Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli. The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.


The binding of bridged bis-pyridinium oximes to DNA and its relevance to the induction of mitochondrial dysfunction in yeast. Bis-pyridium oximes and methoximes from a newly synthesized series are weak DNA binders (K = 3.10(4) M-1 under physiological conditions). From the number of binding sites per phosphate, 0.25, the ionic strength dependence of the binding constant and the negative electric dichroism, it is concluded that monointercalation is the mode of association. In contrast to methoxy compounds, the oxime derivatives are able both to induce the mutated "petite" phenotype in yeast S. cerevisiae and to cause "in vitro" extensive condensation of single stranded DNA. This reaction is postulated to be relevant to the mutational process that leads to "peptide" cells. The absence of nuclear mutation is interpreted in terms of sequestration of the drug in mitochondria under the effect of the organelle inner membrane electrochemical potential.


Regulation of endothelin-1 synthesis in cultured guinea pig airway epithelial cells by various cytokines. To study regulatory mechanisms influencing the synthesis and release of ET-1, a potent bronchoconstrictor, epithelial cells from guinea pig tracheas were cultured to test various cytokines for the synthesis of ET-1 and its precursor, big ET-1. Cytokines tested were divided into 4 groups, based on their potential modes of action. IL-8, TNF alpha and TGF beta transiently increased the synthesis of ET-1, while EGF, PDGF and GM/CSF promoted proliferation of ET-1 synthesizing cells. IL-1 enhanced the synthesis of ET-1 precursor without mitogenesis, whereas IL-2, IL-6 and IGF-1 induced both the synthesis of big ET-1 and mitogenesis. These observations suggest that cytokines involved in damage, inflammation and repair of the airway epithelial layer regulate the synthesis and release of ET-1 by multiple mechanisms, thereby influencing airway muscle tone.


Molecular cloning and chromosomal mapping of a human protein-tyrosine phosphatase LC-PTP. We isolated cDNA clones encoding a protein-tyrosine phosphatase (PTP) from a human T cell PEER cDNA library. The predicted open reading frame encodes a approximately 40-kDa protein composed of 360 amino acids and has no apparent hydrophobic segments, suggesting that it is a nontransmembrane PTP, which was designated as LC-PTP (leukocyte PTP). Northern blot analysis revealed that the LC-PTP mRNA was preferentially expressed in a variety of hematopoietic cells and the transcriptional sizes were approximately 4.0 kilobases and approximately 2.9 kilobases. Fluorescent in situ hybridization revealed that the human LC-PTP gene is located on the chromosome region 1q32.1, which is known to be a site associated with chromosomal deletion in malignant lymphomas, where candidate tumor suppressor genes might be present.


Peptide tyrosine phenylalanine: a novel neuropeptide F-related nonapeptide from the brain of the squid, Loligo vulgaris. A novel nonapeptide, sequence YAIVARPRFamide, was isolated from brain extracts of the squid, L. vulgaris. Designated peptide tyrosine phenylalanine (PYF), the peptide shows marked homology with the C-terminal nonapeptides of pancreatic polypeptide and neuropeptide F (NPF) from a number of sources. If PYF is the C-terminal nonapeptide of squid NPF, then it may be derived by a novel processing mechanism involving specific cleavage between two TYR residues. PYF may be a highly truncated, receptor-active variant of NPF.


Evidence for thiophene-S-oxide as a primary reactive metabolite of thiophene in vivo: formation of a dihydrothiophene sulfoxide mercapturic acid. Urine of rats treated with thiophene contains a very major metabolite which represents about 30% of the administered dose. A detailed analysis of its 1H and 13C NMR spectra and a study of its IR and mass spectra clearly showed that it was a 2,5-dihydrothiophene sulfoxide bearing a N-acetyl-cysteinyl group on position 2. Upon heating, it lost water with formation of N-acetyl-S-(2-thienyl)-L-cysteine. A likely mechanism for the formation of this metabolite should involve the S-oxidation of thiophene as a primary step and the addition of glutathione to the very reactive thiophene-S-oxide. These data provide a first evidence for the intermediate formation in vivo of thiophene-S-oxides as reactive metabolites.


Immunosuppressive molecules produced by Sertoli cells cultured in vitro: biological effects on lymphocytes. In the present study we have analyzed the proteins secreted in vitro by murine Sertoli cells to identify immunosuppressive factors. Our data show that Sertoli cells secrete molecules capable to inhibit proliferation of lymphocytes activated in vitro. Cytophluorimetric analysis indicates that treated cells are arrested in the G1 phase of cell cycle. The inhibitory activity is specific for both B or T lymphocytes but not for other non-lymphoid cells and is associated to proteins, heat and freeze stable, with Mr of more than 30 kDa. Lymphocytes treated with Sertoli immunosuppressive proteins drastically reduce the secretion of interleukin-2.


Cholesterylene, a newly recognized tissue lipid, found at high levels in the cornea. In the course of measuring the concentration of cholesterol in an opacified dog cornea by gas-chromatography, relatively large amounts of an unidentified non-saponifiable lipid were recognized. When the unknown lipid was subjected to gas chromatographic-mass spectral analysis it displayed a major ion at m/z 368 M+. and was identified as cholesta-3,5-diene, cholesterylene, by computer match with mass spectral-registry data. Cholesterylene was then shown to be present in the corneas of normal dogs, cows and humans, accounting for 20-25% of the total steroid-sterol in dog corneas and 5-10% in cow and human. Cholesterylene, which can be considered as an extremely nonpolar dehydration product of cholesterol, has not previously been recognized in animal tissues. Although the source of corneal cholesterylene is unknown, preliminary results suggest non-enzymatic formation from cholesterol.


Interaction of ubiquinone and vitamin K3 with mitochondrial succinate-ubiquinone oxidoreductase. Two new methods have been devised for measuring fumarate reduction by beef heart succinate-ubiquinone oxidoreductase with quinols as electron donors. In one assay the quinone is maintained in the reduced state by coupling fumarate reduction with the DT-diaphorase reaction, in the other assay by the presence of excess dithionite. The advantages of these methods are discussed, along with preliminary characterization of the quinol-protein interaction.


Dichotomy in the laminin-binding properties of soluble and membrane-bound human galactoside-binding protein. Recent studies indicate that galactoside-binding proteins may bind the poly-N-acetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins.


Polyamines in neurotrauma. Ubiquitous molecules in search of a function. In spite of their abundance, the function of PAs in the adult nervous system remains enigmatic. It is postulated that after trauma, the induction of polyamine metabolism (i.e. the polyamine response), which is inherently transient, is an integral part of a protective biochemical program that is essential for neuronal survival. Several functions ascribed to PAs may assume importance in cellular defense. Thus, regulation of the ionic environment, modulation of signal pathways, control of cellular Ca2+ homeostasis, inhibition of lipid peroxidation, and interaction with nucleic acids are all putative sites for PA action. During maturation, the CNS, unlike the peripheral nervous system, undergoes changes which result in the expression of an incomplete polyamine response after trauma. This may be due to an altered pattern of gene expression, and/or restrictive compartmentalization of the PAs and their metabolizing enzymes. Induction of this partial polyamine response after injury results in a sustained accumulation of putrescine, which by itself may be harmful, without the concomitant increase in spermidine and spermine. Administration of exogenous PAs after trauma exerts a neuroprotective effect. Exogenous PAs are postulated to gain access into cells via an induced uptake system after trauma, and function similarly to newly synthesized PAs. Besides the injured neurons themselves, tissues which are connected or associated with these neurons may be potential targets where PAs could act to stimulate neurotrophic factor production. Based on the neuroprotective effects of PAs in laboratory animals and on their proposed role in mechanisms of neuronal survival, the development of PA-based compounds as therapeutic neuroprotective agents should be pursued.


The sensitivity of human tumour cells to quinone bioreductive drugs: what role for DT-diaphorase? 15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(IH-indole-4,7-dione)prop-beta- en-alpha-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for EO9 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.


Demonstration of ryanodine-induced metabolic effects in rat liver. The effects of ryanodine, a plant alkaloid which alters Ca2+ sequestration in the liver, on O2 uptake and gluconeogenesis were measured. Ryanodine administration to perfused rat liver resulted in the stimulation of O2 uptake and of gluconeogenesis. Because ryanodine does not affect directly mitochondrial respiration, its stimulatory effect on O2 uptake in the whole cell is likely to be secondary to the increased cytosolic free Ca2+ levels.


Polarized efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers. We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 microM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 microM). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.


Effects of cysteine and acetaminophen on the syntheses of glutathione and adenosine 3'-phosphate 5'-phosphosulfate in isolated rat hepatocytes. The aim of the present study was to introduce and validate a radioactive tracer method in which adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and glutathione (GSH) are measured simultaneously in isolated hepatocytes. PAPS and GSH are co-substrates in sulphation and GSH conjugation, and both are dependent on sulphur deriving from cysteine. The effect of cysteine on the syntheses was investigated at non-toxic and toxic concentrations of the hepatotoxic drug acetaminophen (AA). Administration of AA trapped radioactivity (35S) in the pre-labelled PAPS and GSH pools by formation of the metabolites, AA-sulphate and AA-GSH. Turnover rates were determined from the decline of AA-sulphate and AA-GSH specific activity. Syntheses of PAPS and GSH were calculated by multiplying the rates with the concentrations of the respective co-substrates. Increasing AA concentration from non-toxic to toxic levels resulted in increased median PAPS and GSH syntheses (8 to 11 and 311 to 2218 nmol/10(6) cells/min, respectively) (P less than 0.05). Addition of cysteine did not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min) but decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min) (P less than 0.05) in experiments with non-toxic AA concentrations. In experiments with toxic AA concentrations opposite effects of cysteine were seen, i.e. median PAPS synthesis was reduced (3 to 2 nmol/10(6) cells/min) (P less than 0.05) while median GSH synthesis was unchanged (23 to 16 nmol/10(6) cells/min). The present method provides a tool in which two important detoxification pathways can be measured simultaneously and the data suggest that the two pathways are regulated by substrate availability.


Induction of cytochrome P450 activities by polychlorinated biphenyls in isolated mouse hepatocytes. Influence of Ah-phenotype and iron. Exposure of cultured primary hepatocytes from Ah-responsive male C57BL/10ScSn mice to a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) at 0.1-20 micrograms/mL for up to 96 hr induced cytochrome P4501AI-mediated activity (ethoxyresorufin O-deethylase, EROD) up to 50-fold. In contrast, pentoxyresorufin O-dealkylase (PROD), which in some circumstances is a measure of phenobarbitone-induced cytochrome P450 isoenzymes, was induced only 5-fold. There were similar findings on EROD activities with the pure compounds 3,3',4,4',5,5'-hexachlorobiphenyl, 3,3',4,4',5,5'-hexabromobiphenyl and 3,3',4,4'-tetrachlorobiphenyl(TCB) and also beta-naphthoflavone but not with 2,2',4,4'-TCB or phenobarbitone. The higher concentrations of Aroclor 1254 were also associated with cytotoxicity as estimated by release of alanine aminotransferase (ALT) into the medium. Unlike in C57BL/10ScSn hepatocytes induction of EROD and cytotoxicity was minimal in hepatocytes from the Ah-non-responsive strain DBA/2. Although in vivo the hepatic toxicity and carcinogenicity of polyhalogenated aromatics are markedly potentiated by iron, no enhancement of the cytotoxicity of Aroclor 1254 towards C57BL/10ScSn hepatocytes by iron was observed in vitro. However, iron caused decreased EROD activities and possibly cytochrome P4501AI (as judged by Western blotting) as in vivo. Even in the presence of iron and the haem precursor 5-aminolaevulinic acid (5-ALA) there was no development of uroporphyria in this system although this occurs with Aroclor in vivo and is enhanced by iron. Accumulation of uroporphyrin did occur after extended culture of C57BL/10ScSn hepatocytes on matrigel for 8 days in the presence of 5-ALA and Aroclor 1254 but again no potentiation by iron was observed. Thus, although culture of Ah-responsive and -non-responsive hepatocytes mimics some aspects of the mechanisms of in vivo toxicity of PCBs, there is some unknown associated influence of iron metabolism which cannot, as yet, be produced in vitro but which is of importance in vivo.


Increased activity of the GABAergic system in selected brain areas after chronic propranolol treatment in spontaneously hypertensive rats. The influence of chronically administered propranolol on the functional state of the gamma-aminobutyric acid-ergic (GABAergic) system in spontaneously hypertensive rats was studied and compared with the effect of dihydralazine. GABA content, synthesis and turnover rate in selected brain areas were assessed. Hypotensive activity of propranolol and dihydralazine after injection of GABA antagonist pictrotoxin was examined in acute experiment. Prolonged administration of propranolol increased GABA content, synthesis and turnover rate in the hypothalamus and the pons-medulla. After chronic injections of dihydralazine there was no change in GABA indices. Antihypertensive activity of dihydralazine in picrotoxin-treated animals remained unchanged. On the contrary, picrotoxin suppressed the propranolol-induced decrease in blood pressure. Our results indicate that propranolol increases GABAergic system activity. Therefore, we conclude that down-regulation of the GABAergic system in hypertension may be compensated by the regulatory action of propranolol.


Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P. A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.


Reduction of alpha-tocopherolquinone to alpha-tocopherolhydroquinone in rat hepatocytes. The contents of alpha-tocopherolhydroquinone (TQH2), alpha-tocopherolquinone (TQ) and alpha-tocopherol (Toc) in isolated rat hepatocytes and liver homogenates were determined by HPLC under anaerobic conditions, because TQH2 easily autoxidizes to TQ under aerobic conditions. The viable hepatocytes were used for the determination without homogenization. The hepatocytes contained 3.1, ND and 5.0, 3.1-9.0, and 31.3-63.2 nmol of TQH2, TQ and Toc/g liver, respectively. However, TQH2 was not detected in liver homogenates because endogeneous TQH2 autoxidizes to TQ during preparation of homogenates under aerobic conditions. The homogenates contained 2.0-23.5 and 36.5-54.9 nmol of TQ and Toc/g liver, respectively. Addition of TQ showed that TQ was reduced and converted into TQH2 in isolated hepatocytes. The TQH2 formation from TQ was also observed in liver homogenates in the presence of either NADPH or NADH. The formation was further analysed and confirmed by HPLC and mass spectrometry. The formation of TQH2 was also found to occur in mitochondria, microsomes and cytosol. The specific activity of NADPH-dependent TQ reductase activity was in the order of mitochondria greater than or equal to microsomes greater than cytosol. Furthermore, NADPH-cytochrome P450 reductase was found to catalyse TQH2 formation from TQ.


Peroxide-dependent amino acid oxidation and chemiluminescence catalysed by magnesium-pyridoxal phosphate-glutamate complex. Magnesium-pyridoxal-5'-phosphate-glutamate (MPPG) has been shown to ameliorate atherosclerotic symptoms in rabbits. In vitro, MPPG in the presence of peroxides such as cholesterolhydroperoxide or cumene hydroperoxide and Mn2+ ions produces "excited states" measurable as chemiluminescence or ethylene release from 1-aminocyclopropane-1-carboxylic acid (ACC). The reactions are stimulated synergistically by unsaturated fatty acids. Pyridoxal phosphate exhibits similar properties, but can be differentiated from the activities of MPPG or the sum of the components present in MPPG.


Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. The thienopyridine clopidogrel is not active in vitro and must be administered i.v. or orally, suggesting that metabolism is necessary for activity. To verify whether the effect after i.v. administration was consecutive to recycling by hepatic bile secretion of clopidogrel or its metabolite(s) in the digestive tract, a catheter was implanted in the choledocus of rats, preventing bile and pancreatic secretions from being excreted into the digestive tractus. Two hours after clopidogrel administration (10 mg/kg, i.v.), blood was withdrawn and platelet-rich plasma aggregation was measured after the addition of 5 microM ADP. Clopidogrel treatment was equally efficient for sham-operated and catheterized animals (% inhibition of platelet aggregation: 76% and 59%, respectively) suggesting that the i.v. effect of clopidogrel was independent of re-absorption of biliary-excreted products and consequently that enteric metabolism is not necessary for activity. The antiaggregating activity of clopidogrel in rats before and after functional hepatectomy by a porto-jugular shunt was then studied. A great difference between treated animals was observed 30 min after i.v. administration of 25 mg/kg of clopidogrel. Per cent inhibition of platelet aggregation was 76% and 6% (P less than 0.001) for sham-operated and hepatectomized animals, respectively. Similar results were obtained after intraduodenal administration of clopidogrel, showing that the treatment was completely ineffective in hepatectomized animals. In isolated, blood-perfused rat livers, clopidogrel inhibited ADP-induced platelet aggregation, thereby supporting the theory that the activity of clopidogrel is highly dependent on hepatic metabolism.


Effect of purification, theophylline and sodium fluoride on histamine release produced by antineoplastic drugs on rat mast cells. A distinctive mechanism of action for carboplatin. The antineoplastic drugs, cytarabine, ifosfamide, vinorelbine, doxorubicin, asparaginase and carboplatin, elicit histamine release from rat pleural and peritoneal mast cells. Both cell populations do not show heterogeneity in their response to stimulation by any of these drugs, with the exception of cytarabine, which activates pleural mast cells at lower concentrations. When these cells are purified with Percoll, L-asparaginase, vinorelbine and cytarabine completely lose their capability of inducing histamine release in both cell populations, while the other drugs still show the same pattern of response as with unpurified cells. These results indicate that some membrane component crucial for the action of vinorelbine, L-asparaginase and cytarabine is lost during the purification procedure. Pretreatment of the cells with theophylline completely inhibits the response to cytarabine, ifosfamide, vinorelbine and asparaginase, and inhibits the response to doxorubicin by up to 75% only. Theophylline does not change the response to carboplatin. Sodium fluoride does not change the response to any of the drugs tested, with the exception of carboplatin, in which case a complete inhibition is observed. In conclusion, carboplatin activates rat mast cells through a completely different mechanism of action with respect to the other drugs studied.


Differential stimulation of the GTPase activity of G-proteins by polylysine. Polylysine, polyornithine and, to a lesser extent, polyarginine were found to stimulate the GTPase activity of the purified recombinant alpha subunit of the human G(i)-3 transducing protein alpha i-3. Optimal stimulation of 4- to 5-fold was obtained with polylysine concentrations between 1 and 20 microM, higher concentrations being inhibitory. Polylysine at similar concentrations stimulated by 50% the GTPase of transducin (GT), the vision transducing protein, but had only a very slight effect on the GTPase of the p21 product of the H-ras protooncogene. The stimulation of the alpha i-3 GTPase caused by polylysine was due to a reduction of the apparent Km for GTP from 3.8 to 1.3 microM. The stimulation by polylysine was observed at free Mg2+ concentrations below 1 microM. These results indicate that polylysine acts in a fashion similar to mastoparan and substance P in mimicking the action of an agonist-bound receptor on G-proteins.


Studies of chlorambucil-DNA adducts.

Studies of chlorambucil-DNA adducts. Chlorambucil (CLB) is a bifunctional nitrogen mustard whose therapeutic and major side-effects are thought to be caused by binding to DNA. HPLC analysis of hydrolyzed DNA from L1210 cells incubated with [14C]CLB generated two peaks of radioactivity, indicating the formation of two or more major adducts. Since DNA incubated with [14C]CLB in a cell-free system gave rise to the same profile, experiments were conducted with DNA from cells exposed to radiolabeled DNA precursors, which was then reacted with CLB. DNA containing [8-14C]guanine gave rise to one peak of radioactivity, while DNA containing [2,8-3H]adenine gave rise to two peaks. These peaks corresponded to the peaks seen in the experiment with intact L1210 cells treated with [14C]CLB. Experiments with DNA containing [5-3H]cytosine indicated that no cytosyl adducts were formed. No adducts were seen in hydrolysates prepared from labeled DNA incubated with drug solvent alone. These data indicate that the majority of adducts induced by CLB are guanyl adducts, but a substantial quantity of adenyl adducts has also been identified.


Primaquine metabolism by human liver microsomes: effect of other antimalarial drugs. A number of drugs have been studied for their effect on the metabolism of the antimalarial drug primaquine by human liver microsomes (N = 4) in vitro. The only metabolite generated was identified as carboxyprimaquine by co-chromatography with the authentic standard. Ketoconazole, a known inhibitor of cytochrome P450 isozymes, caused marked inhibition of carboxyprimaquine formation with IC50 and K(i) values of 15 and 6.7 microM, respectively. This finding and the dependency of metabolite formation on NADPH indicates that cytochrome P450 isozyme(s) catalysed metabolite production. Of compounds actually or likely to be coadministered with primaquine to malaria patients, only mefloquine produced any inhibition (K(i) = 52.5 microM). Quinine, artemether, artesunate, halofantrine and chloroquine did not significantly inhibit metabolite formation. It seems unlikely that the concurrent administration of mefloquine, or other antimalarials, with primaquine will lead to appreciably altered disposition.


Inhibitory potency of some isatin analogues on human monoamine oxidase A and B. Isatin is an endogenous compound which acts as a selective inhibitor of monoamine oxidase (MAO) B. In this study a range of isatin analogues were tested for their in vitro inhibition of human MAO A and B. Most of the analogues were less potent than isatin. Hydroxylation of the aromatic ring changed the inhibitory potency in favour of MAO A, with 5-hydroxyisatin being a potent and selective MAO A inhibitor (IC50 8 microM). Isatinic acid, which is formed reversibly from isatin at alkaline pH, showed no inhibition.


A novel note on the effect of ara-C on the polyamine content of the male accessory organs of the rat. The effects of 1-beta-D-arabinosylcytosine (ara-C) given for 5 days on body weight, on the weights of the prostate, seminal vesicles and testis, and on the polyamine content (putrescine, put; spermidine, spd; spermine, spm) in these organs were examined in rats. The body weights of the ara-C-treated rats showed a decrease of 13% (P less than 0.01) and the weights of the prostate, seminal vesicles and testis were reduced by 20% (P less than 0.05), 13% and 5.0% (P less than 0.1), respectively. However, put, spd and spm contents in the ara-C-treated prostate were increased 1.2, 1.8 and 2.9 times, respectively. The contents of these polyamines in the ara-C-treated seminal vesicles increased about 40% for all polyamines. Both spd and spm in the treated testis showed a significant increase of about 20% (P less than 0.05) as compared with the control. In addition, the total polyamines in the whole organs increased significantly in both the prostate and testis.


Induction of rat hepatic and intestinal glutathione S-transferases by dietary butylated hydroxyanisole. To obtain insight into the protection mechanism of butylated hydroxyanisole (BHA), a widely used food preservative with anticarcinogenic properties, we investigated the effects of dietary BHA on rat hepatic and intestinal glutathione S-transferase (GST) enzyme activity, and GST isozyme levels. In the proximal small intestine and liver, BHA supplementation significantly increased GST enzyme activity as compared with controls (2.3- and 1.7-fold, respectively, P less than 0.05). GST class alpha and mu contents were significantly higher only in the small intestine (1.6-2.1-fold and 1.3-1.5-fold, respectively, P less than 0.05), whereas GST class pi was significantly induced in liver (4.6-fold, P less than 0.05).


Splanchnic uptake of haloperidol and release of reduced haloperidol in vivo in the guinea pig. Splanchnic uptake of haloperidol (HAL) and release of reduced haloperidol (RHAL) were studied in vivo in guinea pigs. Anesthetized animals with implanted cannulae in the aorta, the hepatic vein and the inferior vena cava were infused intravenously with HAL at a rate of 9.6 micrograms/min/animal for 90 min. Plasma HAL and RHAL in samples taken from the arterial and hepatic venous cannulae were measured by HPLC with an electrochemical detector. Contamination of the hepatic venous samples by blood from the inferior vena cava was ruled out by the validation method of tritiated water washout [Huang MT, J Appl Physiol 71: 359-364, 1991]. HAL concentrations plateaued at 70-80 ng/mL in the aorta and 5-7 ng/mL in the hepatic vein during the final 30 min of infusion. Splanchnic extraction of HAL was 91%. Hepatic blood flow was estimated to be 1.95 +/- 0.40 (SD) mL/min/g. If assuming that splanchnic uptake of HAL took place in the liver, a rate of uptake of HAL in the liver of 79.2 +/- 18.6 (SD) ng/min/g could be calculated by the Fick principle. The uptake in the whole liver accounted for 14% of the rate of HAL infusion into the animal. Plasma RHAL in the aorta, 6.4 +/- 6.6 (SD) ng/mL at 60 min and 9.4 +/- 4.6 (SD) ng/mL at the end of HAL infusion, was about 10-12-fold less than aortic HAL. The concentrations of RHAL in the hepatic vein were not significantly different from those in the aorta, indicating that splanchnic tissues including the liver are not responsible for plasma RHAL secretion. The highly efficient uptake of HAL as well as the ketone reductases found previously in vitro in liver microsomes of guinea pigs were probably involved only in biliary excretion of HAL.


Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast. A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, PELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50 = 0.05 microM; Qn IC50 = 4 microM) and O-demethylation (Qd IC50 = 0.05 microM; Qn IC50 = 4 microM). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol, a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.


The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats. Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.


Salicylate-induced cation fluxes across biological membranes. A study of the underlying mechanism. The effect of salicylate on cation transport by lactating rat mammary tissue has been examined. Consistent with previous results, salicylate increased the unidirectional efflux of K+ (Rb+): this increase was dependent on extracellular Ca2+. Lowering the temperature of the incubation medium from 37 degrees to 4 degrees did not attenuate the effect of salicylate on K+ (Rb+). Ca2+ uptake by mammary tissue was stimulated by salicylate, whereas Ca2+ efflux was not greatly affected. Salicylate reduced the ouabain-insensitive uptake of K+ (Rb+). The net K+ content of mammary tissue was reduced by incubating tissue in a medium containing salicylate, whereas net Na+ content was increased. The results are consistent with the hypothesis that salicylate activated a calcium-dependent K+ efflux channel and/or a calcium-dependent non-selective cation channel.


The microsomal demethylation of N,N-dimethylbenzamides. Substituent and kinetic deuterium isotope effects. The metabolism of N,N-dimethylbenzamides by phenobarbital-induced rat liver microsomes results in the formation of N-methylbenzamides and formaldehyde. The reaction proceeds via the formation of an intermediate N-hydroxymethyl-N-methylbenzamide, which, for the microsomal oxidation of N,N-dimethylbenzamide, was isolated and characterized. Confirmation of the N-hydroxymethyl-N-methylbenzamide was obtained by its independent synthesis from N-methylbenzamide and formaldehyde. The intermolecular kinetic deuterium isotope effects for the reaction are 0.9 (+/- 0.1) for Vmax and 1.4 (+/- 0.1) for Vmax/Km. The intramolecular kinetic deuterium isotope effect, determined from the relative amounts of N-methylbenzamide and N-trideuteriomethylbenzamide formed in the microsomal demethylation of N-trideuteriomethyl-N-methylbenzamide, is 6.0 +/- 0.3. There is no correlation of Vmax or Vmax/Km with the substituent in the aromatic ring, nor with the calculated ionization potentials of the benzamides. The results are interpreted in terms of a mechanism in which the benzamide undergoes direct hydrogen atom abstraction to form a carbon centred radical. This carbon centred radical subsequently forms an N-hydroxymethyl-N-methylbenzamide that decomposes to formaldehyde and an N-methylbenzamide. Semi-empirical AM1 self consistent field molecular orbital calculations identify that loss of a hydrogen atom from the E-methyl group is thermodynamically more favourable than from the Z-methyl group by ca. 5 kJ/mol.


Cycloheximide treatment of mouse mast cells inhibits serotonin release. Evidence of a requirement for newly synthesized protein in the exocytotic response. Treatment of mouse peritoneal mast cells and mouse bone marrow-derived cloned mast cells with the protein synthesis inhibitor cycloheximide led to a marked abrogation of serotonin (5-hydroxytryptamine; 5-HT) release induced by a range of activators including antigen, anti-immunoglobulin E (anti-IgE) antibody, calcium ionophore A23187, and the polycation polylysine. Significant inhibition (28%) of IgE-mediated secretion was attained after incubation of peritoneal cells for only 2 hr, and inhibition progressed over a 24 hr period, reaching greater than 80% after 15 hr. When peritoneal mast cells were exposed to cycloheximide and then washed and returned to culture conditions, a substantial recovery of responsiveness to anti-IgE was seen after 5 hr. Under the same conditions to those used in functional studies, cycloheximide inhibited protein synthesis, measured as incorporation of [35S]-methionine, by purified peritoneal mast cells and cloned mast cells to 18.5% and 7.9% of control levels, respectively. These results show that synthesis of new protein over a period of a few hours is required to render mast cells fully responsive to stimuli that act via the IgE receptor, and to certain other stimuli that are receptor-independent.


Effects of oral hypoglycaemic agents on platelet functions. The in vitro effects of three oral hypoglycaemic agents, gliclazide (1-(4-methylbenzensulfonyl)-3-[3-azabicylo(3,3,0)octyl]urea) , glibenclamide (1-[4-[2-(chloro-2-methoxybenzamide)-ethyl]-phenyl- sulfonyl]-3-cyclohexyl-urea) and glimepiride (1-[4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-carboxamide)- ethyl]-phenylsulphonyl]3-(4-methylcyclohexyl)-urea), on functions of human platelets were evaluated. None of these agents up to a concentration of 40 microM inhibited platelet aggregation induced by thrombin. Glibenclamide and glimepiride in the range of 20-40 microM suppressed Ca2+ release from internal Ca2+ stores induced by thrombin. Gliclazide showed no effect on arachidonic acid metabolism of human platelets. Glimepiride selectively inhibited the cyclooxygenase pathway, while the activities of 12-lipoxygenase and phospholipase A2 were unaffected. Glibenclamide inhibited both the cyclooxygenase and 12-lipoxygenase pathways. It also attenuated arachidonic acid release from phospholipase A2. Oral hypoglycaemic agents with inhibitory effects on arachidonic acid metabolism may prove useful for the treatment of diabetic patients with enhanced platelet functions.


Purification and characterization of 3-mercaptolactic acid S-conjugate oxidases. Two enzymes catalysing the oxidative formation of 3-mercaptopyruvic acid S-conjugates from L-3-mercaptolactic acid S-conjugates were purified to apparent homogeneity from rat liver cytosol. The two enzymes, tentatively designated MLO-I and MLO-II, showed a molecular mass of 160 and 250 kDa and were composed of four and six subunits of 41 and 39 kDa, respectively. Both enzymes possessed flavin mononucleotide as prosthetic group and oxidized several aromatic and aliphatic S-substituted L-3-mercaptolactic acids as well as alpha-hydroxy acids such as L-3-phenyllactic acid and L-2-hydroxyisocaproic acid. Glycolic acid and 3-(4-hydroxyphenyl)-lactic acid were the specific substrates for MLO-I and MLO-II, respectively. Neither of the enzymes oxidized beta- and gamma-hydroxy acids such as 3- and 4-hydroxybutyric acid. 2-Hydroxyisobutyric acid, ethyl-2-hydroxybutyrate, malic acid, 1-butanol, benzyl alcohol and L-leucine did not act as substrates for the enzymes. MLO-I and MLO-II exerted their maximum activities around pH 5.5 with Km of 0.5 and 0.25 mM and Vmax of 0.9 and 0.2 mumol/min/mg, respectively, when S-(4-bromophenyl)-3-thiolactic acid was used as substrate. MLO-I was inhibited by sulphydryl-modifying agents, while MLO-II was not. Both enzymes were strongly inhibited by divalent metal ions. These results indicate that MLO-I and MLO-II are different from L-amino acid oxidase (EC 1.4.3.2), malate oxidase (EC 1.1.3.3), L-alpha-hydroxy acid oxidase (EC 1.1.3.15) and glycolate oxidase (EC 1.1.3.1). The present enzymes are likely to be involved in the formation of cysteine conjugates from L-3-mercaptolactic acid S-conjugates in conjunction with cysteine conjugate aminotransferases.


In human monocytes interleukin-1 stimulates a phospholipase C active on phosphatidylcholine and inactive on phosphatidylinositol. Interleukin-1 (IL-1) can initiate the synthesis of prostaglandins which in turn act as endogenous modulators of IL-1 production. The human monocyte/macrophage synthesizes various eicosanoids through the activation of the cellular phospholipase system. Cell stimulation results in the activation of phospholipase A2 (PLA2) whose major substrate is phosphatidylcholine (PC) and the release of the eicosanoid precursor arachidonic acid (AA) from PC. Another pathway is the stimulation of a phospholipase C (PLC) mainly active on phosphoinositides and the resulting formation of inositol phosphates (IPs) and diacylglycerol (DAG). Phospholipids other than phosphoinositides can also be hydrolysed by PLC to give rise to DAG. Studies have shown that IL-1 does not activate the IP pathway, but it primarily stimulates a PLC linked to phosphatidylethanolamine in cultured rat mesangial cells, and a PLC linked to PC in Jurkart cells. We have stimulated human monocytes with IL-1 and calcium ionophore A23187 and we have observed their effect on the phospholipase system. The results indicate that IL-1 does not activate the formation of IPs in cells labeled with [3H]myo-inositol. In contrast, in cells labeled with [3H]AA, IL-1 causes the formation of DAG associated with the hydrolysis of PC. Moreover, after stimulation with IL-1 there is no accumulation of free AA which would indicate that there has been no activation of PLA2, which occurs instead with A23187 stimulation. These data suggest that, in monocytes, IL-1 does not directly stimulate a PLA2 or a PLC active on phosphatidylinositol; instead it primarily stimulates a PLC active on PC.


Cadmium induction of renal and hepatic ornithine decarboxylase activity in the rat. Effects of sex hormones and involvement of the renin-angiotensin system. We investigated the effect of sex hormones on the sex-dependent response of rat kidney ornithine decarboxylase (ODC) activity to cadmium (Cd) administration and the involvement of the renin-angiotensin system in mediating stimulation of the liver enzyme by the metal. The response of renal ODC to Cd, which occurs in intact adult males but not in females, is also detectable in prepubertal and castrated males. Upon treatment with 17 beta-estradiol, the basal levels of enzyme activity in intact or castrated adult males were enhanced and Cd administration failed to increase them further. In adult females the kidney enzyme became responsive after ovariectomy. Also, in prepubertal females renal ODC was induced by Cd, and this was prevented by treatment with 17 beta-estradiol. Under the same conditions, changes in the levels of Cd accumulation within the kidney, that might account for variations in the response of ODC activity, did not occur. Cd caused an increase in renin activity starting minutes after its injection. Captopril, which specifically inhibits the conversion of angiotensin I to angiotensin II, prevented completely the induction of liver ODC by this metal; stimulation of the enzyme by Co was not affected by the drug. A similar inhibitory effect was exerted by propranolol. Adrenalectomy had no influence on the response of hepatic ODC to Cd; the decarboxylase was unaffected by aldosterone administration. It is suggested that Cd may induce liver ODC through the increase in angiotensin II following stimulation of renin by the metal.


Effect of 2,2'-dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone on antioxidant defenses of Acanthocheilonema viteae and its laboratory host Mastomys natalensis. The effect of the macrofilaricidal agent of 2,2'-dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone (C.D.R.I. compound 82/437), on the metabolism of reactive oxygen species (ROs) in Acanthocheilonema viteae and Mastomys natalensis was measured following intraperitoneal administration at therapeutic doses. The recovered worms possessed substantially reduced levels of catalase and glutathione peroxidase (GPx), and thus were less able to detoxify H2O2. Nonetheless, the subcutaneous and adjoining muscle tissues, in which the parasites were lodged, exhibited elevated levels of antioxidant enzymes and reduced glutathione. It is concluded that compound 82/437 kills the filariid by paralysing its H2O2 detoxifying capacity without altering ROs metabolism in the tissue in which the parasite resides. Furthermore, since catalase and GPx of the liver and lungs do not show sign of inhibition, a difference appears to exist in the enzymes of the parasite and the host.


Evidence for a bioactive clonidine-displacing substance in peripheral tissues and serum. Clonidine-displacing substance (CDS) from brain is biologically active in the kidney and stomach and on platelets. To determine whether CDS is contained in these and other peripheral tissues, homogenates of fresh brain, eight other organs and serum from rat were ultrafiltered (less than 10,000 mol. wt only), dried and extracted with methanol. Evaluation by radioimmunoassay (RIA) using antibodies to p-aminoclonidine showed that adrenal gland and gastric fundus (GF) contained significantly greater amounts of CDS-like radioimmunoactivity than brain; intermediate-to-low activity was present in heart, small intestine, serum, kidney and liver; lung and skeletal muscle values were near-background. RIA-positive extracts elicited well-correlated contractile activity in a GF smooth muscle bioassay; contractions persisted in the presence of antagonists of various transmitters and modulators, but were abolished by low concentrations of the calcium channel blocker verapamil. Serum levels of CDS were profoundly reduced following removal of the adrenal glands. We conclude that a CDS-like substance is present not only in brain as previously reported, but also in peripheral organs and in the circulation.


17 alpha-Z-[125I]iodovinyloestradiol and its 3-acetate: chemical synthesis in vivo distribution studies in the rat. Comparison of tissue accumulation and metabolic stability with 17 alpha-E-[125I]iodovinyl and 16 alpha-[125I]iodo oestradiols. Certain oestrogens on substitution with halogens, have been shown to retain receptor binding affinity and accumulate in target tissues and therefore could, when labelled with gamma-emitting halogen isotope, act as radiopharmaceuticals for the imaging of receptor positive breast tumours. In order to evaluate putative radiopharmaceuticals, 17 alpha-Z-iodovinyloestradiol ((17 alpha,20Z)-21-iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diol) and its 3-acetate have been chemically synthesized and labelled with 125I. Tissue distribution studies in female rats have been compared to those obtained using 17 alpha-E-[125I]iodovinyloestradiol ((17 alpha,20E)-21-[125I]iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diol) and its 3-acetate and to 16 alpha-[125I]iodo oestradiol (1,3,5(10)-estratien-16 alpha-[125I]iodo-3,17 beta-diol). All compounds showed a similar tissue distribution with the highest concentrations (cpm/g tissue) in the thyroid greater than liver greater than uterus greater than kidney greater than lung greater than blood greater than heart, spleen. The concentration in the uterus was highest after injection of 17 alpha-Z-iodovinyloestradiols or 16 alpha-iodo oestradiol. Target (uterus) to background (blood) tissue ratios were highest after injection of 17 alpha-Z-iodovinyloestradiol-3-acetate and 16 alpha-iodo oestradiol (6:1). Deiodination in vivo took place to a small degree, amounting to 1.6%, 0.9% and 0.35% of the injected dose after 4 hr with the 17 alpha-Z, 17 alpha-E and 16 alpha compounds, respectively. For reasons of chemical expediency and consideration of the biological results 17 alpha-Z-iodovinyloestradiol-3-acetate is the compound of choice for the detection of oestrogen receptor positive tissues.


Effects of cyclophosphamide and adriamycin on rat hepatic microsomal glucuronidation and lipid peroxidation. The effect of cytotoxic drug administration, as a single dose i.p. to rats (six rats/treatment group), on hepatic microsomal UDP-glucuronosyltransferase (UGT) activity was investigated. Glucuronidation of morphine in microsomes from control rats apparently involved at least two enzymes. Administration of cyclophosphamide (CP; 200 mg/kg 7 days prior to killing) significantly increased the rate of morphine glucuronidation over the range 0.05-10 mM, and significantly increased the apparent Vmax for the high capacity isoenzyme from 1.25 +/- 0.12 to 1.95 +/- 0.39 nmol/mg/min. In contrast, the activity of 1-naphthol UGT was not significantly altered by administration of CP. Rats treated with the same dose of CP 1 day prior to killing showed a significant decrease in microsomal morphine-UGT activity at 0.05 and 2.5 mM morphine, but a significant increase in activity was observed following administration of CP or Adriamycin (AD; 10 mg/kg) 4 days prior to killing. The extent of microsomal lipid peroxidation was significantly increased in microsomes obtained from rats treated with CP or AD 4 days prior to killing, and was positively correlated (P less than 0.001) with the rate of glucuronidation of 0.05 and 2.5 mM morphine. Preincubation of microsomes in the presence of CP (5 mM) and AD (100 microM) significantly decreased the rate of glucuronidation of 2.5 mM morphine. In vitro NADPH-mediated lipid peroxidation significantly increased the activity of both the high and low affinity morphine-UGT isoenzymes. Administration of the cytotoxic drugs CP and AD may alter microsomal morphine-UGT activity via the process of lipid peroxidation, although other mechanisms cannot be excluded.


Evaluation of adriamycin-induced lipid peroxidation. Lipid peroxidation is known to be a mechanism for Adriamycin-induced toxicity. In the present study, two methods which detect fluorescent substances and high molecular weight protein aggregates in peroxidized membranes were applied to Adriamycin-induced lipid peroxidation in liver microsomes. A rat liver microsomal suspension containing an NADPH-generating system was incubated with Adriamycin. Thiobarbituric acid reactive substances (TBA-RS), formed during this incubation, were transferred from the microsomes to the medium. Fluorescent substances determined by the fluorescence emitted from both the microsomes themselves and the chloroform/methanol extracts of the microsomes, were found to be formed during this incubation. High molecular weight protein aggregates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were also formed. Fluorescent substances and high molecular weight protein aggregates were found in microsomal membranes themselves and increased time dependently. These substances retained in membranes can be of great use to delineate the site of Adriamycin-induced lipid peroxidation in vitro and in vivo and to determine how this lipid peroxidation affects the membrane.


A deuterium NMR and steady-state fluorescence anisotropy study of the effects of cholesterol on the lipid membrane-disordering actions of ethanol. We examined the effects of cholesterol on the membrane-disordering action of ethanol by using deuterium nuclear magnetic resonance (2H-NMR) and fluorescence spectroscopy. Specifically, the effects of ethanol were measured on the 2H-NMR spectra of di(perdeuteropalmitoyl)phosphatidylcholine (DPPC-d62) and on the steady-state emission anisotropy of diphenylhexatriene (DPH) incorporated into hydrated egg phosphatidylcholine (eggPC)/cholesterol dispersions. Analysis of the 2H-NMR spectra of DPPC-d62 incorporated into eggPC liposomes showed that the addition of cholesterol up to 30 mol% enhanced the ability of ethanol to disorder methylene groups all along the phospholipid acyl chains. This effect was somewhat greater toward the terminal methyl groups. However, above 30 mol% cholesterol, the bilayer-disordering action of ethanol on both the upper and lower portions of the acyl chains decreased to an apparent constant change up to the highest cholesterol content examined (50 mol%). Analysis of the fluorescence anisotropy of DPH, on the other hand, suggested that cholesterol attenuated the ability of ethanol to disorder the bilayers, which is in agreement with a previous EPR study [Chin and Goldstein, Mol Pharmacol 19: 425-431, 1981]. Re-analysis of our previous fluorescence anisotropy results with DPH incorporated into dispersions of brain-lipid extracts as a percent change [Johnson et al., Mol Pharmacol 15: 739-746, 1979] indicated that the chemical composition of the lipid bilayers also affects the apparent ability of cholesterol to modulate the membrane-disordering action of ethanol, because the addition of cholesterol to brain-lipid extracts had no significant effect on the membrane-disordering action of ethanol. Given the greater likelihood that the 2H-NMR probes accurately monitor bulk phospholipid properties, some caution is required in the analysis of the membrane-disordering actions of drugs using EPR and fluorescence spectroscopy.


Human kidney thiopurine methyltransferase. Photoaffinity labeling with S-adenosyl-L-methionine. Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of heterocyclic and aromatic sulfhydryl compounds such as the thiopurine drug 6-mercaptopurine (6-MP). TPMT activity in human tissue is regulated by a common genetic polymorphism, and "pharmacogenetic" variation in TPMT activity is an important factor in individual differences in thiopurine drug metabolism, toxicity and therapeutic efficacy. Human renal tissue contains two isozymes of TPMT, Peak I and Peak II, that can be separated by ion exchange chromatography. Our experiments were performed to determine whether S-adenosyl-L-methionine (Ado-Met), the methyl donor for the TPMT reaction, could be used as a photoaffinity ligand for these isozymes as one step in the study of the molecular basis for the TPMT genetic polymorphism. When [3H-methyl]Ado-Met and partially purified preparations of either isozyme of human kidney TPMT were exposed to ultraviolet light at 254 nm, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35 kDa protein was the predominant species that was radioactively labeled. The same 35 kDa protein was photoaffinity labeled with [14C-carboxyl]Ado-Met, demonstrating that labeling involved covalent binding of Ado-Met rather than methylation of the protein. TPMT enzymatic activity co-eluted with the 35 kDa protein during sequential DEAE ion exchange, gel filtration and hydroxylapatite chromatography. Inhibitors of TPMT enzymatic activity including S-adenosyl-L-homocysteine, sinefungin, 6-methylmercaptopurine and 3,4-dimethoxy-5-hydroxybenzoic acid inhibited photoaffinity labeling of the 35 kDa protein in preparations of both TPMT Peak I and Peak II isozymes in a concentration-dependent fashion, as did 6-MP, the methyl acceptor substrate for the TPMT reaction. All of these results were compatible with the conclusion that the 35 kDa protein was TPMT. Photoaffinity labeling of TPMT with [3H]Ado-Met should make it possible to purify the enzyme to homogeneity and to study amino acid sequences at or near its active site.


Suicide inhibitors of cytochrome P450 1A1 and P450 2B1. The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.


Studies on the developmental and adrenal regulation of cytosolic and microsomal epoxide hydrolase activities in rat liver. The present study was undertaken to ascertain developmental profiles of microsomal epoxide hydrolase (mEH) and cytosolic epoxide hydrolase (cEH) enzyme activities in rat liver. During development, mEH activity reached an optimum by 6 weeks of age (63 nmol/min/mg protein). Activities decreased thereafter in both sexes although in adult male liver the activity was twice that measured in adult female liver. Thus, the importance of pituitary maturation was suggested from these findings. Since glucocorticoids have been implicated in the regulation of mEH gene expression the effect of adrenalectomy on mEH activity was investigated in adult male rat liver. The procedure increased mEH activity almost two-fold and the increase was reversed by dexamethasone, but not deoxycorticosterone, administration. With respect to hepatic cEH activity, the developmental profiles indicated that enzyme activity was greatest in rats at 1 week of age (12-15 nmol/min/mg protein) and very little activity was detected beyond 4 weeks of age (approximately 5 nmol/min/mg protein); sex differences in cEH activity were not apparent at any age. Thus, the pituitary appears to be important in the developmental induction of mEH but not cEH. Glucocorticoids appear to provide the major hormonal influence on mEH expression. Thus, the hypothalamus-pituitary-adrenal axis is involved in the regulation of mEH but the regulation of the cEH enzyme remains unclear.


Lack of enantio-selectivity in the in vitro antitumour cytotoxicity and membrane-damaging activity of ether lipid SRI 62-834: further evidence for a non-receptor-mediated mechanism of action. SRI 62-834 ([tetrahydro-2-(octadecycloxy)methylfuran- 2-yl]methoxylphosphocholine; CRC 86-05; NSC 614383) is a cyclic antitumour ether lipid (AEL) with a novel, but ill-defined, mechanism of action. AELs are believed to act on membranes and cell signals, but the precise mechanisms of selectivity are unclear. Receptor-mediated mechanisms can often be identified by the differential activity of the individual stereoisomers of a drug. We have therefore compared the R- and S-enantiomers of SRI 62-834 for: (1) cytotoxicity against the human HT29 colon carcinoma cell line using a tetrazolium dye reduction assay and (2) membrane-damaging effects monitored by 51Cr radiolabel release. The tetrazolium assay revealed near-identical mean ID50 values around of 2-3 microM for the R- and S-isomers as well as for the racemic mixture. Moreover, pre- and co-incubation of the cells with the potent platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine;PAF) receptor antagonist WEB 2086BS (3-[4-(chorophenyl)-9-methyl-6H-thieno[3,2- f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4- morpholinyl)-1-propanone) had no effect on the cytotoxicity of either isomer or the racemate. Short-term membrane damage was not evident at low micromolar concentrations and between 139 and 163 microM either lipid was required to release 50% of the incorporated 51Cr label. Again, there was no difference in potency between the enantiomers and the racemate. Coincubation with WEB 2086BS also failed to modulate the membrane-lytic potency of the AELs. These results indicate that the site(s) of cytotoxic action of SRI 62-834 is (are) not stereospecific and also appear to rule out the involvement of a conventional PAF receptor in the mechanism of action of SRI 62-834.


Fluorescence quenching of human serum albumin by xanthines. A study of the fluorescence quenching of human serum albumin (HSA) by caffeine, theophylline and theobromine, based on temperature dependence, has shown that it is predominantly static. This quenching mechanism is due to the formation of a xanthine-HSA non-fluorescent complex. The Stern-Volmer equation let us determine the association constants. It seems that the quenching of the protein fluorescence depends on the number and position of the methyl groups. The temperature dependence of the association constant is used to estimate the values of the thermodynamic parameters involved in the interaction of the drugs with HSA. All three binding processes are exothermic and probably hydrophobic, and hydrogen bonds play a significant role in the stabilization of such complexes. The enthalpy and entropy changes observed appear to compensate each other to produce a relatively small Gibbs free energy.


Heterogeneity of carboxylesterases in rat liver cells. Rat liver cells were separated into parenchymal cells (PC), Kupffer cells (KC) and endothelial cells (EC). The distribution of carboxylesterases (EC 3.1.1.1) between these cell types was investigated by PAGE and chromatogenic substrate staining, and compared with the results for total liver preparation and individual isoenzymes isolated by chromatofocusing. All of the liver carboxylesterase isoenzymes could be detected in the PC, whereas in both KC and EC only those with isoelectric point (pI) 6.4/6.2 could be detected. Use of carboxylesterase inhibitors like bis-(4-nitrophenyl)phosphate and paraoxon, and organophosphorus compound hydrolase inhibitors like 4-hydroxymercuribenzoate and EDTA confirmed that these esterases were of the carboxylesterase type.


Inhibition of enzymes of polyamine back-conversion by pentamidine and berenil. Pentamidine and berenil, clinical antiparasitic amidines, have been found to be potent competitive inhibitors of human spermidine/spermine acetyltransferase (SSAT). Ki values were found to be 2.4 and 2 microM, respectively, with spermidine as substrate. A second enzyme of polyamine back-conversion, murine polyamine oxidase (PAO), was found to be competitively inhibited by pentamidine, with a Ki of 7.6 microM when N-acetylspermine was the substrate. Berenil, on the other hand, was an extremely weak inhibitor (Ki = 120 microM). The implication of the effect of inhibition of polyamine back-conversion on the growth of mammalian parasites is discussed.


Protection by cyclosporin A of cultured hepatocytes from the toxic consequences of the loss of mitochondrial energization produced by 1-methyl-4-phenylpyridinium. Cyclosporin A prevented the killing of cultured rat hepatocytes by 1-methyl-4-phenylpyridinium (MPP+). However, in the presence of both cyclosporin and atractyloside, there was no protection. Cyclosporin had no effect on the depletion of ATP or the loss of mitochondrial energization by MPP+. Cyclosporin, however, did prevent the increase in the molecular order of hepatocyte membranes produced by MPP+. These data suggest that mitochondrial de-energization produced by MPP+ is accompanied by a "permeability transition" analogous to that which occurs in vitro in the presence of calcium. By preventing this transition, cyclosporin protects the cells. By antagonizing this action of cyclosporin, atractyloside restores the cell killing. The mitochondrial transition is causally linked to cell killing by a mechanism that increases the molecular order of the hepatocyte plasma membrane.


Monoclonal antibodies against 4-hydroxybiphenyl-UDP-glucuronosyltransferase. Monoclonal antibodies against purified rat liver 4-hydroxybiphenyl UDP-glucuronosyltransferase were developed using the hybridoma technology. In immunoblot analysis the antibodies specifically reacted with purified 4-hydroxybiphenyl-UDPGT but not with other purified UDPGT enzyme fractions. One single band was detected with microsomes of rat liver and small intestine but not with microsomes of kidney and lung. The reactive protein was also found in dog and human liver microsomes. It could be shown that there was no increase of immunoreactive protein after pretreatment with phenobarbital or 5,6-benzoflavone. This supports the hypothesis that more than one 4-hydroxybiphenyl-UDPGT exist in rat liver which are differently inducible.


PZ-51 (Ebselen) in vivo protection against adriamycin-induced mouse cardiac and hepatic lipid peroxidation and toxicity. Adriamycin (Adr)-induced cardiotoxicity occurs most likely via an oxidative mechanism of action. Moderation of this activity may result in an improved therapeutic index for this compound. PZ-51, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one, is a selenoorganic compound with thiol-dependent, peroxidase-like activity. We tested this compound alone and in combination with N-acetylcysteine (NAC) for its effect on Adr-induced in vivo toxicity in Balb/c mice. These studies demonstrated that PZ-51 protects against Adr-induced lipid peroxidation in heart and liver tissue and Adr-induced toxicity in general, as measured by total serum creatine kinase activity and body weight.


Effects of continuous and pulsed 2450-MHz radiation on spontaneous lymphoblastoid transformation of human lymphocytes in vitro. Normal human lymphocytes were isolated from the peripheral blood of healthy donors. One-ml samples containing (10(6)) cells in chromosome medium 1A were exposed for 5 days to conventional heating or to continuous wave (CW) or pulsed wave (PW) 2450-MHz radiation at non-heating (37 degrees C) and various heating levels (temperature increases of 0.5, 1.0, 1.5, and 2 degrees C). The pulsed exposures involved 1-microsecond pulses at pulse repetition frequencies from 100 to 1,000 pulses per second at the same average SAR levels as the CW exposures. Actual average SARs ranged to 12.3 W/kg. Following termination of the incubation period, spontaneous lymphoblastoid transformation was determined with an image analysis system. The results were compared among each of the experimental conditions and with sham-exposed cultures. At non-heating levels, CW exposure did not affect transformation. At heating levels both conventional and CW heating enhanced transformation to the same extent and correlate with the increases in incubation temperature. PW exposure enhanced transformation at non-heating levels. This finding is significant (P less than .002). At heating levels PW exposure enhanced transformation to a greater extent than did conventional or CW heating. This finding is significant at the .02 level. We conclude that PW 2450-MHz radiation acts differently on the process of lymphoblastoid transformation in vitro compared with CW 2450-MHz radiation at the same average SARs.


A replication study of human exposure to 60-Hz fields: effects on neurobehavioral measures. The purpose of this study was to reproduce and extend an earlier investigation of the effects of human exposure to combined, 60-Hz electric and magnetic fields. This paper presents the neurobehavioral results. Thirty men participated in one training session and four testing sessions. Subjects were randomly assigned to two groups. The 18 subjects in Group I were exposed (9 kV/m, 20 microT) and sham exposed in two counterbalanced orders. In Group II, half of 12 subjects were exposed (9 kV/m, 20 microT) every session, and the remaining half were sham exposed every session. The study was doubly blinded. Measures of cardiac interbeat interval, event-related brain potentials, and performance were obtained before, during, and after exposures. As in the earlier study, exposure to the combined field resulted in a statistically significant slowing of heart rate, in changes in late components of event-related brain potentials, and in decreased errors on a choice reaction-time task. In addition, field effects on several other measures approached statistical significance. The physiological measures obtained during exposure indicated that effects were greatest soon after the field was switched on, and again when it was switched off. The data indicate that changes in exposure level may be more important than duration of exposure for producing effects in human beings.


Residential exposure to 60-Hz magnetic fields from appliances. A model has been developed that permits assessment of residential exposure to 60-Hz magnetic fields emitted by appliances. It is based on volume- and time-averaging of magnetic-dipole fields. The model enables the contribution of appliances in the total residential exposure to be compared with that of other sources in any residence under study. Calculations based on measurements reported in the literature on 98 appliances revealed that appliances are not a significant source of whole-body exposure, but that they may be the dominant source of exposure of the body's extremities.


Preliminary report: modification of cardiac contraction rate by pulsed magnetic fields. Isolated rat hearts and excised canine cardiac tissues were subjected to pulsed magnetic fields. The fields excited in coils by tandem pairings of sinusoidal pulses were presented at various inter-pair delays and repetition rates. The waveform of the magnetic field was a single or multiple sinusoid followed after a variable delay by another single or multiple sinusoid. Small but reliable increases in the beating rate of rat heart were observed. Similar increases occurred in contraction rates of canine tissues. Both preparations exhibited a contraction-rate dependency on the repetition rate of the paired magnetic pulses: 4.5-6 rep/s for canine tissue, and 20-25 and 40-55 reps/s for rat heart. Flux-density thresholds for both preparations approximated 10 mT (100 gauss) rms.


Electric fields induced in rat and human models by 60-Hz magnetic fields: comparison of calculated and measured values. The calculated distribution of electric fields induced in homogeneous human and rat models by a 60-Hz magnetic field is compared with values measured in instrumented mannequins. The calculated values agree well with measured values.


Dose dependence of acetylcholinesterase activity in neuroblastoma cells exposed to modulated radio-frequency electromagnetic radiation. Radio-frequency electromagnetic radiation (RFR) at 915 and 147 MHz, when sinusoidally amplitude modulated (AM) at 16 Hz, has been shown to enhance release of calcium ions from neuroblastoma cells in culture. The dose-response relation is unusual, consisting of two power-density "windows" in which enhanced efflux occurs, separated by power-density regions in which no effect is observed. To explore the physiological importance of these findings, we have examined the impact of RFR exposure on a membrane-bound enzyme, acetylcholinesterase (AChE), which is intimately involved with the acetylcholine (ACh) neurotransmitter system. Neuroblastoma cells (NG108), exposed for 30 min to 147-MHz radiation, AM at 16 Hz, demonstrated enhanced AChE activity, as assayed by a procedure using 14C-labeled ACh. Enhanced activity was observed within a time window between 7.0 and 7.5 h after the cells were plated and only when the exposure occurred at power densities identified in a previous report as being effective for altering the release of calcium ions. Thus RFR affects both calcium-ion release and AChE activity in nervous system-derived cells in culture in a common dose-dependent manner.


Modification of acoustic startle by microwave pulses in the rat: a preliminary report. Single, 1.25-GHz microwave pulses of 0.8- to 1.0-microseconds duration were presented to each of four rats 100 ms before presentation of a startle-inducing acoustic stimulus. This sequential pairing of microwave pulse and acoustic stimulus was found to modify the startle response. At an energy dose to the head of 22-43 mJ/kg per pulse (peak SAR, 23-48 kW/kg), the mean latency to the startle response was longer and the mean amplitude of the response was smaller with respect to control responses that occurred to acoustic stimuli alone. However, at a higher energy dose per microwave pulse in the range of 59-107 mJ/kg (peak SAR, 63-111 kW/kg), the mean latency and amplitude of the startle response were not statistically different from the respective means of control responses.


En bloc radical vulvectomy and lymphadenectomy with modifications of separate groin incisions. A new modification of radical vulvectomy and lymphadenectomy through separate groin incisions involves dissection of the intervening skin bridge and allows an en bloc dissection. The results in 26 women treated with this technique are compared with those of seven treated with separate incisions without an en bloc dissection. All 33 women had squamous carcinoma of the vulva and were treated between 1985-1989. The incidence of advanced disease was high, with nodal metastases present in 52% of cases. Dissection of the tissue beneath the skin bridge did not alter the morbidity of the procedure in terms of the incidence of wound infection, number of units of blood transfused, or duration of hospitalization. The only case of an isolated recurrence in the skin bridge occurred in a woman who did not have an en bloc dissection, although there was no significant difference in the overall local recurrence rate between the groups. Further evaluation with larger numbers is required, but we suggest that an en bloc dissection using separate incisions may reduce the risk of isolated recurrence in the skin bridge, particularly in patients with advanced disease.


Decisions for and against resuscitation in an acute geriatric medicine unit serving the frail elderly. Recent studies indicate continuing controversy over the appropriateness of intensive care in various clinical settings, particularly for very young and very old patients. We studied decisions regarding cardiopulmonary resuscitation (CPR) and the associated clinical course in an acute geriatric unit serving the frail elderly. Despite multiple acute and chronic conditions, advanced age, and functional impairment, patients overwhelmingly preferred CPR. The patients with do-not-resuscitate (DNR) orders were more functionally dependent, more acutely and chronically ill, and less likely to participate in the decision regarding CPR. The majority of DNR orders were made by surrogates, while the majority of CPR directives were made by the patients themselves. Other forms of acute and supportive care, such as parenteral antibiotics, artificial feeding, and transfusions were not withheld from the DNR patients unless a separate decision to limit a specific treatment was undertaken following consultation between the attending physician and the patient or family. Acuity of illness greater than two SDs above the unit mean and the presence of a surrogate decision maker predicted the majority of DNR orders. Length of stay averaged 28 days for all unit patients, 24 days for patients choosing CPR, and 46 days for patients with DNR orders. The four patients who were resuscitated but died stayed an average of 25 days, while the two survivors of resuscitation stayed 20 and 53 days, respectively. The findings indicate that patients and their families considered appropriate clinical criteria including severity of illness when making their decisions about CPR. Nevertheless, the majority chose to be resuscitated in the event of an arrest. Further studies are needed to explore medical decision making by elderly inpatients and their surrogates and to describe the associated clinical course.


Deprivation and general practitioner workload. To examine general practitioner consultations by demographic and socioeconomic variables and to derive a method of measuring the impact of relative deprivation on general practitioner workload. The study was based on general practitioner consultations reported in the general household surveys of 1983-7, covering a sample of 129,987 individuals in Great Britain. Odds ratios for general practitioner consultations were obtained for selected variables among children (0-15 years), men (16-64), women (16-64), and elderly people (greater than or equal to 65). These were then used to derive deprivation indices specific to electoral wards for use in general practice. Great Britain, with particular findings illustrated by English electoral wards and the conurbations of London, Manchester, Merseyside, and the West Midlands. Council tenure increased the likelihood of consultation significantly in all four groups. Odds ratios were raised in children, men, and women with no access to a car. Birth in the New Commonwealth or Pakistan yielded high odds ratios in men, women, and elderly people but not in children. Marginally increased consultation rates were evident in the manual socioeconomic groups in women, elderly people, and children with a single parent mother. The deprivation indices for general practice derived using these odds ratios varied substantially among English electoral wards with, for example, anticipated general practitioner consultations in the electoral ward of Hulme, Manchester, being 24% higher than the average ward in England as a result of local attributes, and consultations in the Cheam South ward of Sutton, London, 11% lower than average. This deprivation index for general practice overcomes several shortcomings expressed about the underprivileged area score, which has been adopted in the 1990 contract as a basis for allocating deprivation supplements to general practitioners. The proposed index can be applied nationwide.


Vaccine coverage: recent trends and future prospects. To assess the feasibility of achieving the target of 95% coverage for the childhood immunisation schedule by 1995 and to determine the influence of sociodemographic factors and information systems on recent trends. Analyses of trends in quarterly vaccination data for diphtheria, pertussis, and measles in health districts between February 1988 and February 1991. District health authorities in England and Wales, and health and social services boards in Northern Ireland. Cohorts of children whose youngest member had reached the target age of 18 months for receiving the third doses of diphtheria and pertussis vaccines and 2 years for receiving measles vaccine. Predicted coverage levels for mid-1995 were in excess of 95% for diphtheria, pertussis, and measles vaccines. In the 118 districts that continuously reported between February 1988 and February 1991 the increase in coverage was 6% for diphtheria and 13% for pertussis and measles vaccines. 1991 coverage depended primarily on 1988 coverage. The additional effects of deprivation, change in computer system, and child population size achieved at most only marginal statistical significance. The government's target of 95% coverage by 1995 is realistic, although projections should be viewed with caution. Several national vaccination initiatives are likely to have contributed to the recent steady increase in coverage. Updating and validation exercises are likely to improve recorded coverage.


Exogenous administration of IL-1 alpha inhibits active and adoptive transfer autoimmune diabetes in NOD mice. Diabetes susceptibility in non-obese diabetic (NOD) mice may involve immune dysregulation resulting from cytokine deficiencies. The cytokine IL-1 plays a role in various immune as well as endocrine responses and may be hypoexpressed in NOD mice. Treatment with low levels of exogenous IL-1 alpha for 22 weeks prevented the naturally occurring insulitis and diabetogenic process in NOD mice during and at least 33 weeks after cessation of IL-1 alpha treatment. Treatment with IL-1 alpha also inhibited insulitis and hyperglycemia induced by adoptive transfer of pathogenic, polyclonal CD4+8- T cells. Even after islet-cell destruction, IL-1 alpha injections in diabetic NOD mice normalized plasma glucose levels when administered in combination with insulin, whereas equivalent levels of IL-1 alpha alone did not. Our studies support the hypothesis that IL-1 alpha suppresses autoimmune diabetes and hyperglycemia in NOD mice by pleiotropic effects on both immune and metabolic systems. Thus, IL-1 treatment could clinically be an effective immunotherapeutic modality for autoimmune diabetes mellitus by suppressing early disease progression or normalize plasma glucose levels when insulin is present.


Monoclonal anti-tumor necrosis factor antibody renders non-obese diabetic mice hypersensitive to irradiation and enhances insulitis development. In attempt to evaluate biological roles of tumor necrosis factor (TNF), we studied the effects of anti-TNF mAb in non-obese diabetic (NOD) mice. Anti-murine TNF mAb rendered NOD mice hypersensitive to the lethal effects of radiation and prevented the reconstitution of lethally irradiated mice with adoptively transferred lymphocytes. While TNF-alpha reduced the incidence of diabetes development in the adoptive transfer system even when given 6 days post-transfer, mAb to TNF could not reduce or increase the incidence of diabetes compared to control mice. Administration of TNF-alpha for 4 or 8 weeks significantly reduced the incidence of spontaneous insulitis in NOD mice, while anti-TNF mAb given for 8 weeks increased the incidence of insulitis significantly.


Thanatology: a new clinical specialty to help confront death. The complexities, widespread ramifications and uncertainties surrounding decisions dealing with the process of dying call for a specialty of clinical thanatology. The need for such a specialty grows out of medical technological progress, which has permitted the prolongation of life without parallel guidelines for its termination. The further functions and roles of a thanatologist would evolve with the passage of time.


Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro. To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.


Anti-AIDS therapy: an update.

Anti-AIDS therapy: an update. This review first examines AIDS as regards its historical aspects and current situation. Attention focuses on some basic etiopathogenic characteristics of this disease. After this point, the main pharmacological aspects are examined and, in particular, attention is paid to the most two interesting targets for pharmacological therapy. Lastly, the dynamic and kinetic profiles of the most promising compounds for therapeutic application are considered.


3,5-Diphenyl-1H-pyrazole derivatives. IX. 2-substituted 4-phenyl-5-(3,5-diphenyl-1H-pyrazol-1-yl) pyrimidines with platelet antiaggregating and other activities. The synthesis of 2-substituted 4-phenyl-5-(3,5-diphenyl-1H-pyrazol-1-yl)pyrimidines 4 a-e by reaction of 1-(1-dimethylaminomethylene-2-oxo-2-phenylethyl)-3,5-diphenyl-1H-pyrazol e with acetamidine, benzamidine, guanidine, guanidinaecetic acid and creatine, respectively, is described. The alpha-aminoacid derivatives 4 d and 4 e gave a series of amides 7 by coupling with primary or secondary amines in dimethylformamide (DMF) solution in the presence of diphenyl-phosphorylazide (DPPA) and triethylamine. Some compounds 4 and 7 showed a platelet antiaggregating activity in vitro superior or comparable to that of acetyl-salicylic acid, as well as moderate antihypertensive, local anesthetic, analgesic and antiinflammatory activities in rats and mice.


Synthesis and evaluation of antimicrobial activity of new 4-nitroso and 4-diazopyrazole derivatives. Some N-(pyrazol-5-yl)-2-nitrobenzamides, variably substituted in the pyrazole nucleus as well as in the amidic group, were reacted in acetic acid media with potassium nitrite and hydrochloric acid. The different chemical behaviour of the reacted pyrazole derivatives in relation to the substitution pattern in both the pyrazole nucleus and the amidic group, was observed. All compounds isolated from the reaction mixtures (4-nitroso, 4-nitro, 4-diazo and 4-chloro derivatives) were evaluated by the agar diffusion method for their "in vitro" growth inhibitory activity against Candida albicans (our collection), Candida tropicalis ATCC 13803, Saccharomyces cerevisiae ATCC 36375, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. The 1-methylpyrazole derivatives showed larger inhibition zones than the 1-phenyl ones in the antimicrobial tests.


Antimicrobial and genotoxic activities of N-substituted 2,2'-dicarboxamidodiphenyldisulfides. This paper describes the in vitro evaluation of antibacterial, antifungal and genotoxic activities of a series of 2,2'-dicarboxamidodiphenyldisulfides. All the studied compounds exhibited antibacterial activity against Bacillus subtilis. Most compounds were also active against Staphylococcus aureus. Several compounds were active against Saccharomyces cerevisiae and, in most cases, also against Candida tropicalis. No effects were observed against Escherichia coli or Aspergillus niger. Antimicrobial activity turned out to be affected by the substituent in the benzene ring and structure-activity relationships were found. The antibacterial and antifungal activities of the studied derivatives were compared with those of the corresponding 1,2-benzisothiazolin-3-ones and in some cases strong differences were observed. None of the tested compounds contained alerting groups or showed genotoxic properties.


1-Acylaminoimidazoles synthesis and antimicrobial activity. Several new 1-acylamino-2-alkyl-4-arylimidazoles were synthesized. The compounds were obtained by reaction of N1-acylacetamidrazones and alpha-bromoketones. The antimicrobial activity of the prepared compounds was tested.


Synthesis and antimicrobial activity of 1,3,4-triaryl-2-azetidinones. A set of substituted 1,3,4-triaryl-2-azetidinones were synthesized and characterized. Their antimicrobial activity, against Gram+ and Gram- bacteria and Fungi, was tested. The compounds 23 and 30 showed remarkable activity against Pseudomonas aeruginosa.


Development of absorption furosemide prodrugs: synthesis, in vitro and in vivo evaluation. Six acyloxymethyl esters of Furosemide were synthesized and the structures were determined by chemical and spectroscopic methods. Lipophilicity parameters were analysed by high performance liquid chromatography (HPLC). Hydrolysis performances in human plasma and intestinal fluids anticipate their properties as absorption prodrugs of Furosemide. A bioavailability study carried out with 8 male Wistar rats with one of the synthesized prodrug (acetyloxymethyl 4-chloro-N-furfuryl-5-sulfamoylanthranilate) showed a greater absorption in relation to Furosemide. The percentages of mean urinary recovery of Furosemide for the prodrug and for the standard solution of the drug were 20.84 and 14.36 respectively. The doses were 10 mg/Kg in Furosemide. The analytical determinations of Furosemide in biological fluids were done by HPLC.


Cytotoxic and DNA binding properties of aminoalkyl derivatives of di- and triazaphenanthrenes. A series of mono- and dicationic derivatives of various di- and triazaphenanthrenes were synthesized and evaluated against both wild-type and amsacrine-resistant P388 leukemia in vitro. Monocationic derivatives of the fully-aromatic benzo[c][1,8]naphthyridine chromophore showed moderate and equal cytotoxicity in both cell lines, suggestive of efficacy against both the alpha and beta isozymes of mammalian DNA topoisomerase II. Derivatives of both the benzo[c][1,8]naphthyridine and benzo[c][1,8]naphthyridine-3(4H)-one chromophores also showed significant in vivo activity against wild-type P388 leukemia.


Photodynamic effects of a cationic mesosubstituted porphyrin in cell cultures. The photobiological activity of mesotetra (4N-methylpyridyl) porphine (T4MPyP) has been studied in a mammalian cell line in vitro. The uptake of T4MPyP to HeLa cells was examined using fluorescence microscopy. Under violet-blue exciting light, a nucleus-selective red fluorescence was detected. The intensity of fluorescence depended on drug concentration and incubation time, with a maximum relative emission at a concentration value of 10(-5) M for 20 min. The photosensitivity of HeLa cells incubated with T4MPyP under previous conditions and subsequently exposed to variable doses of visible light has been examined. Cell toxicity was evaluated as the surviving fraction, using the trypan blue staining test. The extent of cellular damage was found to be dependent on irradiation time, with a lethal damage after 8 min of light exposure. Cellular DNA-photodamage was studied under sublethal conditions (2 min of irradiation) by the use of the micronucleus test. The number of micronuclei, in these conditions, increased with regard to baseline levels.


Conformational aspects of poly(dI-dC).poly(dI-dC) and poly(dG-dC).poly(dG-dC) on binding of the alkaloid, berberine chloride. Interaction of berberine chloride with poly(dI-dC).poly(dI-dC) and poly(dG-dC).poly(dG-dC) has been studied by various spectroscopic methods. Comparative data on binding parameters, fluorescence enhancement and Tm measurements indicate a stronger binding of berberine to the IC polymer than to the GC one. Conformational changes as monitored by circular dichroic spectra indicate change of the unusual circular dichroism of poly(IC) to a conformation similar to A-conformation, while in poly(GC) only a B-conformational change is observed. Extrinsic CD bands developed in the visible absorption region of berberine on interaction with poly(IC) are of much higher intensity compared to poly(GC). It is concluded that berberine induces a conformational change similar to the A-conformation on binding to poly(IC).


Influence on metastasis of the reduction of major histocompatibility complex (MHC) class I gene with an antisense oligonucleotide. Cell surface MHC H-2K antigens expressed on various types of B16 melanoma cells of C57BL/6 mouse origin were stained by the avidin-biotinylated enzyme complex (ABC) technique. The low metastatic B16F1 cell line, which demonstrates high levels of H-2K antigen, stained strongly, while the high metastatic B16F10 cell line with no H-2K antigen was not stained. Metastatic colonies which developed in lungs after intravenous (i.v.) injection proved to be negative in both cases. Therefore, in consideration of decreased expression of H-2K antigen in the metastatic process both in vivo and in vitro, antisense oligonucleotides against the class I H-2K gene were transfected into B16F1 cells with H-2K antigens. Subsequent immunohistochemical assessment revealed lack of H-2K antigen expression in the cells, but nevertheless they still could not metastasize to lungs when i.v. injected. This may suggest that any link between the two is not directly causal.


How immunology was won in Hungary.

How immunology was won in Hungary. To mark the occasion of the 8th International Congress of Immunology, this special issue surveys the latest findings from Hungarian immunology. Here, János Gergely, president of the Congress, traces the origins and development of immunology in Hungary and describes the joys and sorrows of being an immunologist in Hungary today.


Complement research: biosynthesis, genetics, immunoregulatory role and clinical studies. Complementology is one of the major areas of immunological research in Hungary. Here, András Falus and colleagues describe studies on the control of biosynthesis of complement components and on the nature and function of receptors in the complement system.


Susceptibility to autoimmune disease: a multigenic viewpoint. In Hungary, analysis of genetic predisposition to disease has generally been studied from a multigenic point of view. Alongside markers of the HLA system, other blood groups and allotypes of immunoglobulins and complement components have been studied. These have been correlated with functional parameters in different autoimmune conditions.


The pathogenesis of autoimmune disease.

The pathogenesis of autoimmune disease. The mechanisms of autoimmune pathology are normally described on a disease-by-disease basis. Taking a novel angle, Peter Gergely here examines the role of different types of leucocytes across a spectrum of autoimmune diseases, combining the results from a number of Hungarian laboratories.


HIV in Hungary.

HIV in Hungary. HIV infection in Hungary has not reached the frightening proportions that are currently ravaging other parts of the world. Given this relatively fortunate position and the now clear understanding of the ways in which HIV is transmitted, vigorous public education programmes have been undertaken to minimize the spread of the virus. Here, George Füst outlines these efforts, discusses diagnosis and monitoring of infected patients, and describes HIV research in Hungary.


Cellular and humoral suppression in transplantation. Transplantation immunity and tolerance induction are two sides of the same coin. This article describes a couple of very different approaches, one experimental and one clinical, that may improve the understanding of transplantation immunology through analysis of tolerance induction.


Clinical applications of phenotypic analysis. The ability to track lymphocyte differentiation and activation via the expression of cell surface marker molecules has had an enormous impact on diagnosis, monitoring and therapy of a wide range of diseases. Here, Katalin Pálóczi highlights studies from Hungary on the clinical applications of such analysis.