Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine. The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.


Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C. The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PKC) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was 100-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (1 and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC.


Characterization of a rat insulin-like growth factor I gene promoter. Rat IGF-I mRNAs contain one of two alternative 5'-untranslated regions which are encoded by alternative exons (exons 1 and 2) and whose expression is controlled by alternative promoter elements. We investigated the ability of fragments of DNA which contain exon 1 and its 5'-flanking region to regulate transcription of a luciferase reporter gene in transient transfection assays. Maximal promoter activity was obtained with a construct which contained 412 bp of 5'-flanking region, while constructs which contained 1120 and 1690 bp of 5'-flanking region induced approximately 50% less enzymatic activity. Mapping of transcription start sites by RNase protection assay demonstrated that native start sites were used by these constructs, although the relative use of different start sites was different from start site usage by the endogenous gene. These data demonstrate that the 5'-flanking region of exon 1 is capable of regulating transcription of IGF-I mRNAs.


Age-associated oxygen damage and mutations in mitochondrial DNA in human hearts. Some mutations in mitochondrial DNA (mtDNA) causing a number of neuromuscular diseases are suggested to arise spontaneously during the life of an individual. To substantiate the extent and the rate of these somatic mutations, mtDNA specimens from post-mortem human heart muscles of subjects in differing age groups were hydrolyzed. 8-Hydroxy-deoxyguanosine (8-OH-dG), a hydroxyl-radical adduct of deoxyguanosine, in mtDNA, was quantitatively determined using a micro high-performance liquid chromatography/mass spectrometry system. In each specimen, the mtDNA with a 7.4 kilo base-pair deletion was quantified by the kinetic polymerase chain reaction method. In association with age, the 8-OH-dG content accumulated exponentially up to 1.5% with a correlative increase in the content of the deleted mtDNA up to 7%. Clear correlation between the 8-OH-dG content in mtDNA and the population of mtDNA with a deletion (r = 0.93, P < 0.01) gives insight into the mechanism for the generation of a large deletion. These results indicate that accumulation of somatically acquired oxygen damage together with age-associated mutations in mtDNA which lead to bioenergetic deficiency and the heart muscle weakness are inevitable in human life.


Irradiation increases levels of GM-CSF through RNA stabilization which requires an AU-rich region in cancer cells. Granulocyte/macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates a wide range of myeloid hematopoietic cells; RNAs coding for many oncogenes and cytokines including GM-CSF have a very short half-life. The motif of AUUUA is a highly conserved sequence in the 3'untranslated regions (3'UTR) of these transcripts and is repeated a number of times in these short-lived cytokines and oncogenes. These sequences play a major role in controlling stability of these transcripts. Human cancer cells were transfected with a chimeric rabbit beta-globin gene linked to either a 58 bp sequence of the AT-rich region from GM-CSF or a control sequence. We have found that irradiation stimulates accumulation of GM-CSF, interleukin-6 (IL-6), and IL-1 beta RNAs. In addition, this accumulation of GM-CSF was at least, in part, a result of increased stabilization of GM-CSF transcripts. Further experiments showed that irradiation increased levels of the chimeric beta-globin transcripts containing AUUUA sequences from GM-CSF, but not those containing the control sequences. Our results suggest that irradiation increases expression of GM-CSF RNA and that posttranscriptional stabilization requiring AUUUA sequences probably is in part one of the mechanisms producing the increased levels of GM-CSF RNA by irradiation.


Chemotaxis of human blood monocytes toward endothelin-1 and the influence of calcium channel blockers. The adherence of monocytes to the arterial endothelium followed by its migration into the arterial intima is the earliest event in atherogenesis. The vasoconstrictive peptide, Endothelin-1 (ET-1), is elevated in patients with atherosclerosis. We were interested to know whether ET-1 was a chemoattractant for blood monocytes. Using the modified membrane filter technique for chemotaxsis assessment, ET-1 increased monocyte chemotaxis in a dose-dependent manner. Ca2+ channel blockers, Nifedipine, Diltiazem and Verapamil (5 microM), reduced ET-1 chemotaxsis more than 60% (P < 0.001). Aspirin and Indomethacin (1 mM and 100 microM, respectively) reduced migration by 23% (P < 0.05). Alpha-Lipoic acid, Probucol and Neomycin (100 microM) were also migration inhibitory (37%, P < 0.01). These results suggest that ET-1 is a strong chemoattractant for blood monocytes; Ca2+ influx is probably the major stimulus for the accelerated migration induced by ET-1.


Speculations on the substrate structure-activity relationship (SSAR) of cytochrome P450 enzymes. This brief review attempts to define the SSAR of two families of cytochrome P450. With P4502D catalytic competence is achieved by tight ionic binding which gives the enzyme high regioselectivity. In contrast P4503A achieves catalytic competence by a flexible binding site relying on hydrophobic forces that allow chemically vulnerable sites to be the principal sites of metabolism. In general, the different binding mechanism should be reflected in the enzyme, such that substrates of P4502D should have lower Km values than substrates of P4503A. Thus, routes of metabolism catalysed by P4502D may be saturated at substrate concentrations lower than routes catalysed by P4503A. The apparent differences between P4502D and P4503A in terms of substrate specificity bring into question what relationships govern other families of cytochrome P450. Our analysis of data suggests that the other principal form involved, generally, in the metabolism of pharmaceuticals in humans is P4502C9 (possibly 2C8 and 2C10). The enzyme is responsible for the metabolism of phenytoin, tolbutamide, tienilic acid [4], naproxen, ibuprofen, diclofenac [38], the 7-hydroxylation of S-warfarin [39] and the 7-hydroxylation of delta 1-tetrahydrocannabinol [40]. These compounds all have areas of strong hydrogen bond [4] forming potential (Fig. 8), all distanced 5-10A from the site of metabolism. Moreover the carboxylic acid function of naproxen, ibuprofen and diclofenac (pKa 4.5) and the sulfonylurea of tolbutamide (pKa 5.4) render the compounds ionized at physiological pH. The ionised group is positioned 7-11A from the site of metabolism. It is likely, therefore, that hydrogen bonding and possibly ion-pair interactions play a major role in determining the SSAR of the P4502C isoenzymes. These interactions would suggest that the P4502C enzymes are analogous to P4502D rather than P4503A. In this regard it is noteworthy that P4502C9 is selectively and potently inhibited by sulfaphenazole (IC50 of 0.6 microM), a compound that is structurally related (Fig. 8) to the substrates in terms of potential hydrogen bonding regions [4, 41]. Simplistically we suggest that the SSAR of the various P450 enzymes ranges from the highly selective enzymes dealing with endogenous substrates, through the enzymes metabolising exogenous substrates with narrow substrate structure requirements such as P4502D to P4503A with its broad substrate structure range. It would seem logical that animals and humans would evolve such combinations of isoenzymes to deal with the vast array of exogenous xenobiotics.


Effects of altered calcium homeostasis on the expression of glutathione S-transferase isozymes in primary cultured rat hepatocytes. The effects of altered Ca2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class alpha and mu specific antibodies showed an approximately 1.6- and 1.5-fold increase in the class alpha, Ya and Yc subunits, respectively, whereas no significant increase (approximately 1.2-fold) in class mu GST expression was observed following A23187 treatment. Northern blot analysis revealed an approximately 5-fold increase in GST class alpha and an approximately 7-fold increase in class mu GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Immunoblot analysis showed a significant increase in the expression of GST class alpha, Ya and Yc subunits, approximately 1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class mu GSTs. Northern blot analysis showed approximately 3- and 2-fold increases, respectively, in class alpha and mu GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca2+ homeostasis produced by either Ca2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.


Inhibition of alloxan-induced hyperglycaemia by compounds of similar molecular structure. In this study we have shown that a range of compounds that are structurally similar to alloxan are able to protect mice against the diabetogenic effect of alloxan. The compounds include a group of five barbiturates, a group of five hydantoins, the methylxanthines caffeine and theophylline, the related compound uric acid, and ethosuximide. They were injected intraperitoneally prior to intravenous injection of alloxan, and blood glucose concentration was used as an index of alloxan toxicity. The salient structural feature possessed by all of these protective compounds is a pair of carbonyl oxygen atoms separated by a distance of 4.5 A and projecting from an approximately planar heterocyclic five- or six-membered ring; in all cases the carbonyl groups are separated by a ring nitrogen. We suggest that this feature is required for the protective effect of these compounds. In order to test further the requirement for two ring carbonyl groups, we also examined the effects of two compounds containing hydroxyl groups projecting from a six-membered ring, inositol and glucuronic acid. In agreement with previous studies on hexoses, we found that the effects of compounds such as these are unpredictable, with inositol protecting against alloxan toxicity but glucuronic acid not. We are unable to identify the critical difference in structure between these two compounds.


Studies on the uptake of low molecular weight monomeric tris-galactosyl conjugates by the rat liver. We have attempted to direct low molecular weight compounds to the liver via the internalizing asialoglycoprotein receptor on parenchymal cells by conjugation to a monomeric triantennary galactosyl cluster. Acetate and a hypolipidaemic ansamycin were derivatized and the biodistribution of the conjugates was determined 250 sec and 30 min after administration to Wistar rats. The ansamycin conjugate (CGH46) was rapidly cleared from the circulation by the liver; after 250 sec, 64% of the radiolabelled dose was found in the liver compared to 18% in the blood. However, the distribution of the conjugate did not differ significantly from that of unconjugated ansamycin (CGH45). Tris-galactosyl acetate showed no capacity to localize in the liver, with only 2% recovered from that organ 250 sec after administration compared to 38% in the blood and 13-18% in the kidneys, skin and muscle. Extraction efficiency of CGH46 by isolated perfused rat livers was almost 20% of the administered dose and this value was not significantly changed by co-administration of specific inhibitors of the uptake process. It is concluded that derivatization of low molecular weight molecules with monomeric triantennary galactosyl residues is unlikely to increase their affinity for the liver.


Relationships between thermotolerance, oxidative stress responses and induction of stress proteins in human tumour cell lines. Thermotolerance, resistance to oxidative stress and induction of stress proteins were examined in a panel of 10 human tumour cell lines. An inverse relationship was indicated between intrinsic thermotolerance (cell survival after treatment at 43.5 degrees for 3 hr) and thermotolerance induced by pretreatment at 42.5 degrees for 30 min. Similar levels of induction of hsp 70 were found in cell lines with high or low levels of intrinsic thermotolerance; induction of other stress proteins could not be detected. Cell survival following treatment with H2O2 correlated with that following streptonigrin treatment (P < 0.05). Pretreatment with buthionine sulphoximine or diamide synergistically increased the toxicity of heat, H2O2 and streptonigrin whereas reduced glutathione had the reverse effect. No direct correlation was found, however, between tolerance to heat and to oxidative stress, and hsp 70 was not induced by the latter. The stress protein heme oxygenase, detected by immunoblotting with the monoclonal antibody HO, was induced by H2O2 in melanoma cell lines but not in HeLa. Cadmium and arsenite ions, however, readily induced heme oxygenase in HeLa, indicating that in these cells induction of heme oxygenase by oxidative stress involves a different mechanism. Overall, the results suggest that tolerance to heat or oxidative stress in these cell lines may not necessarily be associated with the induction of heat shock proteins or heme oxygenase but that cell survival after both types of stress depends to a certain extent on cellular sulphydryls.


Characterization of apoptosis in thymocytes isolated from dexamethasone-treated rats. The induction of apoptosis by glucocorticoids in isolated thymocytes has been studied extensively. However, it is not known whether or not the same changes occur after in vivo glucocorticoid treatment. In order to investigate this, we have studied the changes occurring in thymocytes isolated from rats, from 2-24 hr after a dose of dexamethasone (1 mg/kg), which caused 50% thymic atrophy. Thymocytes were separated into four fractions by isopycnic Percoll gradients. A loss of cells occurred within 2-8 hr, primarily in only one of the two major fractions of normal thymocytes. This loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, increased DNA fragmentation, internucleosomal DNA cleavage and a "hypodiploid" peak on flow cytometric analysis. Striking differences occurred in the cellular composition of the different Percoll fractions with time. Initially (up to 4 hr), the pattern of changes occurring in vivo resembled those found in vitro. However, at later times, the complex fate of apoptotic cells in vivo, such as phagocytosis, are not observed in the in vitro studies.


Mechanism of cadmium-decreased glucuronidation in the rat. In isolated rat hepatocytes, cadmium (0-200 microM) decreased the overall glucuronidation of both isopropyl N-(3-chloro-4 hydroxyphenyl)carbamate (4-hydroxychlorpropham, 4-OHCIPC) and 4-nitrophenol in a concentration-dependent manner. In contrast, in native rat liver microsomes, glucuronidation of 4-OHCIPC was increased by cadmium through activation of microsomal 4-OHCIPC glucuronosyl transferase. In addition, in rat microsome incubations, the net amount of 4-OHCIPC glucuronide was also indirectly increased by cadmium through a reduction in the activity of beta-glucuronidase. As the effect of cadmium on the activity of 4-OHCIPC glucuronosyl transferase could not account for the decrease in glucuronide formation in intact hepatocytes, the influence of cadmium on the availability of UDP-glucuronic acid (UDPGA) was investigated further. In isolated rat hepatocytes, cadmium depleted the UDPGA content in a dose-dependent manner without a change in the UDP glucose (UDPG) content. Cadmium did not increase the breakdown of UDPGA by microsomal UDPGA pyrophosphatase but strongly decreased (30-66%) the synthesis of the cofactor in the cytosol by inhibiting UDP-glucose dehydrogenase (UDPGDH). Cadmium (10-50 microM) was found to inhibit the purified enzyme from bovine liver (EC 1.1.1.22) non-competitively. In vivo in the absence of a substrate undergoing glucuronidation, cadmium administration, 1.5 and 2.5 mg Cd/kg i.v., to normally fed rats resulted in a 15 and 30% decrease of hepatic UDPGA, respectively. However, in the liver, neither the NAD+/NADH ratio nor the UDPG content was significantly changed following cadmium treatment. Both in vitro and in vivo results support the conclusion that in intact cells the reduction in overall 4-OHCIPC glucuronidation caused by cadmium was due to a decrease in UDPGA availability which results from the inhibiting effect of cadmium on UDPGDH.


Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum. A 31P NMR study. Two pyrophosphate analogues, dichloromethane diphosphonate (Cl2MDP), and 1-hydroxyethane-1,1-diphosphonate (EHDP), at concentrations of 0.5-1 mM, efficiently inhibited the growth of amoebae of the slime mould Dictyostelium discoideum. Cell viability decreased markedly upon incubation with the diphosphonates. The mechanism of toxicity was investigated by in vivo 31P NMR spectroscopy and the formation of analogues of ATP [adenosine 5'-(beta, gamma-dichloromethane triphosphate) and adenosine 5'-(beta, gamma-1-hydroxyethane triphosphate)] was demonstrated. These two compounds were identified from their 31P NMR spectra in perchloric acid extracts prepared from amoebae poisoned with Cl2MDP or EHDP and may have been synthesized by reversible pyrophosphate exchange catalysed by cytosolic aminoacyl-tRNA synthetases.


The consequences of doxorubicin quinone reduction in vivo in tumour tissue. A clear role for quinone reduction in the mechanism of action of doxorubicin has still to be established. There are three possible outcomes of this form of doxorubicin metabolism: (1) drug free radical formation, redox cycling and generation of reactive oxygen species (ROS) resulting in lipid peroxidation and DNA damage; (2) covalent binding of reactive drug intermediates to DNA; and (3) formation of an inactive 7-deoxyaglycone metabolite. In this work, the occurrence of each of these pathways has been studied in vivo in a subcutaneously growing rat mammary carcinoma (Sp 107). Doxorubicin was administered by direct intratumoural injection either as the free drug or incorporated in albumin microspheres (10-40 microns diameter). There was no evidence of an increase in lipid peroxidation over background after either treatment at any time point studied. In fact, doxorubicin administration resulted in a statistically significant reduction in lipid peroxidation at the later time points studied compared to control (no drug treatment), e.g. 24 hr: control, 21.7 +/- 2.8 SD nmol malondialdehyde/g tissue; free doxorubicin (70 micrograms drug), 14.5 +/- 4.0 SD nmol/g (P < 0.01 Student's t-test) and doxorubicin microspheres (70 micrograms drug), 17.4 +/- 1.1 nmol/g (P < 0.05). Covalent binding to DNA was measured by a 32P-post-labelling technique. Low levels of four putative drug-DNA adducts were detected; however, there were no qualitative or quantitative differences in profiles between free drug and microspheres. High 7-deoxyaglycone metabolite concentrations comparable to the parent drug itself were detected after administration of microspheres (3.0 micrograms/g +/- 1.7 SD at 24 hr and 3.1 micrograms/g +/- 1.1 SD at 48 hr). In contrast, these metabolites were present at levels close to the limit of detection of our HPLC assay after free drug (0.04 microgram/g +/- 0.03 SD at 24 hr and 0.02 microgram/g +/- 0.03 SD at 48 hr). Thus, 7-deoxyaglycone metabolite formation can occur in tumour tissue (indicating active drug quinone reduction) without concomitant increases in the level of lipid peroxidation or the levels of drug-DNA adducts. In conclusion, the main biological consequence of doxorubicin quinone reduction in vivo in tumour tissue would appear to be drug inactivation to a 7-deoxyaglycone metabolite rather than drug activation to DNA reactive species or ROS.


The enzymology of doxorubicin quinone reduction in tumour tissue. We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.


Sequential changes in activities of superoxide dismutase and catalase in brain regions and liver during (-)deprenyl infusion in male rats. A continuous s.c. infusion of (-)deprenyl in young male rats at a dose of 2.0 mg/kg/day for 1 week significantly increased total superoxide dismutase (SOD) activities due to increases in both Cu Zn-SOD and Mn-SOD activities in certain brain regions such as the substantia nitra and striatum, but not in the hippocampus or cerebellum, or in the liver. With continuing infusion, enzyme activities of SOD were further increased in the following weeks, reaching a plateau at 3 weeks. In some cerebral cortices the increase became significant at 3 weeks. In contrast to SOD activities, an increase in catalase (CAT) activity became significant only after 2 weeks of infusion, and only in the brain regions where SOD activities were increased earlier. The delay in the increase in CAT activity following deprenyl infusion suggests that this increased CAT activity is an adaptive response to the earlier increase in deprenyl-induced SOD activities rather than a direct effect of deprenyl on CAT activity, although the latter possibility cannot be excluded.


Comparison of ascorbic acid and ascorbic acid 2-O-alpha-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts. Ascorbic acid 2-O-alpha-glucoside (AA-2G) is a stable ascorbate derivative which has vitamin C activity in vivo and in vitro. We studied whether AA-2G exerts a prooxidant action in cultured fibroblasts from chick embryo and human skin, as does ascorbic acid. At concentrations of 0.1-1.0 mM, ascorbic acid markedly reduced the viable cell number of low density cultures within 24 hr, whereas AA-2G had no such effect. The ascorbate cytotoxicity was dependent on the cell density at the time of its addition and it was characteristic of low density cultures. This cytotoxicity was completely prevented by catalase and partially by an Fe3+ ion chelator, desferrioxamine. In the early culture stage at which a morphological change in the fibroblasts began to occur, intracellular ascorbate concentrations in low density cultures after addition of ascorbic acid were much higher than in high density cultures. However, at the same concentrations, AA-2G did not cause an elevation even in low density cultures and it was also effective on collagen synthesis at high and medium densities. These results suggest that the abnormally accumulated ascorbic acid in the cells cultured at low density possibly amplifies the generation of oxygen radicals through the reduction of Fe3+ ions and subsequent oxidative reactions, leading to cell death. Therefore, it is concluded that AA-2G which supplies an adequate amount of ascorbic acid during culture period is a bioavailable ascorbate source without cytotoxicity.


Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents. The antifungal drug miconazole and the cholesterol-lowering agent lovastatin (mevinolin) were used in combination to assess their potency as anti-leishmanial agents. The drug combination was synergistic, being more potent in terms of inhibition of promastigote proliferation, macrophage infection and amastigote numbers. In promastigote cultures the effect was more marked in Leishmania amazonensis than L. donovani. Analysis of the sterol compositions of both promastigote and amastigote cultures revealed the inhibition of sterol 14 alpha-demethylation by miconazole and showed some apparent evidence of inhibition of sterol biosynthesis by lovastatin.


Protective effects of the calcium antagonists diltiazem and TA3090 against hepatic injury due to hypoxia. Recent work has shown that dihydropyridine-type calcium channel blockers such as nitrendipine protect against ischemic liver damage in the rat in vivo (Thurman RG, Apel E and Lemasters JJ, J Cardiovasc Pharmacol 12: S113-S116, 1988), suggesting that calcium antagonists may have clinical value in preventing ischemic and hypoxic hepatic injury. This study was designed to examine the effects of two benzothiazepine-type calcium channel blockers, diltiazem and TA3090, in the hypoxic perfused rat liver. Livers were isolated and perfused briefly with oxygen-saturated buffer, followed by perfusion for 80 min with nitrogen-saturated buffer with diltiazem or TA3090 (20-200 microM), and concluding with 20 min of perfusion with oxygen-saturated buffer. In control preparations, maximal lactate dehydrogenase (LDH) release into effluent perfusate following hypoxia averaged about 1100 U/L. Diltiazem and TA3090 decreased LDH release at all concentrations studied; both drugs were most effective at the 100 microM concentration (71 and 73% inhibition, respectively). Oxygen uptake by control livers decreased 78% following hypoxia; diltiazem and TA3090 reduced this effect markedly, with maximal effectiveness again observed with 100 microM (O2 uptake was decreased by 22% with 100 microM diltiazem and by only 9% with 100 microM TA3090). Histological examination for nuclear uptake of the vital dye trypan blue revealed necrosis of parenchymal cells along with cell shrinking and consequent expansion of the sinusoids in control livers. Perfusion with diltiazem markedly reduced parenchymal cell death but did not alter the pattern of cell damage observed. In contrast, livers perfused with TA3090 during hypoxia had virtually no parenchymal cell damage, although moderate damage to nonparenchymal cells in the sinusoids occurred. The difference in mechanisms responsible for the phenomena which occur with diltiazem and TA3090 is not completely understood; however, these and other calcium antagonists clearly have powerful hepatoprotective effects against ischemia and hypoxia.


Effects of inhibitors of gluconeogenesis on weak organic acid uptake in rat renal tubules. Using inhibitors of gluconeogenesis (phenylpyruvate, alpha-cyano-4-hydroxycinnamate, quinolinate, D-malate, aminooxyacetate), we analysed mechanisms by which the gluconeogenic substrates, lactate and pyruvate, as well as a short-chain fatty acid, acetate, stimulate the uptake of a weak organic acid, fluorescein, in the rat kidney. We have shown that these inhibitors modified both the rate of glucose production from lactate and pyruvate in the renal cortex fragment suspension and the stimulatory effects of the metabolic substrates on fluorescein uptake in superficial proximal tubules in the renal cortex slices. The peculiarities of the effects of lactate and pyruvate on the uptake were correlated with the partial divergence of the pathways of gluconeogenesis from these precursors. The linkage of the weak organic acid uptake with gluconeogenesis is interpreted in terms of the hypothesis that the uptake is controlled by the cytoplasmic pyridine nucleotide redox potential, which is maintained with the participation of certain processes involved in glucose synthesis.


Detection of new metabolites of trifluridine (F3TdR) using 19F NMR spectroscopy. The metabolism of 5-trifluoromethyl-2'-deoxyuridine (trifluridine, F3TdR) in male BALB/C mice has been studied by 19F NMR spectroscopy. Contrary to previous reports, a number of fluorinated metabolites were observed in urine, whole livers and blood samples taken from mice after i.p. injection of F3TdR. The present study describes the identification of two new metabolites in mouse urine using the 19F NMR technique. The NMR of crude urine showed the presence of F3TdR 5-trifluorothymine (F3T), the newly-identified metabolites, 5-trifluoromethyl-5,6-dihydrouracil (DHF3T) and 5-trifluoromethyl-5,6-dihydroxyracil (DOHF3T), and several new, as yet unidentified fluorinate metabolites. These two new metabolites were characterized by comparison to authentic compounds prepared synthetically from F3T.


Effect of epinephrine on glucose metabolism and hydrogen peroxide content in incubated rat macrophages. The effects of epinephrine on glucose metabolism and hydrogen peroxide content were examined in incubated rat macrophages. An increase in the activities of hexokinase and citrate synthase and a reduction in that of glucose-6-phosphate dehydrogenase was found in resident, inflammatory and activated macrophages incubated for 1 hr in the presence of epinephrine. Glucose utilization by incubated resident, inflammatory and activated macrophages was augmented markedly by the addition of epinephrine, whereas lactate formation was depressed. Under the same conditions, there was a 2.6-fold increment of hydrogen peroxide content and of [U-14C]glucose decarboxylation in activated macrophages incubated for 40 min. Similar results were obtained when pyruvate and oxoglutarate was substituted for glucose. These findings suggest that epinephrine may increase hydrogen peroxide production in activated macrophages possibly through a mitochondrial mechanism other than the pentose-phosphate pathway. Between 40 and 90 min of incubation, the content of hydrogen peroxide decreased markedly, and there was no detectable glucose utilization in the presence of epinephrine. These observations are consistent with the idea that this catecholamine stimulates both hydrogen peroxide production and metabolism, the first process being dependent on mitochondrial fuels.


Deconjugating activity for sulfoconjugated dopamine in homogenates of organs from dogs. To clarify the possibility that sulfoconjugated dopamine (DA) may play a physiological role by being converted to active free DA, we examined the deconjugating activity in homogenates of organs from dogs. Each tissue homogenate was incubated with sulfoconjugated DA, and the deconjugating activity of the organs was compared. The kidney and liver exhibited the highest deconjugating activities. In contrast, the intestine and heart showed lower arylsulfatase activities, and almost no activity was found in the brain or skeletal muscle. Moreover, in the heart, the deconjugating activity for sulfoconjugated DA was higher in the atrium than the ventricle. These results indicate that sulfoconjugated DA is converted to active free DA in homogenates of organs from dogs and that the deconjugating activity varies between different parts of an organ. Sulfoconjugated DA must be looked upon as a possible precursor or reservoir for the production of active free DA.


Clotrimazole-induced modulation of hepatic cytochrome P450 enzymes in Syrian and Chinese hamsters. Clotrimazole, an imidazole antifungal drug, is known to induce cytochrome P450 isozymes of the P450IIIA and P450IIB subfamilies in rats. This agent modulated hepatic cytochrome P450 enzymes differently in golden Syrian and Chinese hamsters and also in hamsters and rats. Clotrimazole at a daily intraperitoneal dose of 100 mg/kg for three days increased the amount of cytochrome P450 in the livers of the two hamster strains. In Syrian hamsters, clotrimazole significantly induced the activities of 7-pentoxyresorufin O-dealkylase, coumarin 7-hydroxylase, benzphetamine N-demethylase and testosterone 15 alpha- and 16 alpha-hydroxylases, but reduced those of testosterone 15 beta-, 7 alpha-, 6 beta-, 2 alpha- and 2 beta-hydroxylases. In Chinese hamsters, clotrimazole markedly stimulated the activities of coumarin 7-hydroxylase and testosterone 15 alpha, 16 alpha- and 2 alpha-hydroxylases as well as the formation of androstenedione. Western blot analysis revealed that clotrimazole treatment induced mainly cytochrome P450 isozymes immunorelated to the P450IIB and P450IIA subfamilies in Syrian hamsters and isozymes immunorelated to the P450IIA subfamily in Chinese hamsters. In contrast, in both hamster strains, clotrimazole did not induce the isozymes corresponding to the P450IIIA subfamily.


EB1089: a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro. EB1089 is a novel vitamin D analogue which has been tested for its effects on breast cancer cell growth in vitro, using the established human breast cancer cell line MCF-7, and in vivo on the growth of established rat mammary tumours. Both EB1089 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited MCF-7 cell proliferation with the synthetic analogue being at least an order of magnitude more potent than the native hormone. In vivo anti-tumour effects were investigated using the N-methyl-nitrosourea-induced rat mammary tumour model. Oral treatment with EB1089 was tested at three doses. With the lower dose, significant inhibition of tumour growth was seen in the absence of a rise in serum calcium. The same dose of 1,25-(OH)2D3 had no effect on tumour growth but caused hypercalcaemia. With the higher dose of EB1089, striking tumour regression was seen although serum calcium rose. This report demonstrates that EB1089 possess enhanced anti-tumour activity coupled with reduced calcaemic effects relative to 1,25-(OH)2D3 and thus may have therapeutic potential as an anti-tumour agent.


Enzymatic basis for the non-linearity of hepatic elimination of propranolol in the isolated perfused rat liver. Propranolol (PL) metabolism was studied in the isolated perfused rat liver under single-pass and steady-state conditions. An attempt was made to predict the data observed in the isolated rat liver perfusion at PL infusion rates of 89-1317 nmol/min using the microsomal kinetic parameters obtained in our previous paper (Ishida et al., Biochem Pharmacol 43: 2489-2492, 1992) and the unbound PL fractions in rat liver microsomes and the perfusion medium. The values of kinetic parameters obtained in rat liver microsomes were corrected for the whole liver. Two groups of cytochrome P450 isozymes having high (Km < 0.5 microM)- and low (Km > 20 microM)-affinities participate in the metabolism of PL and sudan III pretreatment induces the low-affinity enzymes rather than the high-affinity enzymes in control rats. Of high-affinity isozyme(s) PL 4-hydroxylase and 7-hydroxylase made a major contribution to the overall activity, while for low-affinity isozymes PL 4-hydroxylase and N-desisopropylase did. A nonlinear relationship between the PL concentrations entering and leaving the liver was predicted from these corrected kinetic parameters using the venous equilibrium model. The outflow concentrations and the metabolic rates of PL for the predicted curves were over-estimated at higher inflow PL concentrations and under-estimated at higher substrate concentrations, respectively. On the other hand, the prediction for them was successfully carried out for the livers whose intrinsic clearance was altered due to the induction of low-affinity enzymes in PL metabolism by sudan III pretreatment. The outflow rates of 4-hydroxypropranolol showed a downward curvature at lower substrate concentrations, followed a linear rise in the livers from control rats, while the outflow rates of 5- and 7-hydroxypropranolol exhibited their respective limiting values. The outflow rates of 4-hydroxypropranolol and N-desisopropylpropranolol were enhanced markedly with increasing the outflow unbound concentration of PL by sudan III pretreatment. These results indicate that non-linear PL first-pass metabolism is due to the saturation of the reactions for the high-affinity enzymes among enzymes engaging in PL ring hydroxylations.


The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).


The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase. A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.


The effect of enzyme induction on the cytochrome P450-mediated bioactivation of carbamazepine by mouse liver microsomes. Predisposition to idiosyncratic toxicity with carbamazepine is thought to be due to a deficiency of the detoxication enzyme, microsomal epoxide hydrolase, although in some cases, concurrent administration of enzyme inducers might be a contributory risk factor, by altering the critical balance between bioactivation and detoxication. In this study, a mouse model has been used to determine the factors affecting carbamazepine bioactivation, using covalent binding and cytotoxicity as markers of bioactivation in vitro. Microsomes prepared from mice pre-treated with phenobarbitone increased (relative to the control microsomes) the formation of cytotoxic (12.3% vs 3.2%), protein-reactive (3.0% vs 2.0%) and stable (33.8% vs 18.1%) metabolites of carbamazepine. Similarly, pre-treatment with dexamethasone also increased the formation of the cytotoxic (24.8% vs 6.7%), protein-reactive (2.8% vs 1.5%) and stable (38% vs 19.8%) metabolites of carbamazepine, while beta-naphthoflavone pretreatment did not increase the formation of either the toxic or stable metabolites of carbamazepine when compared with its control microsomes. Co-incubation with gestodene (10-250 microM) resulted in a dose-dependent inhibition of both the bioactivation of carbamazepine and the formation of its stable 10,11-epoxide. SDS-PAGE and immunoblotting of the microsomes with anti-CYP3A antibody revealed the presence of a 52 kDa protein band in each preparation of microsomes, but the relative intensities of the bands, as measured by laser densitometry, were highest with the phenobarbitone and dexamethasone microsomes. The microsomal oxidation of cortisol to 6 beta-hydroxycortisol was also enhanced by pretreatment of mice with phenobarbitone (6.5% vs 2.7%) and dexamethasone (8.2% vs 4.3%), but not beta-naphthoflavone (2.2% vs 1.6%), when compared with their respective control microsomes, and was inhibited (range 25-68% inhibition), with all the microsomes by gestodene (50 microM). Taken collectively, the data in this study demonstrate that in the mouse, induction of the CYP3A subfamily significantly increases carbamazepine bioactivation. It is likely that in humans inducers of the orthologous form of this enzyme, most notably anticonvulsants, may increase the bioactivation of carbamazepine.


A multispecific uptake system for taurocholate, cardiac glycosides and cationic drugs in the liver. To test the hypothesis of multiplicity in carrier-mediated uptake mechanisms for organic cations in the liver and to study the possible relation with bile acid and cardiac glycoside uptake mechanisms, mutual interaction during uptake of various radiolabeled quaternary amines has been studied in isolated rat hepatocytes. Inhibition patterns at low concentrations (1 microM) of the presumed type I monovalent organic cation tri-n-butylmethylammonium were markedly different from those at relatively high concentrations (25 microM). Both the cardiac glycoside K-strophantoside and the bile acid taurocholate clearly reduced the uptake rate of tri-n-butylmethylammonium at 25 microM whereas these agents completely failed to reduce the uptake at low concentrations of the cation. Subsequently, inhibition of uptake of some multivalent amphipathic organic cations (muscle relaxants) for the type II uptake system was investigated. It was found that the uptake of these muscle relaxants both at tracer concentrations (< 1 microM) and at relatively high concentrations (25 microM) was decreased in the presence of low concentrations of the cardiac glycoside K-strophantoside, while taurocholate only inhibited the uptake at the concentration range > 25 microM of the muscle relaxants. Procainamide ethobromide, a typical type I organic cation, did not affect the uptake either at the low or high concentration range of the muscle relaxants. It is concluded that for each of the type I-like compounds and type II-like compounds tested at least two systems are involved in uptake into hepatocytes: tri-n-butylmethylammonium in a concentration range < or = 1 microM is mainly taken up by the type I uptake system and at concentrations > or = 25 microM also by system(s) that can be inhibited by taurocholate and K-strophantoside. Bulky amphipathic organic (type II) cations at concentrations < 1 microM are also transported by an uptake system that is inhibitable by cardiac glycosides but not by bile salts. At concentrations > 25 microM these compounds are predominantly accommodated by an uptake system that possibly mediates uptake of both cardiac glycosides and bile acids. This concept was supported by the observation that both type II organic cations and bile salts can inhibit ouabain uptake, while type II organic cations as well as the cardiac glycosides reduce taurocholate uptake rate. The present data support the idea that the liver seems to be equipped with a "multispecific" uptake system that transports hydrophobic compounds irrespective of charge, including some type I and type II organic cations at relatively high substrate concentrations.


Fasting increases the susceptibility of rat hepatocytes to the cytotoxic effects of N-hydroxy-acetylaminofluorene. Effects on mitochondrial respiration and membrane potential. Isolated rat hepatocytes were incubated with the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Cells from fasted rats were much more susceptible to the cytotoxic effects of 1 mM N-OH-AAF than cells from fed rats: after approximately 90 min exposure the former were all dead but the latter still viable. Even after 240 min 25% of the "fed" cells were still viable. The loss of viability was preceded by a decrease in mitochondrial membrane potential (MMP) and inhibition of respiration; the mitochondrial respiration as measured in permeabilized cells appeared uncoupled. Addition of 15 mM fructose prevented cell death and the loss of MMP in cells both from fed and fasted rats to a large extent; however, uncoupling was not prevented. After incubation of hepatocytes from fasted rats with 1 mM [3H]N-OH-AAF for 120 min, 12 nmol [3H]N-OH-AAF became bound per mg cell protein. Addition of fructose decreased this to 7 nmol. In cells from fed animals 4 nmol [3H]N-OH-AAF became bound after 120 min, in this case fructose had no effect. Part of the protective effect of fructose might be explained by a decrease in intracellular ATP, which prevents the formation of reactive intermediates of N-OH-AAF resulting in a decrease of covalent binding, in addition, fructose protects via a yet to be determined mechanism.


The influence of pH on the interaction of lipophilic anthracyclines with bovine serum albumin. Quantitative characterization by measurement of fluorescence quenching. We have investigated the interaction of the lipophilic anthracyclines 4'-iodo-4'-deoxydoxorubicin (IDX) and 4-demethoxy-daunorubicin (DDN) with bovine serum albumin by the quantitation of fluorescence quenching. The protein binding of IDX was extremely sensitive to the pH of the solution in which the complex was formed and paralleled the effect of pH on dimerization of the drug. The effect of pH on the protein binding and self-association of DDN was less extensive. Both compounds exhibited curvilinear Scatchard plots indicating apparent cooperativity in the binding process. Because of the self-association of the drugs in aqueous solution, we attempted to resolve this cooperativity in terms of the preferential binding of the dimer to the acceptor. However, we found that similar Scatchard plots could be simulated by using slightly erroneous estimates of the fluorescence yield of the complex, rendering any such analysis inconclusive. Consequently, the relationship between acceptor concentration and the fraction of ligand bound was considered to be fitted adequately in terms of a single acceptor site per albumin molecule. The pH dependence of the association constants for bovine serum albumin was described best by the hydrophobic interaction of neutral drug monomer with a binding site with titratable affinity. We postulate that the pH-dependent binding of some anthracyclines with albumin may lead to their enhanced uptake, relative to that of non-target organs, into tumours with an acidotic extracellular milieu.


Inhibition of proliferation of human leukaemia 60 cells by diethyl esters of glyoxalase inhibitors in vitro. Diethyl esters of the glutathione S-conjugate S-p-bromobenzylglutathione, an inhibitor of glyoxalase I, and S-p-nitrobenzoxycarbonylglutathione, an inhibitor of glyoxalase II, induced growth arrest and toxicity in human leukaemia 60 cells in culture. The median growth inhibitory concentration IC50 values were 8.3 microM (95% C.I. 7.0-9.9 microM) for S-p-bromobenzylglutathione diethyl ester and 56 microM (95% C.I. 36-86 microM) for p-nitrobenzoxycarbonylglutathione. Monoethyl ester and unesterified derivatives were inactive. The diethyl ester derivatives were also toxic to mature human neutrophils under the same culture conditions where the respective median toxic concentration IC50 values were 39.7 (95% C.I. 35.4-44.5 microM) and 127 (95% C.I. 123-132 microM) microM. Diester derivatives may be of future interest in studying the cytotoxicity of glutathione S-conjugates and for the development of cytotoxic anti-tumour agents.


Effects of mono- and divalent ions on the binding of the adenosine analogue CGS 21680 to adenosine A2 receptors in rat striatum. The effect of monovalent and divalent cations on equilibrium binding of the adenosine A2-selective agonist ligand CGS 21680 (2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxami doadenosine) to membranes prepared from rat striatum was examined. Competition experiments with cyclohexyladenosine, 2-chloroadenosine, N-ethylcarboxamidoadenosine and CGS 21680 suggest that at 2 nM [3H]CGS 21680 binds to a single site with the pharmacology of an A2a receptor. Magnesium and calcium ions caused a concentration-dependent increase in binding that reached about 10-fold at 100 mM. Manganese ions had a biphasic effect on binding with a maximal increase at 5 mM. Lithium, sodium and potassium ions all caused a concentration-dependent decrease of binding. Sodium was most potent, potassium least. At 200 mM ion concentration, the inhibition of binding was 88% by sodium, 47% by lithium and 29% by potassium ions. The effect of sodium chloride was the same as that of sodium acetate. The effect of sodium ions was essentially similar to that of Gpp(NH)p. However, sodium ions produced a larger effect than even maximally effective concentrations of Gpp(NH)p. The maximal inhibition by Gpp(NH)p was about 55% at 2 nM radioligand concentration irrespective of the magnesium concentration. The maximal effect of sodium ions was reduced by increasing concentrations of magnesium ions. Increasing magnesium ion concentration from 1 to 100 mM increased the half-maximally effective concentration of Gpp(NH)p almost 10-fold and that of sodium ions less than 2-fold. Furthermore, sodium ions and Gpp(NH)p had additive effects. The binding of an agonist to striatal A2a receptors shows an unusually large dependence on both divalent and monovalent cations that can only partly be explained by a change in the coupling to Gs proteins.


Ketoconazole inhibition of the bifunctional cytochrome P450c17 does not affect androgen formation from the endogenous lyase substrate. The catalytic site remains refractory in the course of intermediary hydroxyprogesterone processing. The inhibition of the bifunctional steroidogenic cytochrome P450c17 (CYP17: steroid-17 alpha-hydroxylase/steroid-17,20-lyase) by the imidazole-type fungicide, [(+/-)-cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl- methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) (ketoconazole), was investigated with the aim of differentiating between effects on androgen formation from exogenously added and endogenously produced 17 alpha-hydroxyprogesterone. Using microsomal membranes from rat testis, turnover of progesterone by P450c17 was competitively inhibited by ketoconazole with KI = 0.40 microM. Ketoconazole did not affect the linear relationship between the ratio of productive events (corresponding to androgen formation rates) versus abortive events (corresponding to 17 alpha-hydroxyprogesterone formation rates) and the sum of catalytic events. This was an indication that this inhibitor did not interfere with intermediate processing by P450c17. Androgen formation from exogenous but not from endogenous 17 alpha-hydroxyprogesterone was competitively inhibited by ketoconazole. The simultaneous conversion of 1 microM each of [3H]progesterone and 17 alpha-hydroxy[14C]progesterone was also reduced by ketoconazole. Calculation of 3H/14C ratios in the 17 alpha-hydroxyprogesterone and androgen fractions revealed that the endogenous 17 alpha-hydroxyprogesterone pool was metabolized to androgens at rates 6.4, 11.6, 17.6 and 21.2-fold faster than the exogenous pool in the presence of 0.5, 1, 2 and 4 microM ketoconazole, respectively; this value was only 4.0 in controls. It is concluded that ketoconazole inhibits turnover of steroid ligands only when they approach the P450c17 active site in a substrate-state and that inhibition of androgen formation from progesterone is due to inhibition of the first catalytic step only. A model is described in which the P450c17 active site is refractory towards ketoconazole when the intermediary steroid is retained and being processed at that site.


Comparative cytoprotective effect of dihydropyridine calcium channel blockers against the toxicity of oxidized low density lipoprotein for cultured lymphoid cells. The ability of dihydropyridine Ca2+ channel blockers (nicardipine, nimodipine and nisoldipine) to inhibit low density lipoprotein (LDL) oxidation and to prevent the cytotoxicity of oxidized LDL for lymphoid cells have been compared. The lipid peroxidation of LDL promoted either by UV radiation or by copper ions was inhibited (antioxidant effect) in a dose-dependent manner by nisoldipine (IC50 values were evaluated at around 10 microM), whereas nimodipine was less potent (IC50 around 50-100 microM) and nicardipine almost inactive. The cytotoxicity of LDL treated (by UV or by copper) in the presence of effective antioxidant concentrations of dihydropyridine Ca2+ channel blockers was less than that of unprotected oxidized LDL (i.e. LDL oxidized in the absence of any dihydropyridine Ca2+ channel blockers). The inhibition of the cytotoxic effect of LDL oxidized in the presence of dihydropyridine Ca2+ channel blockers correlated well with protection from oxidation by these compounds. Beside this indirect protective effect, dihydropyridine Ca2+ channel blockers exhibit a direct protective effect for cells against the toxicity of previously oxidized LDL. Although complete protection cannot be obtained because of the cytotoxicity of the dihydropyridine compounds per se, the IC50 values were 6 +/- 2 and 80 +/- 20 microM for nisoldipine and nimodipine, respectively. The potential relevance to the prevention of atherogenesis is discussed.


Effects of diarylsulfonylurea antitumor agents on the function of mitochondria isolated from rat liver and GC3/c1 cells. Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pKa values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.


Prevention by Ginkgo biloba extract (EGb 761) and trolox C of the decrease in synaptosomal dopamine or serotonin uptake following incubation. Prolonged incubation of synaptosomes in Krebs-Ringer oxygenated medium in the presence of ascorbic acid (10(-4) M) led, after 20 min, to a decrease in [3H]dopamine (DA) (synaptosomes prepared from the striatum) and [3H]serotonin (5HT) (synaptosomes prepared from the cortex) uptake. The decrease was progressive and uptake was virtually abolished after a 60 min incubation period. A concentration-dependent (from 5 x 10(-6) M) role of ascorbic acid in the decrease of [3H]DA or [3H]5HT uptake was demonstrated. This decrease was potentiated by Fe2+ ions and prevented by the ferrous chelating agent desferrioxamine. Thus, the progressive decrease in synaptosomal uptake of either [3H]DA or [3H]5HT could depend on the generation of free radicals by the association of ascorbic acid with Fe2+ ions. The decrease in synaptosomal uptake was prevented, in a concentration-dependent manner, by the Ginkgo biloba extract EGb 761 (4-16 micrograms/mL) and the vitamin E analog trolox C (10(-4) M). The terpenic fraction of EGb 761, Bn 52063 (up to 0.5 microgram/mL), did not prevent the reduction of [3H]amine uptake. In contrast, the flavonoidic fraction, Cp 202, was effective (from 1 microgram/mL) and its efficacy was shared by the flavonoid quercetin (from 0.1 microgram/mL). The prolongation of the ability of synaptosomes to take up [3H]amine elicited by EGb 761, in particular its flavonoidic fraction, as well as by trolox C could be due to their free radical scavenger properties.


Cellular mechanism of thrombin on endothelin-1 biosynthesis and release in bovine endothelial cell. We have studied the cellular mechanism responsible for induction of preproendothelin (preproET)-1 mRNA and release of ET-1 by thrombin in cultured bovine endothelial cells (ECs). Thrombin induced an immediate and dose-dependent formation of inositol-1,4,5-trisphosphate (IP3) with a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). The thrombin-induced ET-1 release was abolished either by a phospholipase C inhibitor, a protein kinase C (PKC) inhibitor, or an intracellular Ca(2+)-chelator, whereas a Ca(2+)-channel antagonist was ineffective. A selective thrombin inhibitor (argatroban) decreased IP3 formation and the increase in [Ca2+]i and ET-1 release stimulated by thrombin. Northern blot analysis revealed that thrombin-induced expression of preproET-1 mRNA was inhibited completely by a PKC inhibitor and partially by argatroban. These data suggest that thrombin is involved in the mechanism of preproET-1 mRNA expression and subsequent ET-1 release, possibly through activation PKC and mobilization of intracellular Ca2+ resulting from the receptor-mediated phosphoinositide breakdown in ECs.


Chemiluminescence-HPLC assay of phosphatidylcholine hydroperoxide generated by ischemia-reperfusion in the liver of rats. To determine cellular damage due to "oxidative stress", we developed a sensitive and specific quantitative assay for phosphatidylcholine hydroperoxide (PCOOH) by coupling HPLC with detection of chemiluminescence (CL). The qualitative and quantitative detection limits of PCOOH by this assay were 0.5 and 2 pmol (based on active oxygen from hydroperoxide). Using this CL-HPLC method, we determined PCOOH levels caused by ischemia-reperfusion in rat livers. The PCOOH levels in livers of control, sham-operated and operated rats with only ischemic treatment were approximately 2 nmol/g wet liver weight. The PCOOH level and several serum parameters of liver injury increased with an increase in the duration of ischemia, and also increased in proportion to the duration of reperfusion. The determination of PCOOH in liver caused by ischemia-reperfusion could be a useful method for investigating liver damage induced by free radicals.


Evidence of multiple forms of rat liver microsomal coenzyme A ligase catalysing the formation of 2-arylpropionyl-coenzyme A thioesters. This study has demonstrated the involvement of multiple forms of rat hepatic microsomal CoA ligases in the formation of 2-arylpropionyl-CoA thioesters. In the presence of (-)R-ibuprofen (0.1 microM-1 mM) two enzymic processes were observed, one of which exhibited enantiospecificity and apparent high affinity for the R enantiomer (Km 0.06 microM) whilst the second, a low-affinity component was non-enantiospecific. An equivalent high-affinity isoform catalysing R-flurbiprofen-CoA formation at concentrations less than 100 microM was not demonstrated. However, at higher substrate concentrations formation of both R- and S-flurbiprofenyl-CoA thioesters occurred. Marked inter-individual variation was observed in the formation of S-ibuprofen-CoA and S-flurbiprofen-CoA in the rats studied.


L-glutamine prevents the L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity. Results presented in this study demonstrate that L-glutamine, a competitive inhibitor of L-histidine uptake, inhibits in a concentration-dependent fashion the L-histidine-mediated enhancement of H2O2-induced cytotoxicity. L-Glutamine also prevents the induction of DNA double strand breaks (DSB) but does not affect the enhancing effect of L-histidine on DNA single strand break induction by H2O2. Taken together, these data demonstrate that L-histidine, in order to allow the formation of DNA double strand breakage and increase the toxicity elicited by the oxidant, has to enter the cell. In addition, these results indicate that the enhancement of DNA single strand breakage is a consequence of the action of the amino acid at the extracellular level and/or outer surface of the plasma membrane and does not appear related to the mechanism whereby L-histidine increases the cytotoxic response to H2O2. The latter mechanism very likely involves the formation of DNA DSB.


Oxidative activation of 2-aminofluorene by human red blood cell cytosol. Purified red blood cell cytosol is able to activate 2-aminofluorene (2AF) to N-hydroxy-2-aminofluorene. Apparent kinetic parameters are determined with and without methylene blue. The latter, which maintains haemoglobin in the reduced form and stimulates NADPH production, increases the affinity of the enzyme for the 2AF. This activity is inhibited by carbon monoxide while cyanide is without effect. The involvement of a peroxidative reaction or a one-electron oxidative mechanism involving free radicals may be excluded.


Activation of aldehyde dehydrogenase at physiological temperatures. The release of NADH from the enzyme.NADH complexes was rate limiting at 37 degrees, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was activation by p-(chloromercuri)benzoate. Activation of enzymic activity may be of importance in vivo.


Does sorbinil bind to the substrate binding site of aldose reductase? With benzyl alcohol as the varied substrate, sorbinil was found to be a competitive inhibitor of aldose reductase, an enzyme implicated in the etiology of secondary diabetic complications. The K(is sorbinil) and the Vmax/Km (V/K) benzyl alcohol decreased at low pH with a pK of 7.5 and 7.7, respectively. These observations suggest that both sorbinil and benzyl alcohol bind to the same site on the enzyme. Active site inhibition by sorbinil is consistent with non-competitive inhibition patterns of sorbinil with nucleotide coenzyme or aldehyde as the varied substrate in the direction of aldehyde reduction.


[Multiple attack in the mechanism of endoglucanase action]. The review deals with the phenomenon of repetitive catalytic acts occurring in one enzyme-substrate encounter of certain endo-glucanases with water soluble polysaccharides. Here are discussed experimental results and theoretical models applied in analysis of data.


[Site-specific endonuclease BstM6I from Bacillus stearothermophilus M6]. A site-specific endonuclease activity was found in extract of Bacillus stearothermophilus M6 isolated from molasses. The functionally pure enzyme designated as BstM6I was obtained by consecutive chromatographies on blue sepharose, hydroxyapatite, and heparin-sepharose. The endonuclease recognizes the nucleotide sequence CC decreases WGG in double-stranded DNA and cleaves it as indicated by the arrow to give one-nucleotide 5'-protruding ends. Consequently, the site-specific endonuclease BstM6I is an isoshizomer of BstNI.


[Site-specific photomodification of nucleic acids with arylazide and perfluoroarylazide oligonucleotide derivatives. II. Specificity in relation to nucleosides]. Oligonucleotide reagents bearing aromatic azido groups of different structures were shown to be suitable for nucleoside specific photomodification of nucleic acids. Modification of the pentadecanucleotide targets d(TAAGTGGAGTTTGGC), d(TAAGTGGAAAAAAAA), d(TAAGTGGACCCCCCC) and d(TAAGTGGATTTTTTT) was investigated with reagents d(UCH2OCH2CH2NHCORCCACTT) carrying a photoactive group R(R1-n-azidotetrafluorophenyl-reagent (I), R2-2-nitro-5-azidophenyl-reagent (II) and R3-n-azidophenyl-reagent (III)) at C-5-modified deoxyuridine. Photomodification did not exceed 5% for the targets in case of reagent (III); the modification extent was 25-50% depending on the target sequence for reagent (II); reagent (I) with perfluoro azido group was the most effective, that provided 60-70% of modification. Reagents (I) and (II) were found to be sensitive to the nucleoside sequence of the target: the most vulnerable sites for reagent (I) and (II) were guanine and cytosine residues, respectively. These bases were modified predominantly when being adjacent to the addressed site of the target.


[Immobilization of phospholipid micelles on modified apoHDL-Sepharose]. There was developed a procedure for immobilization of phosphatidyl cholines (Egg yolk phosphatidyl choline and polyunsaturated soya beams phosphatidyl choline) on the modified apoHDL-Sepharose. The formation of phospholipid micelles was proved by linear dependence of the content of the sorbed phosphatidyl choline versus, the content of apoHDL bound to Sepharose. Incubation of apoHDL/PC-Sepharose with human plasma was shown to change the plasma lipid composition. The apoHDL/PC-Sepharose might be used for correction of the plasma lipid composition on vitro experiments.


[Metabolism of phosphatidylcholine in model lipoproteins. Use of phospholipid micelles immobilized on apoHDL-Sepharose]. A phosphatidyl choline (PC) exchange between apoHDL/PC micellar complexes in solution and the same complexes immobilized on Sepharose was studied. The PC exchange in buffer was represented in terms of pseudo first order reversible process. The first order constants for the unidirectional efflux of PC from apoHDL/PC-Sepharose (k1) and for the unidirectional efflux of PC from apoHDL/PC complexes (k2) were equal to (0.45 +/- 0.2) x 10(-3) and (1.35 +/- 0.2) x 10(-3) min-1, respectively. The k1 values showed the Arrhenius dependence on the temperature within range 278-323 K. Plasma serum proteins facilitated the PC efflux from apoHDL/PC-Sepharose being additional acceptors of PC. These data allow use of apoHDL/PC-Sepharose for correcting lipid plasma composition in vitro.


[Synthesis of new pyrimidine 5'-H-thiophosphonates]. Thymidine and 3'-deoxythymidine 5'-H-thiophosphonates were synthesized by interaction of 3'-O-acetylthymidine or 3'-deoxythymidine and ammonium phosphinate in the presence of pivaloyl chloride and following oxidation in situ by sulfur of the intermediate phosphinates. The compounds seemed to be 1:1 mixture of diastereomers with chiral phosphorus. The prepared compounds were tested as potential inhibitors of HIV production.


Activation of gold-reactive T lymphocytes in rheumatoid arthritis patients treated with gold. To assess the role of T lymphocyte sensitization in the etiology of side effects of gold therapy in patients with rheumatoid arthritis (RA). Lymphocyte proliferation induced by gold(III) and gold(I) salts was measured in 53 subjects: 30 RA patients with gold-induced side effects (17 with dermatitis, 9 with proteinuria, 3 with hematologic complications, and 1 with colitis), 9 RA patients without side effects despite prolonged chrysotherapy, 4 RA patients who had never received gold, and 10 healthy controls. Peripheral blood lymphocytes were cultured with the different gold salts and proliferation was measured by 3H-thymidine incorporation. Thirteen of the 17 RA patients who developed gold-induced dermatitis showed significant T lymphocyte proliferation in response to gold(III) salts, and this proliferation could be completely blocked by monoclonal antibodies directed at the HLA-DR molecule. Such proliferative responses were not seen in patients with other gold-induced side effects, in patients who had never received gold, or in healthy controls. Only 1 of 9 patients who had not developed side effects despite long-term maintenance chrysotherapy showed significant lymphocyte activation with gold(III) salts. Lymphocyte proliferation could not be induced with gold(I) salts or with other metal salts. Patients with RA who develop dermatitis following treatment with sodium aurothiomalate [gold(I)] have T cells which proliferate in an HLA-DR-restricted manner in response to HAuCl4 [gold(III)]. We believe this observation can lead to more accurate diagnosis and treatment of side effects, which currently limit the use of one of the most effective antirheumatic drugs.


Lymphocyte responses to DR4/1-restricted peptides in rheumatoid arthritis. The immunodominant T cell epitope on the 19-kd Mycobacterium tuberculosis protein. Peptides presented by DR4/1 may be involved in the pathogenesis of rheumatoid arthritis (RA). T cell responses to DR4/1-restricted peptides unrelated to the causative antigen may be altered in RA. Thus, DR4/1-restricted lymphocyte responses in healthy volunteers and patients with RA were determined. Peripheral blood lymphocytes (PBL) and synovial lymphocytes were cultured with synthetic peptides spanning the 19-kd Mycobacterium tuberculosis (MT) protein. 3H-thymidine uptake by PBL from 5 of 7 healthy individuals and 5 of 7 RA patients increased in response to the N-terminal peptide (residues 1-20). Eleven fresh synovial fluid and 4 fresh synovial tissue (ST) lymphocyte samples did not proliferate in response to any of the peptides. However, the same T cell epitope was identified by ST lymphocytes when these were precultured. The N-terminal peptide was not a common antibody-binding site, unlike several of the other peptides. Similar responses by RA and normal PBL to a DR4/1-restricted immunodominant T cell epitope on the 19-kd MT protein were observed. The responses were more readily detected in PBL than in synovial lymphocytes. These observations may be relevant for assessing unrelated synthetic peptides in the development of DR4/1-restricted peptide immunotherapy.


Auranofin-induced vasomotor reaction.

Auranofin-induced vasomotor reaction. Vasomotor reactions after administration of gold sodium thiomalate (GST) are well recognized, but such reactions have not been reported to occur in association with oral gold treatment. We describe a woman with rheumatoid arthritis who experienced typical nitritoid reactions after treatment with GST, and later, with auranofin.


Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. RA SF and ST T cells were found to be markedly enriched in CD45RAdim, CD45RO+, CD45RBdim mature memory cells, whereas in the PB, CD45RAbright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RBdim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3-stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RBdim phenotype and B cell help, CD45RBdim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RBdim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RBdim T cells with mitomycin C. In contrast, healthy control sorted CD45RBbright or sorted CD4+, CD45RO+, CD45RBbright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. RA synovium is enriched in differentiated CD45RBdim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.


Ankylosing spondylitis in Rochester, Minnesota, 1935-1989. Is the epidemiology changing? To determine trends in the incidence and clinical presentation of ankylosing spondylitis first diagnosed between 1935 and 1989 among residents of Rochester, Minnesota, and in the survival of the patients. Population-based descriptive study. The overall age- and sex-adjusted incidence rate was 7.3 per 100,000 person-years (95% confidence interval 6.1-8.4). The rate tended to decline between 1935 and 1989, but there was little change in the age at symptom onset or diagnosis over the 55-year study period. Overall survival was not decreased up to 28 years following diagnosis. These data indicate that there is a constancy in the epidemiologic characteristics of ankylosing spondylitis and suggest that previously study results indicating changes may have been due to biases in patient selection and study design.


Antibodies to retroviral proteins in autoimmune connective tissue disease. Relation to clinical manifestations and ribonucleoprotein autoantibodies. To study the relationship between antibodies that recognize human retroviral proteins and the presence of clinical features and ribonucleoprotein antibodies in patients with autoimmune connective tissue diseases (CTDs). Antibodies against native human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia/lymphoma virus type I, recombinant HIV-1 Nef protein, and ribonucleoprotein antigens were determined by immunoblot of sera from 65 prospectively studied patients with definite or suspected CTDs of autoimmune type. Antibodies to retroviral proteins (ARP), most frequently to HIV Gag proteins p55 and p24, were found in 64% of 22 patients with systemic lupus erythematosus (SLE), in 63% of 8 patients with discoid LE (DLE), in 75% of 8 patients with mixed connective tissue disease (MCTD), and in 26% of 19 individuals with chronic biologically false-positive (CBFP) seroreactions, but not in 8 patients with subacute cutaneous lupus erythematosus. No clear correlation of ARP with antibodies to any specific small nuclear RNP antigen was observed. The most striking finding was that recurrent infections, both in LE patients and in those with CBFP reactions and widespread, acral discoid skin lesions, occurred significantly more often in ARP-positive patients. The occurrence of antibodies reacting with human retroviral proteins is associated with severe skin lesions and recurrent infections in SLE, DLE, and MCTD patients, and with a disposition toward developing systemic disease in CBFP reactors.


Association of dry eyes and dry mouth with anti-Ro/SS-A and anti-La/SS-B autoantibodies in normal adults. To evaluate the extent to which seemingly healthy, mature adults with mild symptoms of dry eyes or dry mouth share the immunologic features found in patients with Sjögren's syndrome. Of 705 subjects in Malmö, Sweden (age range 52-72 years) who responded to a questionnaire, 35% reported some symptoms of dry eyes or mouth. A random subgroup of the symptomatic subjects (n = 77) and an age- and sex-matched control group from among the asymptomatic subjects (n = 32) were evaluated objectively by serologic testing and by various measures of exocrine gland function. The symptomatic subjects had relatively impaired exocrine gland function and elevated levels of anti-Ro and anti-La (1.54-2.88-fold increase compared with the asymptomatic subjects, 95% confidence interval [CI] 1.41, 4.03). The 2 autoantibodies correlated with each other (r = 0.64, 95% CI 0.49, 0.78) as well as with selected clinical measures of glandular function. The association between self-reported symptoms of dry eyes or dry mouth and anti-Ro and anti-La, found in more than one-third of mature adults in this study, suggests that the immune abnormalities and exocrine gland dysfunction found in Sjögren's syndrome affect a substantial proportion of the general population.


Interactions of synovial fluid immunoglobulins with chondrocytes. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with 125I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by 51Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF: Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.


Induction of synthesis and release of interleukin-8 from human articular chondrocytes and cartilage explants. The activation of neutrophils in the joint space may contribute to the destruction of cartilage matrix observed in rheumatoid arthritis. The capacity of articular chondrocytes to synthesize and secrete interleukin-8 (IL-8) and GRO alpha, two potent neutrophil chemoattractant peptides, was investigated to determine whether cartilage itself could serve as a source of these small cytokines. Induction of IL-8 and GRO protein was studied both at the messenger RNA (mRNA) and the protein level by reverse transcriptase/polymerase chain reaction and metabolic labeling, respectively. Strong induction of IL-8 was observed in primary cultures of articular chondrocytes as well as in cartilage explants stimulated with IL-1 beta. The increased secretion of the IL-8 protein was accompanied by corresponding increases in mRNA levels. In contrast to other connective tissue cells, a peptide corresponding in molecular size to the GRO proteins was only weakly induced in cartilage explants or primary chondrocyte cultures. However, mRNA for all 3 members of the GRO family was easily detectable in cultured chondrocytes following stimulation with IL-1 beta. In explanted cartilage, mRNA for only GRO gamma was found to be induced. Newly synthesized IL-8 was slowly released from cartilage explants over a prolonged time in culture. The results suggest that synthesis and secretion of the diverse members of the IL-8/GRO family is regulated in a tissue-specific or cell-specific manner. The slow release of IL-8 from articular cartilage following induction by IL-1 beta could establish a chemotactic gradient toward the articular surface and mediate the migration and attachment of neutrophils and lymphocytes to this tissue.


ATP-induced chondrocalcinosis.

ATP-induced chondrocalcinosis. To determine whether adult articular cartilage mineralizes in the presence of ATP. Intact adult porcine articular cartilage and monolayers of chondrocytes were cultured in physiologic media containing ATP, and mineralization was measured as retention of 45Ca. Cartilage was analyzed by electron microscopy. Articular cartilage sequestered 45Ca when incubated with 100 microM ATP: Use of the ATP analog alpha, beta-methylene ATP did not promote mineralization and addition of pyrophosphatase inhibited mineralization, indicating that hydrolysis of ATP to AMP and inorganic pyrophosphate is necessary for the process to occur. Mineral was concentrated in articular cartilage vesicles in the perichondral area. Adult articular cartilage mineralizes in the presence of ATP, in a manner similar to that found with isolated matrix or articular cartilage vesicles. This supports the notion that these structures have a role in chondrocalcinosis.


Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts. To study the plasminogen activator inhibitor(s) (PAI) produced in vitro by human synovial fibroblast-like cells. Human synovial cell explant cultures were established using cells from nonrheumatoid donors. PAI-2 and PAI-1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. The synovial fibroblasts produced both PAI-2 and PAI-1. Interleukin-1 (IL-1) increased PAI-2 but decreased PAI-1 formation, both at the protein and the mRNA levels. Using cyclooxygenase inhibitors, evidence was obtained that an endogenous cyclooxygenase product(s) in the IL-1-treated cultures inhibited formation of both PAIs; exogenous prostaglandin E2 (10(-7) M) reversed the effect of cyclooxygenase inhibition. The glucocorticoid dexamethasone (10(-6) to 10(-7) M) inhibited IL-1-stimulated PAI-2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. PAI-2 formation and PAI-1 formation can be regulated independently in human synoviocytes, illustrating the complexity of the modulation of the net PA activity expressed by these cells.


Development of a sensitive enzyme immunoassay for the determination of vinpocetine in human plasma. An Enzyme immunoassay for the quantitative determination of vinpocetine (CAS-42971-09-5) in human plasma has been developed. The lower limit of quantification is 0.1 ng/ml plasma. The assay shows no cross reactivity with the major metabolite apovincaminic acid. Because of a strong unspecific binding of vinpocetine to plasma proteins an extraction step was necessary. The inter- and intra-assay reproducibility of the test (coefficient of variation) is in a range of 1.1 and 18.3%.


Studies with new ergoline derivatives on the effects of central and peripheral 5-hydroxytryptamine receptors. The antiserotonin properties of a series of new ergoline derivatives were investigated in several pharmacological test systems which have been proposed for the characterization of putative antagonists at central and peripheral 5-HT2 receptors. In radioligand binding studies with [3H]ketanserin among the new ergolines only 1-methyl-2-brom-9,10-dihydrolysergic acid-bis(beta-acetoxyethyl)-amide (AWD 52-336) showed high affinity at cortical 5-HT2 receptors (Ki-5.4 nmol/l). In 5-HT-amplified ADP-induced aggregation of human platelets 6-nor-6-propyl-9,10-dihydro ergometrine (AWD 52-227) and 9,10-dihydrolysergic acid-di-ethanol-amide (AWD 52-302) were potent inhibitors of 5-HT response. Comparison of this two in vitro tests demonstrated a significant correlation (r = 0.636; p < 0.05) between the ability of the ergolines to block the 5-HT aggregation mediated by platelet 5-HT2 receptors and their affinity to [3H]ketanserin-labelled binding sites in rat cortical membranes. In the used in vivo tests (tryptamine tremor, 5-HTP-induced head twitches) 1-methyl-9,10-dihydrolysergic acid-bis(beta-acetoxyethyl)-amide (AWD 52-83) and AWD 52-336 were found to antagonize the behavioural responses with comparatively moderate potency. The results suggest, therefore, that AWD 52-83 and AWD 52-336 may be both central and peripheral acting 5-HT2 antagonists, whereas AWD 52-227 and AWD 52-302 seem to be potent blockers at peripheral 5-HT2 receptors. Furthermore, the obtained results allow to reveal structure-activity relationships of ergolines. Substitution in position 1 in the tetracyclic ergoline ring system may be important with respect to the efficacy at central 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiovascular effects of the novel potassium channel opener (3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo- 1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate. Cardiovascular effects of the novel potassium channel opener (3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6- phenylsulfonylchromane hemihydrate (Hoe 234, CAS 132014-21-2) were investigated in rats, dogs and monkeys. In all species and independent of the route of administration Hoe 234 lowered systemic blood pressure accompanied with increases in heart rate. In rats after intravenous (i.v.) application Hoe 234 was 3 times more potent than cromakalim and its effects were reduced by pretreatment with the potassium channel blocker glibenclamide. Following intraduodenal application again Hoe 234 was more potent but mean arterial blood pressure (MAP) decreased more slowly and maximal effects were obtained later than after cromakalim. Oral administration of either single or repeated doses, however, revealed a somewhat higher potency for cromakalim. In anesthetized dogs Hoe 234 i.v. reduced MAP more potently than cromakalim whereas changes in heart rate were less pronounced. Cardiac output was increased and total peripheral resistance decreased for either agent. These results show that Hoe 234 is a novel potassium channel opener lowering blood pressure in animals due to peripheral vasodilation. It compares favourable with known potassium channel openers except for oral administration.


Pharmacokinetics of the organic nitrates trans-2-amino-2-methyl-N-(4- nitroxycyclohexylmethyl)-propionamide in dogs, and of 4-(2-nitroxyethyl)-piperidine in dogs and in monkeys. The plasma concentrations of the organic nitrates (nitric acid esters) trans-2-amino-2-methyl-N-(4-nitroxycyclohexylmethyl)-propionamide (BM 12.1200) and of 4-(2-nitroxyethyl)-piperidine (BM 12.1173, CAS 129999-77-5) were determined in dog plasma by capillary gas chromatography with electron capture detection (GC-ECD). Intra- and interassay variation coefficients of the gas chromatographic analysis lay between 2.9 and 8.8%; recoveries amounted to 50-62%. Both nitric acid esters were absorbed quickly and attained maximum plasma levels within 1 h. The total bioavailability of BM 12.1200 is > 55%, and that of BM 12.1173 is 63%; their elimination half-lives are, respectively, 4.2 h and 1.3-1.4 h. Thus, BM 12.1173 and the reference substance IS-5-MN (isosorbide-5-mononitrate) show corresponding elimination half-lives. These results regarding the pharmacokinetic parameters of BM 12.1173 were confirmed by cross-over application of BM 12.1173 and IS-5-MN to Macaca Arctoides. In comparison to all species which have been treated with IS-5-MN thus far, the shortest elimination half-life (t1/2 = 0.6 h) is found in monkeys.


Metoprolol and propranolol treatment in carbon tetrachloride-induced hepatic injury. The effects of beta 1-selective metoprolol (CAS 37350-58-6) and the nonselective beta-adrenoceptor antagonist propranolol (CAS 525-66-6) were investigated in healthy and CCl4 damaged male rats. Treatments were performed for 16 days, orally with dosages reducing heart beat/min by 25% (metoprolol 10 mg/kg, propranolol 1 mg/kg). Liver glycogen content was not influenced by either beta-blocker in healthy animals. CCl4-induced loss of glycogen was equally moderated by metoprolol and propranolol. Blood glucose increased in metoprolol treated healthy and liver damaged rats after a single dosage likewise following prolonged treatment. Propranolol was without effect on blood glucose level. Cytochrome P-450 decline in microsomal fraction was greater in metoprolol, than in propranolol treated healthy animals. However, the severe fall elicited by liver injury was moderated by metoprolol and normalised by propranolol. Cytochrome b5 seems to be involved in metoprolol metabolism. Cytochrome P-450-dependent aminopyrine-N-demethylase was impeded by metoprolol in animals with healthy liver. The serious inhibition caused by CCl4 was moderated by metoprolol and with a better result by propranolol. The high serum bilirubin level in liver lesion was lowered by metoprolol and particularly by propranolol. Neither phase I metabolic process aminopyrine-N-demethylation nor phase II glucuronidation were normalised. A comparison of this data with the results of a previous 12-day treatment schedule indicates that no changes in efficacy occurred with longer treatment. The present results pertain to the importance of the selection of beta-adrenoceptor blocker in liver lesion.(ABSTRACT TRUNCATED AT 250 WORDS)


General pharmacological properties of the new vasodilator flosequinan. Pharmacological effects of a new vasodilator, flosequinan (7-fluoro-1-methyl-3-(methylsulfinyl)-4(1H)-quinolone, BTS 49 465, CAS 76568-02-0) on the central nervous system, somatic nervous system, autonomic nervous system and smooth muscle, digestive system and miscellaneous organs were investigated. 1. The central nervous system: Flosequinan inhibited acetic acid-induced writhing at doses of more than 30 mg/kg p.o. and decreased body temperature and tended to decrease spontaneous movement slightly in mice at a dose of 100 mg/kg p.o. However, flosequinan had little effect on hexobarbital-induced hypnosis, reserpine-induced hypothermia and motor coordination and lacked anticonvulsant and analgesic activities in mice. Flosequinan had little effect on general behavior in rats and did not have any effect on spontaneous EEG and EEG arousal response in rabbits. 2. The somatic nervous system: Flosequinan did not cause muscle relaxation in mice and had little effect on neuromuscular transmission in cats. No local anesthetic activity was exhibited through inhibition of the corneal reflex in guinea pigs. 3. The autonomic nervous system and smooth muscle: Flosequinan produced a relaxation of the isolated trachea of guinea pigs at concentrations of more than 3 x 10(-5) mol/l, but its potency was very weak in comparison with that of isoproterenol (isoprenaline). Flosequinan inhibited spontaneous motility of the isolated uterus of pregnant rats at concentrations higher than 10(-4) mol/l and the motility of the uterus of non-pregnant rats in vivo was inhibited at 30 mg/kg i.v. Flosequinan does not seem to exert any on norepinephrine, serotonin, acetylcholine or histamine. This is supported by the fact that at concentrations of 10(-4)-3 x 10(-3) mol/l non-competitive inhibition was observed with regard of the contractions of the isolated aorta and vas deferens of rats induced by norepinephrine, the contraction of isolated rat stomach induced by serotonin, the contraction of isolated guinea-pig ileum induced by acetylcholine, histamine and barium chloride and the contraction of the isolated uterus of non-pregnant rats induced by oxytocin. However, flosequinan was more potent as a relaxant of vascular than of these other smooth muscles. The drug was slightly inhibitive at a high dose of 30 mg/kg i.v. with regard of the contraction of nictitating membrane induced by stimulation of preganglionic sympathetic nerve in cats. 4. The digestive system: Flosequinan at 100 mg/kg p.o. inhibited intestinal propulsion in mice and inhibited spontaneous motility of stomach and duodenum of rats at a dose of 30 mg/kg i.v.(ABSTRACT TRUNCATED AT 400 WORDS)


[Postprandial lipemia under treatment with Allium sativum. Controlled double-blind study of subjects with reduced HDL2-cholesterol]. Postprandial Lipaemia under Treatment with Allium sativum/Controlled double-blind study in healthy volunteers with reduced HDL2-cholesterol levels. The effectiveness of a standardized garlic powder preparation (Sapec, Kwai) on alimentary hypertriglyceridaemia after intake of a standardized fatty test meal containing 100 g butter was analyzed in a randomized placebo-controlled double-blind study. 24 volunteers with HDL2-cholesterol concentrations in plasma of less than 10 mg/dl (men) respectively 15 mg/dl (women) participated in the study. The volunteers received 3 times 1 tablet daily over a period of 6 weeks equivalent to a daily dosage of 900 mg garlic powder in the active treated group. Control measurements were made on the 1st, 22nd and 43rd day of treatment and 0, 3 and 5 h after intake of the meal. The postprandial increase of triglycerides was clearly reduced under garlic medication as compared to placebo treatment. The determined AUC-values for the triglycerides were up to 35% lower in the garlic group compared to the placebo group. The regular intake of the garlic preparation over the period of 6 weeks showed a significant lowering of the fasting values of triglycerides in comparison to placebo. Under garlic medication HDL2-cholesterol increased more than under placebo in tendency.