-receptor mediated signal transduction is a significant contributory factor to AZT-induced erythroid toxicity.


The pharmacokinetics of 125I-atrial natriuretic factor in anaesthetized rats. Effects of neutral endopeptidase inhibition with candoxatrilat and of ANF-C receptor blockade.

The effects of candoxatrilat (cis-4-([2-carboxy-3-(2-methoxyethoxy)propyl]-1-cyclopentanecarbonyla mino)- 1-cyclohexane carboxylic acid) and the ring-deleted atrial natriuretic factor (ANF) analogue C-ANF4-23 (des[Gln18, Ser19, Gly20, Leu21, Gly22]ANF4-23-NH2) on the clearance of (3-[125I]iodotyrosyl28)ANF (125I-ANF) were studied in both intact and nephrectomized anaesthetized rats. HPLC analysis was used to verify that the 125I-labelled material isolated by solid phase extraction of rat plasma was intact ANF. In intact animals, clearance of 125I-ANF was biphasic with a T1/2 alpha of 17 sec and T1/2 beta of 95 sec. Volume of distribution (Vd) was 564 mL/kg and plasma clearance (Clp) 248 mL/min/kg. Candoxatrilat, over the dose range 0.01-10 mg/kg i.v., increased T1/2 beta (by a maximum of 56%) and decreased Clp (by up to 52%) with no effect on T1/2 alpha or Vd. C-ANF4-23 (10 micrograms/kg+1 microgram/kg/min i.v.) reduced Vd (by 57%) and Clp (by 54%) with no effect on T1/2 beta, whilst abolishing the T1/2 alpha phase in over 50% of animals. Increasing the dose of C-ANF4-23 did not increase the effect on any of these parameters, apart from a small increase in T1/2 beta. Combining the two agents resulted in a substantial decrease in Clp (76%) whilst the reduction in Vd and increase in T1/2 beta were comparable to those seen with C-ANF4-23 and candoxatrilat alone, respectively. In nephrectomized rats, the pharmacokinetics of 125I-ANF and the changes induced by candoxatrilat were similar to those observed in intact animals, whilst the effects of C-ANF4-23 alone were greater than in intact animals. The combination of C-ANF4-23 and candoxatrilat again produced a substantial increase in T1/2 beta (153%) and decreases in Vd (55%) and Clp (78%) in nephrectomized animals, although these changes could not be distinguished from those seen in intact animals treated with the same combination. Our studies indicate that neutral endopeptidase and ANF-C receptors are both major, and approximately equal, clearance mechanisms for 125I-ANF, together accounting for at least 75% of the total clearance of this peptide in the rat.


Studies of in vivo iron mobilization by chelators in the ferrocene-loaded rat.

The oral efficacy of the oral iron chelators 1,2-dimethyl-3-hydroxypyrid-4-one (CP20), 1,2-diethyl-3-hydroxypyrid-4-one (CP94) and desferrioxamine B (DFO) has been compared with intraperitoneal DFO in an experimental model of iron overload with similar biochemical and biophysical characteristics to those observed for human genetic haemochromatosis. The hepatic iron stores in the ferrocene-loaded rat were relatively stable and did not decrease at the end of the loading period. In contrast, the iron dextran rat model showed a rapid depletion of its iron stores 2 weeks after cessation of intraperitoneal injection. When CP20 and CP94 were administered to the ferrocene-loaded rat model in combination with an iron-free diet there were significant decreases in (i) total homogenate iron and (ii) hepatic ferritin iron when compared to the iron-loaded rat receiving the iron-free diet alone. Desferrioxamine, when administered by gavage, only showed chelation of ferritin iron, while intraperitoneal injection of desferrioxamine showed significant depletion of iron both in the total homogenate and ferritin. Subcellular fractionation of the hepatic organelle clearly showed that where there was depletion of homogenate iron there was a net decrease in the lysosomal fraction, while changes in ferritin iron were reflected by decreases in the cytosolic iron content. Although no assessment of net iron excretion was made, we suggest that the use of this animal model should ascertain the site of chelation by iron chelators.


Competition between paraquat and putrescine for uptake by suspensions of rat alveolar type II cells.

Paraquat and the structurally similar polyamines, such as putrescine and spermidine, are accumulated actively and selectively by the alveolar type II cells via the polyamine uptake system. We report the uptake kinetics of paraquat and putrescine and their mutual inhibition in freshly isolated rat type II cell suspensions. The uptake of paraquat by type II cells exhibited saturation kinetics and could be inhibited in a concentration-dependent manner by putrescine. By applying enzyme kinetic analysis to our experimental data it was demonstrated that the uptake of paraquat or putrescine is inhibited in a partially competitive manner by the respective inhibitor. Thus, we postulate that the polyamine uptake pathway in type II cells for paraquat and putrescine has two separate sites, one for each substrate, and that binding of one leads to a conformational change in the other.


Cytotoxic effects of phenyl-hydroquinone and some hydroquinones on isolated rat hepatocytes.

The cytotoxic effects of phenyl-hydroquinone (PHQ) and some other hydroquinones on freshly isolated rat hepatocytes were investigated. Addition of PHQ (0.5 or 0.75 mM) to the hepatocytes elicited dose-dependent cell death accompanied by losses of intracellular glutathione (GSH), protein thiols and ATP. These effects were related to both PHQ loss and phenyl-benzoquinone (PBQ) formation in the cell suspension. The cytotoxicity of PHQ was prevented by sulphydryl compounds such as cysteine and GSH. In Krebs-Henseleit buffer without cells, loss of PHQ (0.5 mM; initial concentration) and formation of PBQ, monitored by spectral measurements, were inhibited by addition of 50 microM GSH. Further, the oxygen consumption owing to autoxidation of PHQ (0.5 mM) in Krebs-Henseleit buffer without cells was depressed by addition of 50 microM GSH. Among all the hydroquinones tested (at 0.5 mM), tert-butyl-hydroquinone and PHQ were most toxic, followed by hydroquinone and 2,5-di(tert-butyl)-1,4-benzohydroquinone. However, accumulation of cellular malondialdehyde was not affected by these hydroquinones. The toxicity was related to the rate of oxygen consumption by each hydroquinone in the buffer. These results suggest that hydroquinone-induced cytotoxicity is dependent on the rate of oxidation of these compounds as well as the loss of protein thiols.


Effects of snake venom phospholipase A2 toxins (beta-bungarotoxin, notexin) and enzymes (Naja naja atra, Naja nigricollis) on aminophospholipid asymmetry in rat cerebrocortical synaptosomes.

The effects of snake venom phospholipase A2 (PLA2) toxins (beta-bungarotoxin, notexin) and PLA2 enzymes (Naja nigricollis, Naja naja atra) on aminophospholipid asymmetry in rat cerebrocortical synaptic plasma membranes (SPM) were examined. Incubation of intact synaptosomes with 2 mM 2,4,6-trinitrobenzene sulfonic acid (TNBS) for 40 min, under non-penetrating conditions, followed by SPM isolation, allowed us to calculate the percentage of phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the outer leaflet of the SPM, while incubation with disrupted synaptosomes provided total labeling values with the difference representing labeling of the inner leaflet. We found that 30% of the PE and 2% of the PS were in the outer leaflet, with 54% of the PE and 80% of the PS in the inner leaflet; 16% of the PE and 18% of the PS was inaccessible to TNBS. PLA2 toxins and enzymes increased in a concentration-dependent manner the percentage of PS and, to a lesser extent, the percentage of PE in the outer leaflet of the SPM, due to a redistribution from the inner to the outer leaflet. There was no correlation between the PLA2 enzymatic activities and the increased percentage of PS in the outer leaflet of the SPM induced by the PLA2 toxins and enzymes. Alteration of aminophospholipid asymmetry does not explain the greater presynaptic specificity and potencies of the PLA2 toxins as compared to the PLA2 enzymes, but may be associated with the increased acetylcholine release from synaptosomes induced by both the toxins and enzymes.


Gold complexes and activation of human polymorphonuclear leukocytes. Dissociation of changes in membrane potential and oxidative burst.

The effects of the gold compounds on the alteration of membrane potential of polymorphonuclear leukocytes (PMN) in response to various stimulants have been compared with their effects on the oxidative burst. The present studies have shown that gold complexes [auranofin (AF), aurothiomalate (Autm), aurocyanide (Au(CN)2-)] have contrasting effects on the membrane potential of 3,3'-dipentyloxacarbocyanine [di-O-C5(3)] loaded PMN. Au(CN)2- at concentrations which inhibit the oxidative burst of PMN did not affect the membrane depolarization after activation of PMN by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP); Autm slightly stimulated the oxidative burst but had no effect on the depolarization of PMN. In contrast, AF inhibited the depolarization of stimulated PMN to an extent depending upon the concentration of AF, the time of preincubation and the stimulus. The membrane depolarization of PMN caused by PMA, FMLP and concanavalin A (ConA) was inhibited by AF (5 microM) but the depolarization induced by calcium ionophore (A23187) was not affected. AF at the same conditions inhibits the oxidative burst of PMN induced by all these single stimuli including the calcium ionophore. Dissociation of membrane depolarization and superoxide generation caused by AF was also seen in PMN activated by two stimuli. AF (5 microM) had little initial inhibitory effect on the oxidative burst of PMN stimulated by combinations of PMA and ConA or PMA and FMLP whereas it almost totally blocked the depolarization caused by these combinations. Preincubation of cells with 5 microM AF for less than 5 min prior to the addition of PMA allowed membrane depolarization which was followed rapidly by repolarization. None of the gold complexes studied had any effect on the resting membrane potential of PMN.


Inhibition of biliary lipid and protein secretion by cyclosporine A in the rat.

We investigated the effect of cyclosporine A (CyA) administered as a single i.v. dose of 20 and 40 mg/kg body wt, on biliary secretion of cholesterol, phospholipid, bile acid, and lysosomal marker and canalicular plasma membrane marker enzymes in anaesthetized Wistar rats. CyA reduced the concentration and biliary secretion of cholesterol, phospholipid and bile acid to a considerable extent; the inhibitory effect of CyA on the biliary secretion of phospholipid and bile acid was greater than that on cholesterol. The biliary outputs of acid phosphatase (AcP) and gamma-glutamyltransferase (gamma-GT) were also diminished by the drug, all these effects being dose-dependent. Maximum decreases in bile acid secretion were observed 10 min after administration, whereas those of cholesterol and phospholipid were delayed. Bile acid concentrations and secretion returned to pretest values at 30-50 min after CyA injection whereas those of cholesterol and phospholipid remained significantly reduced at this time point. The greater inhibitory effect of CyA on the biliary outputs of phospholipid and bile acid relative to cholesterol secretion together with the asynchronous fall and recovery of bile acid, cholesterol and phospholipid concentrations and secretion alter the cholesterol/bile acid, phospholipid/bile acid and cholesterol/phospholipid molar ratios as well as the lithogenic index, thus suggesting that CyA would uncouple biliary lipid secretion from bile acid secretion. Since under physiological conditions biliary lipid and gamma-GT secretion is related to and dependent upon bile acid secretion, we propose that the CyA-induced inhibition on lipid and gamma-GT secretion is, at least partly, secondary to the fall in bile acid output caused by the drug. However, since CyA inhibits secretory processes independent of the hepatobiliary flux of bile acid, such as the exocytic discharge of AcP, and because it also uncouples biliary lipid from bile acid secretion, other mechanisms and factors involved in lipid and protein secretion (such as intracellular transport, canalicular membrane fluidity and/or intracanalicular events) might also be altered by this drug.


In vitro DNA synthesis by DNA polymerase I and DNA polymerase alpha on single-stranded DNA containing either purine or pyrimidine monoadducts.

The capacity of the large fragment of DNA polymerase I from Escherichia coli and of DNA polymerase alpha from Drosophila embryo to replicate single-stranded M13mp10 DNA containing either purine or pyrimidine monoadducts was compared. The monoadducts were respectively induced by cisplatinum and by furocoumarin photoaddition. For both types of lesions, it is observed that the eukaryotic enzyme is more inhibited than the prokaryotic one. By mapping the arrest sites produced by furocoumarin monoadducts on the synthesis catalysed by DNA polymerase alpha, we show that, in contrast with the photoreaction observed with double-stranded DNA, these compounds do not show a strong sequence specificity in reacting with single-stranded DNA.


Events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced release of mitochondrial Ca2+ and their association with cytotoxicity to renal cells.

Previous studies showed that S-(1,2-dichlorovinyl)-L-cysteine perturbs intracellular Ca2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455-461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced mitochondrial Ca2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)-L-cysteine-induced Ca2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meta-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK1 cells, elevation in the cytosolic Ca2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca2+- and Mg(2+)-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)-L-cysteine-induced cytotoxicity in LLC-PK1 cells is blocked by chelation of cytosolic Ca2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that S-(1,2-dichlorovinyl)-L-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca2+ release. Elevated Ca2+ concentrations may activate Ca(2+)-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.


Heart and liver membrane phospholipid homeostasis during acute administration of various antitumoral drugs to the rat.

The purpose of this study was to investigate in the rat heart and liver the effects of an acute administration of three anthracyclines, doxorubicin, epirubicin and pirarubicin, and an anthracenedione, mitoxantrone, on the membrane peroxidative status, which was estimated by the composition of polyunsaturated fatty acids (PUFA), and on the activities of the enzymes involved in membrane repair processes and lipid hydroperoxide detoxification. Rats were injected for four consecutive days with the drugs or saline (control) and killed 24 hr after the last injection. All the drugs induced an increase in plasma thiobarbituric reactive substances and alpha-tocopherol concentrations, both expressed per milligram of plasma lipids. Plasma vitamin A was decreased by about a factor of two by all the drugs. The fatty acid profile in the heart lipids showed that the polyunsaturated species (20:4 n-6, 22:6 n-3) remained at the same or even higher levels after anthracycline treatment. This can be explained by the fact that the activities of the enzymes involved in either the recycling of membrane phospholipids, such as phospholipases A1 and A2 (EC 3.1.1.4 and EC 3.1.1.32), lysophospholipases (EC 3.1.1.5) and acylCoA:lysophosphatidylcholine acyltransferases (EC 2.3.1.23), or hydroperoxide detoxification, such as selenium-dependent glutathione peroxidase (GSH-PX, EC 1.11.1.9) and glutathione S-transferases (GSH-T, EC 2.1.5.18), were maintained at the same level of activity after the antitumoral treatment. In liver, membrane phospholipid levels of PUFA were maintained as well as the activities of phospholipid-metabolizing enzymes. GSH-PX activity was not affected whereas that of GSH-T was slightly lowered by the drugs. These results suggest that during acute antitumoral-induced lipid peroxidation of membranes, the multi-enzymatic complex of the immediate processes of repair and detoxification is fully operational, allowing the membrane to rapidly recover its functional status. The results are discussed in the context of the equivocal relationships between antitumoral-induced lipid peroxidation and cardiac disturbances.


Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.


Ethylbenzene-mediated induction of cytochrome P450 isozymes in male and female rats.

Male and female Holtzman rats were exposed to ethylbenzene, and the effect on liver microsomal activities was studied. Hydrocarbon- and sex-dependent effects on P450-dependent metabolism of drugs and aromatic hydrocarbons were investigated. Hydrocarbon treatment produced two patterns of induction in cytochrome P450-dependent activities: (1) induction common to both sexes; and (2) induction exclusively in females. Benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, p-nitroanisole O-demethylation and aromatic hydroxylation of toluene were induced in both sexes after rats were exposed to ethylbenzene. The rate of benzphetamine N-demethylation increased 4-fold in females and nearly doubled in males. The increase in O-deethylation of 7-ethoxycoumarin was 3-fold in females and doubled in males, while p-nitroanisole O-demethylation increased 4-fold in both sexes after exposure to ethylbenzene. Ethylbenzene had its greatest effect upon the formation of aromatic hydroxylated metabolites of toluene. Ethylbenzene exposure increased the rate of o-cresol formation by 4- and 9-fold in female and male rats, respectively. The formation rate of p-cresol was undetectable in either sex prior to hydrocarbon exposure; however, after the rats were given ethylbenzene, rates increased to 0.4 nmol/min/mg protein in females and to 0.9 nmol/min/mg protein in the males. Ethylbenzene exposure selectively induced aminopyrine demethylation, aniline hydroxylation, N,N-dimethylnitrosamine N-demethylation (DMNA) and aliphatic hydroxylation of toluene in females. Rates for aminopyrine, aniline, and DMNA were increased 50% over controls, while formation of benzyl alcohol from toluene was enhanced to 260% of control. Western immunoblotting indicated that ethylbenzene treatment induced cytochrome P450 2B1/2B2 to a greater extent in male rats and cytochrome P450 2E1 only in females. Ethylbenzene exposure did not affect significantly the level of cytochrome P450 1A1.


Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver.

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.


The role of lipid peroxidation in the nephrotoxicity of cisplatin.

The possible role of lipid peroxidation in the nephrotoxicity of the antitumour drug cisplatin was studied in vitro. In contrast to Adriamycin, cisplatin did not induce lipid peroxidation in rat kidney microsomes containing a NADPH-generating system. Pretreatment of rat kidney microsomes with cisplatin did not reduce the activity of a microsomal glutathione (GSH)-dependent protective factor against lipid peroxidation induced by Fe(2+)-ascorbate. However, pretreatment of rat kidney microsomes with 0.1 mM N-ethyl maleimide (NEM) did reduce this GSH-dependent protection. Cisplatin also did not reduce the activity of a cytosolic GSH-dependent protective factor against Fe(2+)-ascorbate-induced lipid peroxidation. The results of our experiments indicate that, in contrast to Adriamycin, cisplatin does not induce lipid peroxidation in vitro in various test systems. It also does not destroy microsomal and cytosolic GSH-dependent protective factors against lipid peroxidation.


Peptidyl fluoromethyl ketones as inhibitors of cathepsin B. Implication for treatment of rheumatoid arthritis.

Peptidyl fluoromethyl ketones (FMKs), with the amino acid sequence Phe-Ala held constant but with variable N-terminal groups, were synthesized and tested for inhibition of the cysteine proteinase cathepsin B. The FMKs were effective in inhibiting cathepsin B activity in vitro. The inhibition was time dependent and was not reversed by dialysis, suggesting covalent modification of the enzyme. Cathepsin B activity present in livers and kidneys of rats treated with FMKs was reduced by 22-91% 4 hr after a single oral dose of 25 mg/kg. The FMKs inhibited the severity of inflammation and the extent of cartilage and bone damage in adjuvant-induced arthritis. These effects were seen during the late-stage of the disease with no effect on onset or incidence of disease. This is consistent with inhibition of protease-mediated damage. These FMKs or derivatives may be of clinical value in the treatment of arthritis.


Increase of brain endogenous monoamine oxidase inhibitory activity (tribulin) in experimental audiogenic seizures in rats: evidence for a monoamine oxidase A inhibiting component of tribulin.

Brain tribulin activity in rats with an inherited predisposition to audiogenic epilepsy was studied after seizures of different intensity were induced by an electric bell. Weak seizures (from 0 to 2 arbitrary units) did not produce any changes in endogenous inhibitory activity towards either monoamine oxidase (MAO) A or B. Moderate seizures were characterized by increases in both MAO A and MAO B inhibitory activity (up to 1.9-fold). Complete tonic epileptiform seizures with total areflexia (4 arbitrary units) induced further augmentation (up to 2.5-fold) of MAO A but not of MAO B inhibitory activity. This dissociation between the two inhibitory activities points to the existence of a separate MAO A-inhibiting component of brain tribulin which is different from isatin.


Inhibition and induction of cytochrome P450 2B1 in rat liver by promazine and chlorpromazine.

Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro, the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O-depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O-deethylase was also observed following administration of both drugs to rats, suggesting induction of P450 1A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.


Microsomal cytochrome P450 in human brain regions.

Cytochrome P450 (P450) levels were quantitated in microsomes from human brain regions obtained at autopsy. The reduced carbon monoxide binding spectra of cortical microsomes showed two absorption maxima at 449 and 425 nm. On solubilization of the microsomes, essentially a single peak was observed at 449 nm. The P450 levels in human brain cortical microsomes varied from 0.03 to 0.12 nmol/mg protein among the seven samples examined. The concentration of the hemeprotein present as nmol/g tissue was highest in the brain stem and cerebellum and lowest in the striatum and hippocampus.


Reduced carnitine and ketogenesis in the pivampicillin treated rat.

Pivampicillin (630 mg/kg body wt) given daily by stomach tube induced carnitine deficiency in the rat. The carnitine concentrations after 24 days were significantly reduced to (mean +/- SD) 34 +/- 2, 27 +/- 7, 70 +/- 18, 75 +/- 16 and 49 +/- 4% of controls in plasma, liver, muscle, heart and kidney, respectively, without any further reduction after 36 days. Pivampicillin treatment reduced the carnitine concentrations in the liver of the 48 hr fasted rat to about 1/2 of the controls after 6 days. The concentration of beta-hydroxybutyrate was significantly reduced up to 14 days of treatment, and again increased. There was no significant difference in the free fatty acid concentrations between treated and control rats. Thus, pivampicillin treatment induced carnitine deficiency in the rat, but not as pronounced as seen in humans. This is possibly caused by adjustment of bacterial flora in the gut or altered renal mechanisms. The pivampicillin-treated rat, therefore, is not a useful model for pronounced carnitine deficiency in humans.


Active-site directed irreversible inhibition of diamine oxidase by a homologous series of aziridinylalkylamines.

Three electrophilic homologous aminoalkylaziridine analogues of putrescine, cadaverine, and 1,3-diaminopropane were synthesized and found to represent a mechanistically distinct class of irreversible inhibitors of diamine oxidase. The putrescine analogue, N-(4-aminobutyl)aziridine gave the lowest calculated IC50 value, whereas N-(3-aminopropyl)aziridine, an analogue of the poorest substrate of the series, showed the highest IC50. The findings suggest that the aziridinylalkylamines tested are site-directed agents that form irreversible complexes at the active site of diamine oxidase. Affinity of the inhibitors for the active site appeared to be dependent on alkyl chain length, suggesting that binding promotes the reactivity of the aziridinyl group.


Effect of various flavonoids on lysosomes subjected to an oxidative or an osmotic stress.

When a light mitochondrial fraction (L fraction) of rat liver is incubated in the presence of an oxygen free radical generating system (xanthine-xanthine oxidase), the free activity of N-acetylglucosaminidase (NAGase) increases as a result of the deterioration of the lysosomal membrane. Various flavonoids are able to prevent this phenomenon, others are ineffective. Comparative activity studies suggest the importance of the presence of two OH groups in orthosubstitution in the B ring and of an OH in the 3 position. Flavan-type flavonoids behave like their related flavonoids; d-catechin also opposes lysosome disruption. Kaempferol, quercetin, 7,8-dihydroxyflavone and d-catechin inhibit lipoperoxidation occurring in an L fraction incubated with the xanthine oxidase system as ascertained by malondialdehyde (MDA) production. For kaempferol and quercetin, such an inhibition parallels the prevention of NAGase release; this is not the case for the two other compounds where inhibition of NAGase release takes place at a flavonoid concentration lower than that required to oppose MDA production. Morphological observations performed on purified lysosomes confirm the biochemical results. Some flavonoids are also able to prevent release of NAGase caused by the incubation of an L fraction in isoosmotic glucose. Only flavone and hydroxyflavones are effective. It is proposed that the protective effect of flavonoids on lysosomes subjected to oxygen free radicals does not only originate from their scavenger and antilipoperoxidant properties; a more direct action on lysosomal membrane making it more resistant to oxidative aggression has to be considered. The prevention by some flavonoids of lysosome osmotic disruption in isoosmotic glucose could be the result of an inhibition of glucose translocation through the lysosomal membrane.


Stereoselective conjugation of 2-bromocarboxylic acids and their urea derivatives by rat liver glutathione transferase 12-12 and some other isoforms.

Glutathione (GSH) conjugation of the separate enantiomers of five 2-bromocarboxylic acids and some of their urea derivatives by rat liver GSH transferases (GSTs) was studied. The liver cytosolic fraction conjugated all compounds, except for (R)-2-bromoisovaleric acid, with a variable degree of stereoselectivity. A GST pool, prepared by S-hexyl-GSH affinity chromatography, conjugated the urea derivatives at a somewhat higher rate but had very little activity towards the carboxylic acids, indicating that much activity towards the latter substrates was due to transferases not bound by the affinity column. Therefore, the activity was studied of some pure GSTs that are bound only slightly by the affinity column towards the separate enantiomers of 2-bromovaleric acid (BV), its urea derivative and 2-bromo-3-phenylpropionic acid (BPP). No activity was detected with transferases 5-5 and 8-8. Transferase 1-1 was active towards all compounds with high activity towards the urea derivatives. Transferase 12-12 showed high, stereospecific activity towards the R enantiomers of BV, its urea derivative and BPP.


Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.


Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth.

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.


Is there an association between an increase in c-myc RNA steady state levels and c-myc methylation in HL-60 cells treated with 3-deaza-(+/-)-aristeromycin, an indirect inhibitor of methylation?

Alteration in gene expression of the proto-oncogene c-myc in HL-60 cells is associated with differentiation of these cells. We have studied the steady state levels of c-myc transcripts, the levels of transmethylation metabolites S-adenosylmethionine and S-adenosyl-homocysteine and the methylation pattern of the c-myc gene after treatment of HL-60 cells with the transmethylation inhibitor and granulocytic inducer, 3-deaza-(+/-)-aristeromycin. A transient increase in c-myc RNA levels after 45 min of drug exposure was observed which was accompanied by changes in the ratio of transmethylation metabolites in both whole cells and nuclei. The changes in transmethylation metabolites in whole cells, although compatible with levels frequently associated with hypomethylation of cellular components, caused no changes in methylation of c-myc DNA sequences of the HL-60 cells as detected by HpaII or MspI digestion and Southern blotting.


The effects of ibuprofen enantiomers on hepatocyte intermediary metabolism and mitochondrial respiration.

In vivo and in vitro (-)R-ibuprofen is inverted to the (+)S antipode via stereoselective formation of an R-ibuprofenyl-CoA intermediate. In this study the effects of (-)R- and (+)S-ibuprofen on metabolism and respiration were studied using isolated rat hepatocytes and mitochondria. R-Ibuprofen significantly increased the lactate to pyruvate ratio, perturbed mitochondrial ketogenesis as evidenced by alterations in the beta-hydroxybutyrate to acetoacetate ratio and uncoupled mitochondrial oxidative phosphorylation. In addition, substantial dose- and time-dependent sequestration of reduced CoA (CoASH) occurred in the presence of the R enantiomer. Similarly, S-ibuprofen altered both the cytosolic and mitochondrial redox states although the magnitude of the effect was substantially less than that observed with the R enantiomer. In contrast to R-ibuprofen, S-ibuprofen did not uncouple oxidative phosphorylation or sequester hepatocyte CoASH. It is proposed that the perturbations observed in hepatocyte intermediary metabolism and mitochondrial function are attributable to a combination of the direct effects of R-ibuprofen per se and the sequestration of CoASH as R-ibuprofenyl-CoA during the process of chiral inversion. On the basis of these results, R-ibuprofen should be considered more in terms of metabolism to a reactive acyl-CoA intermediate rather than as a pro-drug for the pharmacologically active S-enantiomer.


Effect of dietary fat on the in vitro hepatotoxicity of paracetamol.

In the present study, we have examined the effect of dietary fat on paracetamol-induced liver injury in an in vitro rat liver slice model. Rats were fed, for 7-10 days, diets containing either butter or polyunsaturated vegetable margarine, two fat sources commonly consumed in the human diet. Liver slices were then exposed to paracetamol for 2 hr and further incubated for 4 hr without paracetamol. Cell damage in the slices was quantified at 6 hr by measuring leakage of lactate dehydrogenase, increase in water content and potassium loss. Covalent binding of radioactive paracetamol to liver and the membrane fatty acid composition of the liver were also measured. Liver slices from rats fed butter diets were significantly more sensitive to the toxic effects of paracetamol than those from margarine fed rats. The membrane lipid composition of the livers also reflected the differing fatty acid content of the two diets.


Rapid chloroquine efflux phenotype in both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. A correlation of chloroquine sensitivity with energy-dependent drug accumulation.

Recent reports suggest that lower levels of chloroquine accumulation in chloroquine-resistant isolates of Plasmodium falciparum are achieved by energy-dependent chloroquine efflux from resistant parasites. In support of this argument, a rapid chloroquine efflux phenotype has been observed in some chloroquine-resistant isolates of P. falciparum. In this study, no relationship was found between chloroquine sensitivity and the rate of [3H]chloroquine efflux from four isolates of P. falciparum with a greater than 10-fold range in sensitivity to chloroquine. All the isolates tested displayed the rapid efflux phenotype, irrespective of sensitivity. However, chloroquine sensitivity of these isolates was correlated with energy-dependent rate of drug accumulation into these parasites. Verapamil and a variety of other compounds reverse chloroquine resistance. The reversal mechanism is assumed to result from competition between verapamil and chloroquine for efflux protein translocation sites, thus causing an increase in steady-state accumulation of chloroquine and hence a return to sensitivity. Verapamil accumulation at a steady-state is increased by chloroquine, possibly indicating competition for efflux of the two substrates. Increases in steady-state verapamil concentrations caused by chloroquine were identical in sensitive and resistant strains, suggesting that similar capacity efflux pumps may exist in these isolates. These data suggest that differences in steady-state chloroquine accumulation seen in these isolates can be attributed to changes in the chloroquine concentrating mechanism rather than the efflux pump. It seems likely that chloroquine resistance generally in P. falciparum, results at least in part from a change in the drug concentrating mechanism and that changes in efflux rates per se are insufficient to explain chloroquine resistance.


Potencies of protein kinase C inhibitors are dependent on the activators used to stimulate the enzyme.

The aim was to examine systematically the potencies of protein kinase C inhibitors as a function of the kinase activator. Protein kinase C is activated by at least four stimulators: calcium plus phosphatidylserine (Ca/PS), phorbol 12-myristate 13-acetate plus PS (PS/PMA), arachidonic acid plus calcium (Ca/AA) and the synthetic peptide activator PCK530-558. With histone or GS1-12 as substrates, protein kinase C was maximally activated by Ca/PS, or to maxima of 62%, 89% or 82% with PS/PMA, Ca/AA or PKC530-558, respectively. One group of inhibitors, including H-7 and staurosporine, were equipotent, regardless of the activator. All other inhibitors showed variable selectivity, dependent upon the activator. A second group of inhibitors, including sphingosine and lipophosphoglycan, were eight or 200 times more potent for inhibition of PS/PMA-stimulated activity (relative to Ca/PS) and a third group, including retinal and palmitoylcarnitine, were 14 or 262 times more potent towards Ca/PS-stimulated activity. A final group (rhodamine 6G) was nine times more potent when Ca/AA was the activator. Similar results were obtained using the endogenous substrates dephosphin or MARCKS in synaptosol. Phosphorylation of MARCKS was stimulated by PS/PMA or Ca/PS, while phosphorylation of dephosphin was stimulated only by Ca/PS. The phosphorylation of either by Ca/PS-activated kinase was nine times more potently inhibited by palmitoylcarnitine, while phosphorylation of MARCKS by PS/PMA-activated kinase was 10 times more potently inhibited by sphingosine. H-7 inhibited both at similar concentrations. A model encompasses these differences in potency if the inhibitors are divided into four groups (A-D) according to their competitive inhibition with the appropriate activator or at the active site. The non-selective inhibitors interact at the active sites of protein kinase C (group A). The compounds which preferentially inhibit PS/PMA-activated kinase (sphingosine and lipophosphoglycan) are competitive inhibitors of PMA and 1,2-diacylglycerol (group B), those selective for Ca/PS-activated kinase (palmitoylcarnitine and retinal) are competitive with PS (group C) and those selective for Ca-AA activation (rhodamine 6G) are likely to be competitive with fatty acid (group D). Therefore, the effectiveness of protein kinase C inhibitors is dependent upon the activator employed.


Induction of mutations in V79-4 mammalian cells under hypoxic and aerobic conditions by the cytotoxic 2-nitroimidazole-aziridines, RSU-1069 and RSU-1131. The influence of cellular glutathione.

Incubation of the 2-nitroimidazole-aziridine, RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridinyl)-2-propanol], and its monomethylaziridine analogue, RSU-1131 [1-(2-nitro-1-imidazolyl)-3-(1-(2-methylaziridinyl))-2-propanol], with V79-4 mammalian cells for 2 hr under aerobic or hypoxic conditions induces mutations as measured at the hypoxanthine phosphoribosyl transferase locus. The ability of these agents to induce mutations is increased by a factor of 12-14 under hypoxic conditions. The increased cytotoxicity of these agents under hypoxic conditions was confirmed following a 2 hr incubation period. Decreasing the glutathione (GSH) content of the cells with buthionine-(S,R)-sulphoximine to < 1% of the control generally results in an increase in the cytotoxicity and mutagenicity of these agents under both aerobic and hypoxic conditions. Since these agents do not modify the cellular GSH levels, it is inferred that the thiols partially detoxify through removal of a reactive metabolite of the agents, under hypoxic conditions, or removal of known DNA adducts, and not through their interaction with the agents themselves. Under aerobic conditions, the formation of mutations is consistent with the established monofunctional action of these agents whereas under hypoxic conditions the bifunctional action predominates for mutation induction, based upon the large differential aerobic:hypoxic effect. From a comparison of the number of mutations per lethal event, the effect of thiol depletion is more pronounced for cytotoxicity than for mutation induction by these agents. In summary, these agents are considered to be weak mutagens towards V79-4 cells under aerobic conditions when compared with other DNA alkylating agents, although they are more potent under anoxic conditions.


The relationship between the activity of methionine synthase and the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the brain and other tissues of the pig.

Using nitrous oxide to inactivate methionine synthase in vivo, the relationship of the activity of methionine synthase to the S-adenosylmethionine (AdoMet)/S-adenosylhomocysteine (AdoHcy) ratio was examined in neural and other tissues of the pig. Pigs were exposed to 15% nitrous oxide for varying intervals of up to 7 days or studied at varying intervals of recovery in air after 7 days nitrous oxide inhalation, and the rate of inactivation or resynthesis of methionine synthase was related to the corresponding AdoMet/AdoHcy ratios. The rate of inactivation of enzyme during nitrous oxide exposure was considerably faster in the liver and kidney than in the brain and spinal cord with activity levelling off between 10% and 20% of control values. The AdoMet/AdoHcy ratio fell in all tissues during nitrous oxide treatment, the fall being most marked in the brain and spinal cord where a 10-fold change occurred. This change was attributed mainly to a rise in AdoHcy levels. The recovery pattern of methionine synthase was broadly linear but was slower in the spinal cord (0.10 +/- 0.03% per hr; mean +/- SEM) than in any other tissue examined including brain (0.35 +/- 0.04% per hr). Correspondingly, the recovery of the AdoMet/AdoHcy ratio was also significantly slower in the spinal cord. When values for exposure and recovery were combined there was a significant correlation between the activity of methionine synthase and the AdoMet/AdoHcy ratio in both the brain (r = 0.90; P < 0.001) and the spinal cord (r = 0.92; P < 0.001). These results support the concept that the AdoMet/AdoHcy ratio is closely related to the pathogenic process which produces the neurologic lesions associated with a reduction in methionine synthase activity.


Evaluation of iron binding and peroxide-mediated toxicity in rat hepatocytes.

A novel assay was developed to determine subnanomolar amounts of Fenton-reactive iron (FRI) in biological tissues. FRI represents that pool of iron that is redox active and capable of participating in a model Fenton reaction. The FRI was used to identify a kinetically-defined cellular iron binding site. This site displays positive cooperativity, with apparent kinetic constants of Kd = 10.6 microM, Bmax = 20.7 nmol/mg protein, and the Hill coefficient = 1.4. After addition of exogenous ferrous ammonium sulfate to hepatocytes, binding occurred within a few seconds and was stable for at least an hour. Free extracellular iron, but not bound iron, stimulated lipid peroxidation in hepatocytes. In contrast, bound but not free iron produced a concentration-dependent increase in tert-butyl hydroperoxide (TBH)-mediated toxicity, suggesting the toxicological relevance of bound, rather than free iron. Furthermore, the hydroxyl radical scavengers mannitol and 2-deoxyribose inhibited Fe2/TBH-mediated lipid peroxidation, but not cell killing, suggesting that hydroxyl radical may not be involved in the critical toxic event. The divalent cations Mn2+ and Co2+ inhibited iron-mediated hepatocyte killing in the presence of TBH, but only if added prior to Fe2+. Mn2+, but not Co2+, inhibited Fe(2+)-mediated lipid peroxidation regardless of the order of addition. These results indicate the existence of a specific, kinetically-defined cellular iron binding site. Such binding is involved in peroxide-mediated toxicity, but independent of lipid peroxidation. The specific nature of this site and involvement with other forms of chemical intoxication or cellular iron homeostasis are unknown.


Depletion of a discrete nuclear glutathione pool by oxidative stress, but not by buthionine sulfoximine. Correlation with enhanced alkylating agent cytotoxicity to human melanoma cells in vitro.

The existence of a distinct pool of glutathione in the nucleus of cultured human melanoma cells was demonstrated. Melanoma cell nuclei contained 13-35 pmol of glutathione/10(6) nuclei, or approximately 0.4-1.3% of the total cellular glutathione. This nuclear glutathione pool resisted depletion by buthionine sulfoximine, an agent that inhibits glutathione synthesis, but was rapidly and reversibly depleted by subtoxic concentrations of Adriamycin plus carmustine, two agents that promote oxidation of glutathione without permitting its regeneration through enzymatic reduction of glutathione disulfide. The ability of Adriamycin plus carmustine to deplete this small but significant pool of glutathione in the cell nucleus may explain why these agents potentiate the cytotoxic effects of the DNA-alkylating agent melphalan to a much higher degree than does buthionine sulfoximine at concentrations that are equipotent in depleting cytosolic glutathione.


Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro.

A disruption of calcium homeostasis, leading to a sustained increase in cytosolic calcium levels, has been associated with cytotoxicity in response to a variety of agents in different cell types. We have observed that administration of a single high dose or multiple lower doses of the carcinogenic nephrotoxin ochratoxin A (OTA) to rats resulted in an increase of the renal cortex endoplasmic reticulum ATP-dependent calcium pump activity. The increase was very rapid, being evident within 10 min of OTA administration and remained elevated for at least 6 hr thereafter. The increase in calcium pump activity was inconsistent with previous observations that OTA enhances lipid peroxidation (ethane exhalation) in vivo, a condition known to inhibit the calcium pump. However, no evidence of enhanced lipid peroxidation was observed in the renal cortex since levels of malondialdehyde and a variety of antioxidant enzymes including catalase, DT-diaphorase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were either unaltered or reduced. In in vitro studies, addition of OTA to cortex microsomes during calcium uptake inhibited the uptake process although the effect was reversible. Preincubation of microsomes with NADPH had a profound inhibitory effect on calcium uptake but inclusion of OTA was able to reverse the inhibition. Changes in the rates of microsomal calcium uptake correlated with changes in the steady-state levels of the phosphorylated Mg2+/Ca(2+)-ATPase intermediate, suggesting that in vivo/in vitro conditions were affecting the rate of enzyme phosphorylation.


Binding of 2-acetylpyridine-5-[(2-chloroanilino)thiocarbonyl] thiocarbonohydrazone (BW348U87) to human serum albumin.

Dissociation constants and rate constants for the binding of 2-acetylpyridine-5-[(2-chloroanilino)thiocarbonyl]thiocar bonohydrazone (I, BW348U87), an agent that enhances the antiherpetic efficacy of acyclovir, to human serum albumin have been determined. I quenched the fluorescence of human serum albumin, whereas the absorbance of I at 370 nm increased upon binding to the protein. The absorbance change was attributed to preferential binding of anionic I (pKa 7.0). Titration data indicated multiple binding sites for I. The dissociation constant of the high-affinity site was 0.11 microM. The time course for binding of I to 100 nM human serum albumin was biphasic. The early and late phases were described by first-order rate constants that had maximal values of 100 and 11 sec-1, respectively. The rate constant for the dissociation of I from human serum albumin was estimated to be 6 sec-1. Dodecyl sulfate and octanoate displaced I from human serum albumin.I with rate constants of 4.5 and 7.3 sec-1, respectively. Since the fluorescence emission spectrum of human serum albumin and the absorption spectrum of I overlapped significantly (the spectral overlap integral, J, was 2.6 x 10(-14) M-1 cm3), the possibility of Förster dipole-dipole energy transfer was considered. However, a significant fraction of the fluorescence quenching by I resulted from a conformational change in the protein upon binding of I and was not the result of dipole-dipole energy transfer. Nonetheless, the distance between one of the binding regions for I on human serum albumin and a tryptophan residue in the protein was estimated to be 31 A by this method. The high affinity of I for albumin could be related to its low hematological toxicity.


Suppressive effects of the endothelin receptor (ETA) antagonist BQ-123 on ET-1-induced reduction of lipoprotein lipase activity in 3T3-L1 adipocytes.

Endothelin (ET)-1 reduced heparin-releasable lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes in a concentration-dependent manner. However, a selective ETB receptor agonist, [Ala1,3,11,15]ET-1, did not act like ET-1. The ET-1-induced decrease in LPL activity was suppressed by a selective ETA receptor antagonist, BQ-123: the concentration-response curve for the ET-1 reduction of LPL activity was shifted to the right in the presence of BQ-123 in a concentration-dependent manner. This antagonistic effect of BQ-123 clarifies that the ETA receptor is responsible for the ET-1-induced reduction of LPL activity in 3T3-L1 adipocytes, which suggests that there is therapeutic potential for ETA antagonists in LPL-related lipoprotein disorders.


In vitro and in vivo effects of the anti-inflammatory peptides, antiflammins.

The anti-inflammatory synthetic polypeptides referred to as antiflammins are thought to be inhibitors of phospholipase A2 (PLA2) (EC 3.1.1.4). These peptides are from the sequence of amino acids of greatest similarity between uteroglobin and lipocortin I. The effect of these peptides was studied on PLA2 activation in rat platelets and on acute inflammatory models after local or parenteral administration of drug. We found that antiflammins decreased collagen-induced platelet activation, but had no effect when arachidonic acid was used as activator. The peptides were able to inhibit acute inflammatory processes induced by carrageenan or phorbol esters when administered locally or parenterally. However, antiflammins had no effect on inflammation induced by exogenous PLA2 administration. These results indicate that the antiflammins may have a direct inhibitory effect on PLA2 activation but not on the enzyme or enzyme-substrate interaction.


Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides.

The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD+ enhanced YADH inactivation at NAD+/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD+, which might counteract the protective effect of NADH. Under similar conditions, NADP+ did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD+ couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD+ ratios.


Effect of serum-free medium on cytochrome P450-dependent metabolism and toxicity in rat cultured hepatocytes.

Cytochrome P450 (P450)-dependent activities in homogenates of rat hepatocytes cultured for 96 hr in serum-free and serum-containing medium were compared. Benzphetamine and erythromycin N-demethylases, 7-methoxy- and propoxy-coumarin O-dealkylases, p-nitrophenol hydroxylase and 7-ethoxy- and pentoxy-resorufin O-dealkylases were all maintained at higher levels in hepatocytes cultured in serum-free medium, although there was some selectivity with respect to the extent of the maintenance relative to the activities in fresh cells. The toxicities of coumarin, precocene I and precocene II to 24 hr hepatocyte cultures, determined as decreased survival, were also shown to be increased in serum-free medium. However, the magnitude of the difference between media with respect to the toxicity of precocene II was decreased in hepatocytes cultured for 72 hr. The observed increase in toxicity is consistent with the improved maintenance of P450 in hepatocytes cultured in serum-free medium, although there is still a selective decline in P450 activities and toxicity with increased time in culture. The activity of alcohol dehydrogenase and the toxicity of allyl alcohol were similar in hepatocytes cultured in serum-free and serum-containing medium for 96 hr. The absence of serum did not affect the non-protein sulphydryl content of the cultures.


Impairment of mitochondrial 5-aminolevulinic acid synthase activity in Gunn rat liver.

Gunn rats are characterized by hereditary hyperbilirubinemia and a decrease, when compared to Wistar rats, in hepatic heme pool size which could result from an alteration of mitochondrial functions. Unconjugated bilirubin present in Gunn rat liver did not modify either the ultrastructural morphology or oxidative metabolism of the mitochondria as compared to those in Wistar rat liver. However, 5-aminolevulinic acid synthase activity is reduced by nearly 40% in Gunn rat liver mitochondria, thus explaining the reduced size of the hepatic heme pool.


Influence of palmitate and benzoate on the unidirectional chiral inversion of ibuprofen in isolated rat hepatocytes.

The influence of benzoic acid, a typical substrate of medium-chain acyl-CoA synthetase, and of palmitic acid, a substrate of long-chain acyl-CoA synthetase, on the metabolic chiral inversion of ibuprofen was investigated in freshly isolated hepatocytes. It was shown that the conjugation of benzoid to hippuric acid does not influence the chiral inversion of ibuprofen. In contrast, palmitic acid inhibited markedly the R-to-S inversion of ibuprofen. It was concluded that this inhibition is due to competition between (R)-ibuprofen and palmitic acid for long-chain acyl-CoA synthetases.


Oxidative metabolism of cinnarizine in rat liver microsomes.

The oxidative metabolism of cinnarizine (CZ) [1-(diphenylmethyl)-4-(3-phenyl-2-propenyl)-piperazine] to 1-(diphenylmethyl)piperazine (M-1), 1-(diphenylmethyl)-4-[3-(4'-hydroxyphenyl)-2-propenyl]piperazine (M-2), benzophenone (M-3) and 1-[4'-hydroxyphenyl)-phenylmethyl]-4-(3- phenyl-2-propenyl)piperazine (M-4) has been studied in rat liver microsomes. In Wistar rats, kinetic analysis revealed sex differences (male > female) in the Km values for formation of all the metabolites and the Vmax values for the formation of M-1, M-3 and M-4. The reactions required NADPH, and were inhibited by carbon monoxide and SKF 525-A. Only M-2 formation was suppressed by sparteine or metoprolol, and was significantly lower in female Dark Agouti rats than in Wistar rats of both sexes. The results suggest that CZ is oxidized by cytochrome P450, and M-2 formation is related to debrisoquine/sparteine-type polymorphic drug oxidation.


Glutathione and bile acid synthesis. Effect of GSH content of HepG2 cells on the activity and mRNA levels of cholesterol 7 alpha-hydroxylase.

Cholesterol 7 alpha-hydroxylase (CH-7 alpha) activity in HepG2 cells depleted of glutathione (GSH) was reduced significantly (P < 0.05) compared to that in untreated controls. Northern blot analysis of poly A+ mRNA isolated from GSH-depleted and control HepG2 cells showed that there was a reduction in mRNA for CH-7 alpha in treated HepG2 cells that was commensurate with the reduction in CH-7 alpha activity. The fact that total RNA, rRNA, and mRNA for beta fibrinogen were unaltered by the depletion of GSH suggests that the change in steady-state CH-7 alpha mRNA content is specifically sensitive to GSH content. This observation represents the first demonstration, for human liver cells, that there is an interaction between GSH levels and the regulation of CH-7 alpha mRNA levels.


Qualitative differences in [Ca2+]i increases and InsP3 generation following stimulation of N1E-115 cells with micromolar and millimolar ATP.

Addition of ATP (100 microM) to populations of the neuronal cell line N1E-115 caused a transient increase in intracellular calcium ([Ca2+]i) which rapidly reached a peak (maximum of 243 +/- 7 nM above basal) and returned to basal after approximately 50 sec. The response was concentration-dependent (EC50 21 +/- 4 microM) and was unchanged when calcium was omitted from the extracellular medium. Transient increases in D-myo-inositol-1,4,5-trisphosphate levels (InsP3) were also observed over a similar time period. Addition of millimolar ATP, however, produced characteristically different responses; [Ca2+]i again increased rapidly (reaching a maximum of 639 +/- 23 nM above basal) but returned to a new maintained plateau (274 +/- 34 nM) which was abolished by the inorganic calcium channel blocker, nickel (1 mM), and omission of calcium from the extracellular medium. InsP3 levels were also maintained but were, however, unaffected by nickel or removal of extracellular calcium. The qualitative difference in the mechanism of calcium elevation produced with millimolar ATP, compared with lower concentrations, suggests that the N1E-115 cells might also contain a low affinity P2 receptor coupled with a calcium channel.


Differing response of the glutathione system to fasting in neonatal and adult guinea pigs.

Adult, term neonatal and 3 day preterm neonatal guinea pigs were fasted for 48 hr, and the glutathione concentrations of the liver and lung assessed. In adult animals, glutathione concentration decreased by 43% in the liver and 29% in the lung with respect to fed controls. The decrease in liver glutathione was associated with a 75% reduction in the hepatic activity of tau-glutamyltranspeptidase (tau GGT). Conversely, both liver and lung glutathione levels in preterm pups remained unchanged following 48 hr food restriction. Likewise, hepatic tau GGT, glutathione reductase (GRed) and glutathione peroxidase (GPx) activities were unchanged by fasting in preterm pups. Fasting increased pulmonary GPx activity by 27% in these pups. In fasted, term animals, substantial increases in both lung (65%) and liver (80%) glutathione concentrations were observed, with concomitant increases in GPx and GRed activities. Hepatic tau GGT activity was significantly reduced (57%) in term pups. These results may suggest that the neonatal guinea pig can maintain tissue glutathione status during periods of nutrient stress, through an increased capacity for recycling oxidized glutathione and a decrease in turnover of the tripeptide. Guinea pig neonates are therefore able to resist starvation-induced decreases in tissue glutathione levels seen in adult rodents. If this is a general neonatal response it may have important clinical implications in the treatment of preterm babies.