Direct inactivation of herpes simplex virus type-2 by rat epidermal protein. Proteins were extracted from corneocytes of skin of 2-day-old rats and fractionated by gel filtration and cation exchange column chromatography. The different protein fractions were tested for direct inactivation of herpes simplex virus infectivity as determined by reduction of plaque formation. The most effective protein fractions against herpes simplex virus were a neutral pH buffer soluble and mol. wts. ranging from 20 K to 30 K. Amino acid composition of the proteins were virtually identical to epidermal histidine-rich proteins. The activity was significantly (P less than 0.001) stronger against type-2 than type-1. The activity was most stable at pH 7.2 and the rate of inhibition increased in a time-dependent manner up to 4 h. The 50% effective dose was estimated as 1.1 micrograms protein/ml.


The mannose-specific plant lectins from Cymbidium hybrid and Epipactis helleborine and the (N-acetylglucosamine)n-specific plant lectin from Urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro. A series of four mannose(Man)-, three N-acetylglucosamine (GlcNAc)n-, ten N-acetylgalactosamine/galactose(GalNAc/Gal)-, one 5-acetylneuraminic acid (alpha-2,3-Gal/GalNAc)- and one 5-acetylneuroaminic acid(alpha-2,6-Gal/Gal-NAc)-specific plant agglutinins were evaluated for their antiviral activity in vitro. the mannose-specific lectins from the orchid species Cymbidium hybrid (CA), Epipactis helleborine (EHA) and Listera ovata (LOA) were highly inhibitory to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in MT-4, and showed a marked anti-human cytomegalovirus (CMV), respiratory syncytial virus (RSV) and influenza A virus activity in HEL, HeLa and MDCK cells, respectively. The 50% effective concentration (EC50) of CA and EHA for HIV ranged from 0.04 to 0.08 micrograms/ml, that is about 3 orders of magnitude below their toxicity threshold (50% inhibitory concentration for MT-4 cell growth: 54 to 60 micrograms/ml). Also, the (GlcNAc)n-specific lectin from Urtica dioica (UDA) was inhibitory to HIV-1-, HIV-2-, CMV-, RSV- and influenza A virus-induced cytopathicity at an EC50 ranging from 0.3 to 9 micrograms/ml. The GalNAc/Gal-, alpha-2,3-Gal/GalNAc- or alpha-2,6-Gal/GalNAc-specific lectins were not inhibitory to HIV or CMV at non-toxic concentrations. CA, EHA and UDA proved to be potent inhibitors of syncytium formation between persistently HIV-1- and HIV-2-infected HUT-78 cells and CD4+ Molt/4 (clone 8) cells (EC50: 0.2-2 micrograms/ml). Unlike dextran sulfate, the plant lectins CA, EHA and UDA did not interfere with HIV-1 adsorption to MT-4 cells and RSV- and influenza A virus adsorption to HeLa and MDCK cells, respectively. They presumably interact at the level of virion fusion with the target cell.


Comparison of antiviral assay methods using cell-free and cell-associated varicella-zoster virus. Assay methods for varicella-zoster virus (VZV) susceptibility to acyclovir (ACV) of VZV were compared by using cell-free (CF) and cell-associated (CA) virus of 6 x plaque-purified VZV. The 50% effective doses (ED50) of ACV, as required to reduce virus plaque formation by 50%, were about 8 times higher for CA virus than for CF virus. Also, the ED50 of 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU) for CA-VZV was higher than for CF-VZV, and fresh clinical isolates of VZV gave higher ACV ED50 values than CF virus. CA virus prepared at various times after CF virus infection showed a gradual increase of the ACV ED50 with time, ranging from the ED50 for CF virus to that for CA virus.


Flow cytometric analysis of effects of cytokines on the expression of varicella-zoster virus glycoproteins. Varicella-zoster virus (VZV)-infected human embryonic fibroblast (HEF) cells were stained with monoclonal antibodies directed against VZV glycoprotein I, II and IV, and then labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. The cells were analyzed by flow cytometry. VZV-infected cells expressing VZV glycoproteins were clearly distinguished from uninfected cells. This method was useful for analyzing expression of VZV glycoproteins in different experimental conditions. Interferon alpha, beta, and gamma and tumor necrosis factor (TNF)-alpha reduced the percentage of positive cells and the mean fluorescence intensity of the cells expressing VZV glycoproteins. Interleukin(IL)-1 beta, IL-6 and TNF-beta had little effect on the expression of VZV glycoproteins.


Structure-function relationship of myotoxin a using peptide fragments. Myotoxin a, a small basic polypeptide isolated from the venom of prairie rattlesnake (Crotalus viridis viridis), has been shown to bind to sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The attachment of myotoxin a to Ca(2+)-ATPase is believed to cause uncoupling of the calcium pump. In order to further elucidate which portion of myotoxin a is important for the uncoupling action, five peptides were synthesized and two peptide fragments were obtained by chemical cleavage. These peptides correspond to discrete portions of the primary sequence of myotoxin a. The peptides are equivalent to the primary sequence of myotoxin a from 1 to 16 residues, 7 to 22 residues, 13 to 28 residues, 19 to 34 residues, and 25 to 42 residues. Chemically produced fragments are equivalent to 1 to 28 residues and 29 to 42 residues of myotoxin a. Peptides of the sequences "YKQCHKKGGHCFPKEK" and "LGKMDCRWKWKCCKKGSG" of myotoxin a inhibited 45Ca uptake into isolated SR and bound to Ca(2+)-ATPase. The same peptides caused weak skeletal muscle vacuolization similar to that caused by native myotoxin a and increased serum creatine kinase activity. The active peptides correspond to the N-terminal and C-terminal portions of myotoxin a. The inactive or less active peptides have sequences which correspond to the middle sequence of myotoxin a. From this study, both the N-terminal and the C-terminal regions of primary sequence of myotoxin a are required to express myotoxin a's biological activity.


Ammonia assimilation in S. cerevisiae under chemostatic growth. Glutamate, glutamine, and ammonia pool size have been determined in two S. cerevisiae strains (GOGAT+ and GOGAT-) growing under ammonia excess and limitation at a dilution rate of 0.10/h. The biomass levels and glutamate dehydrogenase NADPH-dependent (NADPH-GDH) activities were also measured for both strains. The strain that lacks GOGAT activity showed lower levels of metabolites under both media and lower levels of biomass under carbon limitation (ammonia excess) compared to the GOGAT+ strain. Under nitrogen limitation, the biomass level was the same for both strains, but GOGAT- changed from rounded to ellipsoidal cells.


Immobilization of invertase through its carbohydrate moiety on Ocimum basilicum seed. Yeast invertase, a glycoprotein, was covalently coupled to Ocimum basilicum seeds either through its protein or carbohydrate moiety. Of the various methods investigated, binding of the enzyme through its carbohydrate moiety resulted in the retention of considerably higher amounts of enzyme activity. Immobilized invertase showed a shift in the pH optimum toward the alkaline side without appreciable change in temperature optimum. However, the immobilized preparation was more thermostable than the free enzyme. Invertase bound to the seeds could be used repeatedly for the hydrolysis of sucrose syrups in a batch process without appreciable loss in activity. The seeds could serve as an inexpensive, ready-to-use, natural pellicular polysaccharide support for immobilizing enzymes.


Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression as determined by increased CYP1A1 mRNA levels and ethoxyresorufin O-deethylase (EROD) activity in mouse Hepa 1c1c7, rat hepatoma H-4II E and human Hep G2 cancer cell lines. In contrast, treatment of these cell lines with either alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) at concentrations as high as 10(-6) M resulted in only minimal induction of CYP1A1 mRNA levels or EROD activity. Cotreatment of the cells with 10(-9) M TCDD plus different concentrations (10(-8)-10(-6) M) of MCDF or alpha NF resulted in a concentration-dependent decrease in TCDD-induced CYP1A1 mRNA levels and EROD activity in the three cell lines. Moreover, using 10(-9) M [3H]TCDD, it was shown that the alpha NF- and MCDF-mediated antagonism of TCDD-induced CYP1A1 gene expression was paralleled by a decrease in levels of the nuclear [3H]TCDD-Ah receptor complex as determined by velocity sedimentation analysis of the nuclear extracts. The binding of nuclear extracts from the treated cells to a synthetic consensus dioxin responsive element (DRE) (a 26-mer) was determined by gel retardation studies using 32P-DRE. In cells treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M alpha NF, the concentration-dependent decrease in TCDD-induced CYP1A1 gene expression by alpha NF was also paralleled by decreased levels of a retarded band associated with the nuclear Ah receptor-DRE complex. In contrast, the results of the gel shift assay of nuclear extracts treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M MCDF indicated that there were relatively high levels of nuclear MCDF-Ah receptor complex in the cells co-treated with TCDD plus the antagonist but this was not accompanied by induced CYP1A1 gene expression. The results suggest that alpha NF and possibly MCDF compete with TCDD for cytosolic Ah receptor binding sites; however, MCDF may also inhibit the induction response by competing for and/or partially inactivating genomic binding sites.


Peroxynitrite formation from macrophage-derived nitric oxide. Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.


Reversal by phosphatidylglycerol and cardiolipin of inhibition of transcription and replication by histones in vitro. We examined the effects of phospholipids on transcription and replication in vitro in the presence of histones. Phosphatidylglycerol and cardiolipin were shown to reverse the inhibitory effects of histones in both random RNA synthesis with purified RNA polymerase II and accurate transcription initiated from the adenovirus 2 major late promoter in a nuclear extract. Phosphatidylserine, phosphatidic acid, and phosphatidylcholine did not activate RNA synthesis although they bound as strongly as phosphatidylglycerol and cardiolipin to histones. Phosphatidylglycerol and cardiolipin also reversed the in vitro inhibition of SV40 DNA replication by histones. Unsaturation of the fatty acid residues was shown to be necessary for the restoration of transcription and replication by phosphatidylglycerol.


Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium. The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.


Lithocholate binding by Y and Y' proteins in bovine small intestine. Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.


Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells. Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.


Stimulation of microsomal production of reactive oxygen intermediates by rifamycin SV: effect of ferric complexes and comparisons between NADPH and NADH. Rifamycins are antibacterial antibiotics which are especially useful for the treatment of tuberculosis. Reactive oxygen intermediates are produced in the presence of rifamycin SV and metals such as copper or manganese. Experiments were carried out to evaluate the interaction of rifamycin SV with rat liver microsomes to catalyze the production of reactive oxygen species. At a concentration of 1 mM, rifamycin SV increased microsomal production of superoxide with NADPH as cofactor 3-fold, and with NADH as reductant by more than 5-fold. Rifamycin SV increased rates of H2O2 production by the microsomes twofold with NADPH, and 4- to 8-fold with NADH. In the presence of various iron complexes, microsomes generated hydroxyl radical-like (.OH) species. Rifamycin SV had no effect on NADPH-dependent microsomal .OH production, irrespective of the iron chelate. A striking stimulation of .OH production was found with NADH as the reductant, ranging from 2- to 4-fold with catalyst such as ferric-EDTA and ferric-DTPA to more than 10-fold with ferric-ATP, -citrate, or -histidine. Catalase and competitive .OH scavengers lowered rates of .OH production (chemical scavenger oxidation) and prevented the stimulation by rifamycin. Superoxide dismutase had no effect on the NADH-dependent rifamycin stimulation of .OH production with ferric-EDTA or -DTPA, but was inhibitory with the other ferric complexes. In contrast to the stimulatory effects on production of O2-., H2O2, and .OH, rifamycin SV was a potent inhibitor of microsomal lipid peroxidation. These results show that rifamycin SV stimulates microsomal production of reactive oxygen intermediates, and in contrast to results with other redox cycling agents, is especially effective with NADH as the microsomal reductant. These interactions may contribute to the hepatotoxicity associated with use of rifamycin, and, since alcohol metabolism increases NADH availability, play a role in the elevated toxic actions of rifamycin plus alcohol.


Regulation of hepatic glycolysis and gluconeogenesis by atrial natriuretic peptide. Recently we reported the presence of both the guanylyl cyclase-linked (116 kDa) and the ANF-C (66 kDa) atrial natriuretic peptide receptors in the rat liver. Since ANF 103-125 (atriopeptin II) stimulates cGMP production in livers and because cGMP has previously been shown to mimic the actions of cAMP in regulating hepatic carbohydrate metabolism, studies were performed to investigate the effects of atriopeptin II on hepatic glycolysis and gluconeogenesis. Additionally, employing analogs of atrial natriuretic hormone [des-(Q116, S117, G118, L119, G120) ANF 102-121 (C-ANF) and des-(C105,121) ANF 104-126 (analog I)] which bind only the ANF-C receptors, the role of the ANF-C receptors in the hepatic actions of atriopeptin II was evaluated. In perfused livers of fed rats atriopeptin II, but not C-ANF and analog I, inhibited hepatic glycolysis and stimulated glucose production. Moreover, analog I did not alter the ability of atriopeptin II to inhibit hepatic glycolysis. Atriopeptin II, but not C-ANF and analog I, also stimulated cGMP production in perfused rat livers. Furthermore, while atriopeptin II inhibited the activity ratio of pyruvate kinase by 30%, C-ANF did not alter hepatic pyruvate kinase activity. Finally, in rat hepatocytes, atriopeptin II stimulated the synthesis of [14C]glucose from [2-14C]pyruvate by 50% and this effect of atriopeptin II was mimicked by the exogenously supplied cGMP analog, 8-bromo cGMP. Thus atriopeptin II increases hepatic gluconeogenesis and inhibits glycolysis, in part by inhibiting pyruvate kinase activity, and the effects of atriopeptin II are mediated via activation of guanylyl cyclase-linked ANF receptors which elevate cGMP production.


Generation of hydroxyl radical by crocidolite asbestos is proportional to surface [Fe3+]. Differences among fibrous silicates to effect injury in biological systems have been postulated to reflect oxidant generation by structural iron within the crystal lattice of amphiboles. Iron is also coordinated to the surface of all silicates in concentrations which depend on the density of acidic functional groups. We tested the hypothesis that oxidant generation by crocidolite is proportional to surface-complexed iron rather than variance in the lattice concentrations of this transition metal. Surface iron was quantified after its reduction to Fe2+ and chelation by citrate. Thiobarbituric acid (TBA) reactive products and dihydroxybenzoic acid products of salicylate were employed as indices of nonspecific oxidant and hydroxyl radical generation, respectively. Surface iron, TBA reactive products, and dihydroxybenzoic acid products all diminished after pretreatment of crocidolite with the metal chelator deferoxamine in concentrations varying from 0 to 250 mM. Inclusion of deferoxamine in the reaction mixture provided similar results of diminishing both TBA reactive products and dihydroxybenzoic acid generation. We conclude that oxidant generation by crocidolite is proportional to surface concentrations of iron which can be chelated using deferoxamine. The design of synthetic fibers without health effects after exposure will likely necessitate decreasing the number of surface acidic functional groups to diminish the capacity to complex iron (i.e., minimize the percentage SiO2).


Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets. The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.


Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons. A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.


Study of a Fenton type reaction: effect of captopril and chelating reagents. The purpose of this study was to determine if captopril, an angiotensin-converting enzyme inhibitor, could interact with iron ions and so modify a Fenton type reaction. Results indicate that different degrees of thiobarbituric acid-reactive substance from deoxyribose are obtained in an ascorbate-driven Fenton system depending on the order of addition of captopril and iron to the incubation medium. Similar results were obtained with the chelating reagents ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, indicating that the buffer solution plays a relevant role when a particular iron complex is formed with a chelating agent. These metal complexes produce oxidizing species in a Fenton type system whose nature is discussed.


[Type C hepatitis and hepatocellular carcinoma]. Recently the number of hepatitis C virus antibody (HCV-Ab) positive male cases with hepatocellular carcinoma (HCC) is increasing only in Japan mainly for two reasons. One is that cases with liver cirrhosis are surviving longer than before. The other is the increasing number of HCC cases receiving blood transfusions at operations approximately 30 years ago. The prognosis of NS' 4 positive cases was worse than NS' 4 negative cases with HCV-Ab positive chronic hepatitis. The rate of HCV-Ab and HBsAg positive cases among 113 ones with HCC was about 70 percent and 25 percent, respectively. Some 239 cases with cirrhosis were followed for 6 years. Consequently HCV-Ab positive HCC cases were found to have a yearly incidence rate of 7 percent. The rate of development to HCC with HCV-Ab and HBsAg positive cases was significantly higher than that of both HCV-Ab and HBsAg negative ones. The integration of HCV-RNA was not found both in cancerous and non-cancerous region, different from the very high integration rate of HBsAg positive cases. Pathoepidemiologically, HCV is closely related to HCC. However, the role of HCV in the development of HCC remains unknown.


Isolation and characterization of the human glutathione S-transferase A2 subunit gene. We have isolated and characterized a second human liver glutathione S-transferase (GST) subunit gene. The nucleotide sequence of this gene indicates that it encodes the alpha class subunit A2, with a coding region of about 13 kb. Using reverse transcription assays it could be shown that the A2 subunit gene is expressed in human liver and HepG2 cells. The transcription initiation site has been determined by primer extension analysis. A "TATA"-sequence was found 26 nucleotides upstream from the transcription start site. A comparison of the structure of the A2 subunit gene with that of the A1 subunit gene shows significant sequence identity between the two genes. Southern blot analysis of restriction endonuclease digests of human DNA indicates that there may be several more human alpha class GST genes.


[Pharmacokinetic study and side effects of chronic daily administration of oral etoposide]. Eleven inoperable patients with non-small cell lung cancer were treated as a maintenance therapy with oral etoposide 25 mg daily. The toxicity appeared during the chemotherapy were assessed in all cases, but the blood concentration of the drug were measured in 5 cases on the first and the seventh day of treatment. While the peak plasma level (Cmax) was 0.92 +/- 0.43 microgram/ml on the first day and 1.02 +/- 0.30 micrograms/ml on the seventh day of chemotherapy, AUC was 12.3 +/- 5.41 micrograms.hr/ml and 11.9 +/- 4.52 micrograms.hr/ml on the first and the seventh day, respectively. Cumulative effect of the drug did not exist, since in any of these two measurements there was no significant statistical difference between values obtained on the first and on the seventh day. Regarding the toxicity of the drug, bone marrow suppression with abnormal reduction of peripheral white blood cells was observed. Though grade 2 adverse reaction was found in 6 cases, stopping drug administration for 2 weeks, enabled to re-administer the drug. Alopecia and liver or renal injury were not observed, and in spite of the presence of nausea and anorexia in one case, maintenance therapy could continue in all cases. Based on these results we concluded that etoposide can be safely administered as a maintenance therapy on out-patient basis.


[Non-Hodgkin lymphoma with Ki-1 antigen: a case report]. An 89-year-old female with fever, lymphoadenopathy and swelling of both upper extremities was admitted. The pathological specimen of enlarged lymph node showed diffuse anaplastic large cell lymphoma, reacted with the antibody Ki-1 (CD 30), and was diagnosed as Ki-1 lymphoma. She was successfully treated with CPM 700 mg (day 1), ADM 20 mg (day 1), VCR 1 mg (day 1) and PSL 60 mg (day 1-5). She showed improved quality of life. This case can be a model for treatment of elderly patients.


[Cell culture and its application--in vitro evaluation of anticancer activity using human tumor cell lines]. Selective toxicity against cancer cells is a most important determinant for anticancer agents. Therefore, we have preferably evaluated anticancer effects in vivo using murine tumor models for several decades. Approximately 50 anticancer agents are currently available for clinical therapy, but very few agents are effective against some types of cancer. Much progresses in cell culture techniques resulted in establishment of various human tumor cell lines. Currently, we are able to use human tumor lines as well as murine ones for the examination of drug sensitivity. A number of assay methods to evaluate anticancer activity have been developed. In the beginning, growth inhibitory activity was evaluated by counting cell numbers after drug exposure. Then, human tumor clonogenic assay (HTCA) was designed to measure only proliferative cells. Recently colorimetric MTT assay and SRB assay in 96-well microplates were developed, which were adopted in the screening system in the NCI, based on a new idea, that is, disease-oriented screening (DOS) using about 60 human tumor cell lines. In this paper outline of each method was described, adding especially several comments on disease-oriented screening.


Thymopentin treatment in severe atopic dermatitis--clinical and immunological evaluations. An open clinical trial of thymopentin was conducted on 16 children with severe atopic dermatitis. The patients were treated with injections three times a week of 50 mg thymopentin for six weeks. They were then divided randomly into two groups: group A continued thymopentin for an additional six weeks, and group B were treated with normal saline. Clinical parameters and immunological function were evaluated serially. The total severity score started to decline from baseline significantly three weeks after treatment, and continued throughout the study period in group A but began to flare up in group B two weeks after stopping thymopentin. All the eight patients in group A completed the trial but three out of eight in group B dropped out because of flaring up of skin lesion. In vitro production of interleukin-4 tended to decrease and that of interferon gamma tended to increase, but total serum IgE, in vitro IgE synthesis, and abnormally low CD8+ CD11b+ suppressor T cells remained unchanged. Histamine releasing factor (HRF), plasma histamine, and respiratory burst activities of polymorphonuclear leucocytes were appreciably decreased after thymopentin treatment. It is concluded that the clinical efficacy of short term thymopentin treatment very possibly results from the decreased production of HRF and decreased release of polymorphonuclear leucocyte derived inflammatory mediators and may have no relation with antigen-IgE immune reaction.


Epidermal and splenic antigen-presenting cell function in a retrovirally induced murine immunodeficiency syndrome (MAIDS). Since alterations of epidermal Langerhans cells (LC) have been observed in humans infected with HIV, we investigated the morphology and function of these cells in murine acquired immunodeficiency syndrome (MAIDS), a murine model closely resembling human AIDS. The number as well as the shape of dendritic MHC class II+ cells from ear skin of C57BL/6 mice were similar in normal and infected animals. In mixed epidermal cell (EC) lymphocyte cultures, EC from infected mice and from normal mice stimulated allogeneic T cell proliferation to the same extent. In contrast to T cells from normal mice, however, T cells from infected mice did not respond to allogeneic spleen cells, confirming the presence of a T-cell defect in MAIDS. Subcutaneous injection of syngeneic mice with trinitrophenyl-modified MAIDS EC resulted in delayed ear swelling responses after challenge that were equivalent to those induced by hapten-modified EC from normal mice, suggesting that the contact sensitivity inducing potential of MAIDS LC was preserved. To investigate antigen presenting and processing function, EC and spleen cells were tested with the ovalbumin-specific IAb-restricted T cell hybridoma BO.17.10 and either ovalbumin 323-339 peptide or intact ovalbumin protein. MAIDS spleen cells had a reduced antigen presenting capacity compared with normal spleen cells, whereas EC from these mice showed the same processing and presenting capacity as normal controls. In summary, our results demonstrate that the frequency, morphology, level of MHC class II antigen expression and ability to process and present antigen is normal for LC from mice with MAIDS whereas the function of splenic T cells and APC from infected mice is significantly impaired.


Effect of dihomogammalinolenic acid and its 15-lipoxygenase metabolite on eicosanoid metabolism by human mononuclear leukocytes in vitro: selective inhibition of the 5-lipoxygenase pathway. The purpose of the present study was to determine the effect of the n-6 fatty acid, dihomogammalinolenic acid (DGLA, 20: 3, n-6) on arachidonic acid (AA) (C20: 4) metabolism by human peripheral mononuclear leukocytes (HPML). After incubation of HPML with A23187 (5 microM) and DGLA, the cyclooxygenase (CO) and lipoxygenase (LO) products were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with radioimmunoassay. DGLA led to no change in PGE2 formation, but at similar concentrations there was a dose-dependent decrease in LTB4 formation (IC50 = 45.0 microM). The inhibition of LTB4 formation by DGLA was associated with a dose-dependent increase in its 15-LO metabolite 15-hydroxyeicosatraenoic acid (15-HETrE) and its CO metabolite prostaglandin E1 (PGE1). Incubation of HPLM with 15-HETrE (0-1.5 microM) alone did not result in a change in PGE2 formation, whereas 15-HETrE was a much more potent inhibitor of LTB4 formation (IC50 = 0.5 microM) than DGLA. These results show that the addition of DGLA to HPML results in a selective inhibition of LTB4 formation, presumably via its metabolite (15-HETrE).


In situ hybridization for human papillomavirus as a method of predicting the evolution of cervical intraepithelial neoplasia. Twenty-four women with cervical condylomata which were immunohistochemically positive for human papillomavirus (PV-Ag) (15 with CIN 1 and 9 with CIN 2) were followed for a period of 2-65 months. Fifty-seven biopsies were studied by the in situ hybridization (ISH) procedure for the detection of HPV 6/11 and 16/18 DNA. ISH positivity was found in 13/24 cases (54.2%); HPV 16/18 was evident in 7/9 CIN 2 (77.8%) as against 3/15 CIN 1 (20%) (P = 0.017) and in 8/13 cases with koilocytosis affecting up to 2/3 of the epithelial thickness (61.5%) as against 2/11 cases with koilocytosis affecting more than 2/3 of the epithelial layer (18.2%) (P = 0.03). Progression to CIN 3 occurred in 4 cases (2 CIN 1 and 2 CIN 2), the degree of dysplasia remained static in 5 cases (1 CIN 1 and 4 CIN 2) and regression occurred in 15 cases (9 CIN 1 and 6 CIN 2). The immunoperoxidase (IP) positive staining for PV-Ag persisted in 5/24 cases and disappeared in 19/24; 6/13 ISH positive cases maintained ISH positive and 7/13 became negative. The progression of dysplasia was significantly related to disappearance of the IP positivity (P less than 0.0001), to the ISH positivity (P = 0.05), to the persistence of ISH positivity (P = 0.008) and to HPV 16/18 positivity (P = 0.01). We believe that ISH positivity for HPV 16/18 in CIN 1 or 2 with low degrees of koilocytosis and conversion from PV-Ag positive to negative indicate a high risk of progression to CIN 3.


Oral contraceptives and human papillomavirus infection in cervical intraepithelial neoplasia. We report about 142 patients from whom colposcopically directed cervical punch biopsies were taken which showed condylomatous lesions with or without cervical intraepithelial neoplasia (CIN). Fifty-six (39.4%) of these women used oral contraceptives (OC) for at least two years before examination. We used DNA in situ hybridization on all biopsies for detection of human papillomavirus (HPV)-DNA. Among OC users a significant trend towards higher HPV infection rates in high grade CIN (odds ratio 2.9, P less than 0.05) was found, whereas non-users of oral contraceptives had the highest HPV infection rate in condylomatous lesions without CIN (odds ratio 0.5, P less than 0.05). Thus in OC users HPV infection was about 24 times more likely in CIN III as in condyloma, while among non-users the trend was the other way round (7-fold likelihood of HPV positivity in condyloma compared to CIN III). Other known risk factors for cervical carcinoma did not influence HPV infection rates in either group.


Detection of human papillomavirus in vulvar carcinoma. A study by in situ hybridisation. Fourty-four specimens of invasive cancers of the vulva, including 38 primary invasive squamous carcinomas, were analysed by in situ hybridisation with biotinylated HPV 6/11, 16 and 18 DNA probes. Four (9%) of the 44 carcinomas were positive for HPV DNA: three (7%) for HPV 16 DNA and one (2%) for HPV 6/11 DNA. HPV DNA was found only in squamous carcinomas. Of the 38 primary squamous carcinomas, 11% were positive (8% HPV 16, 3% HPV 6/11). The overall 5-year survival was 48.7%, 48.5% for the squamous carcinomas and 50.0% for the HPV DNA positive patients.


Absence of a specific effect of free radicals on HLA-B27. The spondylitis associated HLA-B27 epitope includes a characteristic unpaired cysteine at amino acid position 67. On some B27 molecules the thiol (-SH) side chain of this residue seems to be available for chemical interactions. The possibility that free radicals produced during inflammation might specifically affect this group was investigated in this work. Cells bearing HLA-B27 were exposed to free radicals generated by ultraviolet irradiation or hydrogen peroxide, and HLA antigens were then measured by flow cytometry. Binding of monoclonal antibodies to B27 was not affected. These results do not support a specific susceptibility of HLA-B27 to damage by free radicals, despite its apparently vulnerable structure.


Growth hormone regulates amino acid transport in human and rat liver. Human growth hormone (GH) has been shown to improve nitrogen balance in surgical patients and to decrease urea production. This has been thought to be due primarily to an increase in protein synthesis in skeletal muscle. Little attention has focused on the liver as a possible site where GH may modulate amino acid uptake and thereby divert nitrogen away from urea-genesis. The authors hypothesized that GH regulates amino acid transport in hepatocytes at the plasma membrane level. They studied hepatic amino acid transport in 20 healthy surgical patients that received saline, low-dose GH (0.1 mg/kg/day), or high-dose (0.2 mg/kg/day) GH for 3 days before operation. At operation, a 5- to 10-g wedge biopsy of the liver was obtained, and hepatocyte plasma membrane vesicles were prepared by Percoll density gradient centrifugation. Vesicle transport of [3H]-MeAIB, a highly selective system A substrate, and [3H]-glutamine, a selective system N substrate, was measured, employing a rapid mixing/filtration technique. Hepatocyte plasma membrane vesicles were also prepared from 14 rats treated with saline or one of three different GH treatment regimens: (A) 12 hours after chronic GH treatment (6 mg/kg every 12 hours x 4 doses); (B) 4 hours after acute (1 dose) GH treatment; and (C) 4 hours after chronic GH treatment. In human liver vesicles, low-dose GH resulted in a 13% decrease in system A activity (p = not significant), whereas high-dose GH caused a marked 79% decrease (6.7 +/- 1.7 pmol/mg protein/10 seconds in control patients versus 1.4 +/- 0.7 in GH, p less than 0.05). System N was unaffected. Kinetic analysis of MeAIB transport by vesicles from high-dose GH patients showed the reduction in transport to be due to a 63% decrease in the Vmax (maximal transport velocity) with no alteration in the transport Km (carrier affinity). Vesicles from rats treated chronically with GH using a protocol similar to that used for human subjects exhibited decreased system A transport activity (10.4 +/- 0.4 pmol/mg pro/10 seconds in controls versus 7.5 +/- 0.2 in GH, p less than 0.05) secondary to a 59% reduction in the transport Vmax. Chronic growth hormone treatment decreases the activity of system A in both human and rat hepatocytes. This may be one mechanism by which GH diminishes hepatic urea-genesis and spares amino acids for peripheral protein synthesis.


Neurotensin expression and release in human colon cancers. Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonic mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]i) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]i mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers.


Continuing evolution of the pelvic pouch procedure. The results of the pelvic pouch procedure were reviewed to assess the surgical complication rate and outcome of patients who had had the procedure performed with a stapled ileo-anal anastomosis with and without a defunctioning ileostomy. Between December 1982 and March 1992, 483 patients underwent a pelvic pouch (PP) procedure. Patients were divided into three groups: group I consisted of 325 patients (178 men and boys and 147 women and girls) who underwent a PP procedure with a handsewn ileoanal anastomosis (IAA) with a defunctioning loop ileostomy. In group II, there were 87 patients (47 men and boys and 40 women and girls) who had a stapled IAA with a defunctioning ileostomy. Group III patients consisted of 71 patients (43 men and boys and 28 women and girls) who had a stapled IAA with no covering ileostomy. Assessment was made of the IAA leak rate, the surgical complications, the reoperation rate, and functional outcome. Early surgical complications included 40 (12%) IAA leaks in group I patients compared with only six (7%) leaks in group II patients who had a stapled IAA (p < 0.05). In group III patients, who had a stapled IAA but no covering ileostomy, there were 13 leaks (18%). Eleven of these 13 leaks healed spontaneously with tube drainage; one patient remains with a rectal tube in place 6 weeks after operation, and only one patient has required a reoperation (defunctioning ileostomy). Functionally, all patients with a healed IAA after a leak have had an excellent result comparable to those without a leak. Patients who were male, older than age 40, on steroids, and had had a true one-stage PP procedure, had a greater risk of developing an IAA leak. In two patients, there was intraoperative difficulty, and one of these patients had an IAA leak after operation. Disease activity at the resection margin and patient weight did not affect the leak rate. Our results suggest that the IAA leak rate is significantly reduced in patients with a stapled IAA with an ileostomy compared with those with a handsewn IAA. Omission of the defunctioning ileostomy is associated with a higher IAA leak rate, but spontaneous healing occurs in almost all patients without impairment of functional results. In patients in whom the ileostomy is omitted, the IAA leak rate is greatest in male patients who have undergone a true one-stage PP procedure, are on steroids, and are older than age 40.


Inhibition of carbohydrate uptake of Onchocerca volvulus infective larvae (L3) by effector cell constituents. The in vitro killing of Onchocerca volvulus infective third stage larvae (L3) by components of their human hosts' defence mechanisms is not well documented, as no suitable assay exists. Motility is inappropriate as a criterion of larval viability because of the unsteady winding movements of L3. In the present study, a metabolic parameter for larval viability, the uptake of [3H]2-deoxy-D-glucose, was evaluated. To demonstrate the reproducibility and validity of this test, the oxygen radical hydrogen peroxide (H2O2) was applied to viable L3 and the death of L3 demonstrated by a 90% reduction in glucose uptake. The incorporation of glucose by the filarial larvae was also determined after in vitro exposure to lysates of the putative effector cells, i.e. eosinophilic and neutrophilic granulocytes and monocytes. Effector-cell-derived components led to a 30-80% dose-dependent decrease in deoxy-glucose uptake, with a half-maximal effect at about 500 micrograms ml-1. These experiments demonstrate, for the first time, the deleterious impact of effector cell constituents on the metabolic activity of O. volvulus L3. The assay could be used to evaluate the effect of distinct natural or recombinant effector molecules on the viability of O. volvulus infective larvae and to investigate the effect of parasite molecules which interfere with effector mechanisms.


Surgical treatment for higher stage non-small cell lung cancer. Locally advanced lung cancer (stage IIIa, IIIb) in which the primary tumor is proximal (T3) or has invaded adjacent structures (T3) or organs (T4) or in which mediastinal lymph nodes are involved (N2, N3) worsens the prognosis significantly. However, in stage IIIa (T3 or N2), when surgical treatment results in total removal of the primary tumor and involved lymph nodes, there still is a reasonable chance for ultimate cure. On the other hand, total excision can be very rarely performed in T4 or N3 tumors. Therefore, this group (stage IIIb) usually indicates unresectability. Disseminated lung cancer with distant metastasis (stage IV) is still considered to be incurable. Nevertheless, solitary metastatic sites (M1), especially brain, have been treated on occasion by resection of the primary tumor and removal of the solitary metastasis. This appears to improve median survival and does yield 5-year survival in selected patients. The results after surgical treatment in these patients with higher stage lung cancer reported over the last 10 years are reviewed.


[Antibiotic therapy of intestinal infections complicated by toxicosis and exicosis in children with immunodeficiency syndromes]. Clinical processes of acute intestinal infections complicated by toxicosis and exicosis and some host immunological parameters were studied in infants with secondary immunodeficiency. A special treatment scheme was developed including combined use of antibiotics, human immune globulin administered intravenously and cytochrome C. The scheme provided a decrease in the treatment duration by 8.6 +/- 1.1 days. Advanced and chronic diseases and fatal outcomes were absent. It was concluded that the developed scheme increased host immunological reactivity and efficacy of antibiotic therapy. It was recommended for wide clinical use in pediatric clinical care.


[Proteins induced by various types of double-stranded RNA and interferon type I in human fibroblasts. Correlation with antiviral activity of the substances]. Protein induction by new antiviral preparations of dsRNAs (larifan, ridostin, rifastin and poly(A).poly(U)) and recombinant beta-interferon in human fibroblasts (M19) was studied. The common gene products: 88, 80, 68, 58, 56, 52, 50 and 26 kD were detected in the spectra of the induced cytoplasmic polypeptides. At the same time the sets of the induced proteins had individual distinctions in various preparations. Induction of the 56-kD protein was more essential in the action of dsRNAs than that of interferon. The antiviral activity of dsRNAs and interferon preparations correlated with a relative increase in the synthesis of proteins with molecular weights of 88, 80 and 58 kD. The study results are in agreement with the fact that the dsRNAs have interferon-independent pathways of antiviral action with participation of 56- and 58-kD protein genes.


[Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]. The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.


Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype. We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured. The brilliant cresyl blue test reliably detects couples at risk for hemoglobin Bart's hydrops fetalis. Measurement of the number of inclusion bodies differentiates the alpha-thalassemia genotypes in the absence of a coincident beta-chain synthesis deficiency, such as hemoglobin E or beta-thalassemia. The test appears to identify patients, such as those with the Thai and Filipino deletion variants, whose alpha-thalassemia cannot be definitively characterized by DNA testing when only alpha- and zeta-globin probes are used in the analysis. We also found evidence of elevated serum ferritin levels in many patients with deletion of two or three alpha-globin genes. This study shows that most routine screening for alpha-thalassemia can be performed with three simple tests: (1) the brilliant cresyl blue inclusion study, (2) erythrocyte indices, and (3) iron studies. Analysis with DNA probes is needed in only some circumstances.


Müllerian adenosarcomalike tumor of the seminal vesicle. A case report with immunohistochemical and ultrastructural observations. A müllerian adenosarcomalike tumor of the seminal vesicle is described in a 49-year-old man. The tumor occupied the entire right seminal vesicle and adhered to the right lobe of the prostate, in the area adjacent to the seminal vesicle. The tumor was also adherent to the rectum. Microscopically, the cells were seen to invade the prostatic tissue. The tumor consisted of a highly cellular stroma with spindle-shaped pleomorphic cells, suggesting the diagnosis of a low-grade sarcoma. In addition, dilated cystic spaces lined by columnar epithelium were seen. Immunohistochemically, most tumor cells showed positivity for vimentin, desmin, and muscle-specific actin, suggesting smooth-muscle cell differentiation. Furthermore, electron microscopy also demonstrated smooth-muscle differentiation of the tumor cells. The patient has been disease-free for 48 months since undergoing a cystoprostatetectomy.


Synthesis of pyranoid analogues of the anti-HIV active 3'-deoxy-2',3'-didehydrothymidine (D4T). The primary hydroxy group of ethyl 2,3-dideoxy-alpha-D-erythro-hex-2-enopyranoside (1) was selectively protected and the secondary hydroxy group was deoxygenated via the dithiocarbonate 3 from which ethyl 6-O-(4-methoxybenzoyl)-2,3,4-trideoxy-alpha-D-glycero-hex-2-eno pyranoside (4) and its regioisomer (5) were produced. These were converted into didehydro nucleosides by glycosylation of silylated heterocyclic bases in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst. The configurations of the anomeric products were assigned by 1H-NMR analysis of the corresponding saturated compounds which were obtained by hydrogenation of the double bond in the carbohydrate moiety. The compounds 9a,b,d, 10a,b, 14a,b,e,f, and 15a,b,e,f did not show any significant activity against HIV or HSV-1.


Fine-needle aspiration biopsy causing peritoneal seeding of a carcinoid tumor. A rare complication of fine-needle aspiration biopsy (FNA) is needle tract seeding. Peritoneal seeding from FNA in the clinical setting is rarely reported. To our knowledge, this is the first case of peritoneal seeding of a neuroendocrine carcinoid tumor as a result of FNA. Peritoneal contamination and tumor seeding can occur with FNA and they probably do occur more often than is recognized clinically. Therefore, we caution against the use of FNA in the assessment of potentially curable intra-abdominal tumors, and recommend that this technique be used only when a cytological diagnosis is required before initiation of palliative therapy.


[Hormones and aggression].

[Hormones and aggression]. This work is a review about psychoendocrinologic aspects of aggressive behavior. We have considered two approaches, the classical view focused on hormonal influences in the regulation of aggression and the most recent view which accounts for the influences of aggressive behavior and social status on hormonal secretion. In this review we differentiate the organizational from the activational effects. The main conclusions are referred to the gonadal steroids effects both organization and activation of the neural subtract of aggression. Among them it seems that aromatizable androgens have the most important effects. On the other hand, the testosterone would be predictive for dominant social status, the corticosterone would be for submission and finally, the ACTH would be predictive for the aggressive behavior but not for submission. In addition to that the literature point that the defeat experience is the main factor in submission. Referred to the effects of behavior on hormonal secretions, all the studies point out that social interaction elicits sympathetic-medullar and gonadal activation in the dominant animals and cortico-adrenal activation in the submissive ones.


A pilot study on the influence of a corticotropin (4-9) analogue on Vinca alkaloid-induced neuropathy. In a randomized, double-blind, placebo-controlled pilot study, we examined the effect of Org 2766--a corticotropin (4-9) analogue--on neurotoxicity in 28 patients with lymphoma who were treated with combination chemotherapy containing Vinca alkaloids (vincristine and vinblastine). The patients received a total dose of 12 mg of vincristine in the case of non-Hodgkin's lymphoma and a total dose of 16 mg of vincristine in the case of Hodgkin's disease. Moreover, the patients with Hodgkin's disease received a mean total dose of 84 mg of vinblastine. Subcutaneous injections of 2 mg of Org 2766 or placebo were administered to patients with non-Hodgkin's lymphoma on days 1 and 10 of each chemotherapy course and to patients with Hodgkin's disease on days 1 and 8 of each chemotherapy course. The first injection was always given before the administration of vincristine. Assessment of neurologic symptoms and signs and measurement of sensory thresholds (vibration sense and temperature sense) were performed on day 1 of the first, fourth, and sixth (or eighth) courses and 6 weeks after cessation of chemotherapy. Thirteen patients (mean age, 44.7 years) received Org 2766 and 15 patients (mean age, 54.7 years) received placebo. More symptoms occurred in the placebo group, but only numbness and autonomic complaints occurred significantly more often in the placebo group. Motor deficit and sensory disturbances were more severe and also occurred significantly more often in the placebo group. There was no difference with respect to reflex examination findings and sensory thresholds.(ABSTRACT TRUNCATED AT 250 WORDS)


The treatment of neurosarcoidosis with cyclosporine. Six patients with refractory neurosarcoidosis were enrolled in a 12-month open-label trial to investigate the safety and efficacy of cyclosporine therapy. Patients were stabilized on a corticosteroid dose, randomized to a low-dose or high-dose cyclosporine group (with appropriate target whole blood cyclosporine levels) for 6 months, and assessed by prospectively defined studies. The corticosteroid dose was adjusted as clinically tolerated. We found that the corticosteroid dose could be lowered to 30% to 58% of the initial stabilization dose in conjunction with cyclosporine therapy, at the time of maximal clinical and laboratory improvement. However, four patients deteriorated while using corticosteroids and cyclosporine; one of these patients died. At the time of clinical deterioration, the prednisone dose ranged from 6 to 22.5 mg daily (or the equivalent). No serious toxic effects developed from cyclosporine therapy. Cyclosporine treatment is a reasonably safe and effective adjunct to corticosteroid therapy for patients with refractory neurosarcoidosis, although clinical deterioration can occur despite combination therapy.


Na,K-ATPase in resting and stimulated submandibular salivary glands in cats, studied by means of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity. Sections from normal resting submandibular glands were incubated in a p-nitrophenylphosphate substrate mixture to demonstrate Na,K-ATPase activity by light and electron microscopy. Dense deposits of reaction product occurred within the basal and lateral infoldings of striated duct cells. A similar, less heavy deposition occurred in demilune cells. Only scattered, infrequent pockets of staining occurred in association with the basal plasma membrane of central acinar cells, supporting the belief that these cells do not contribute greatly to the secretion of fluid. No reaction was detected on luminal membranes. Glands tested after sympathetic or parasympathetic nerve stimulation showed no obvious differences from resting glands. These results contrast with the previously reported paucity of immunostaining in resting demilunes with an antibody to Na,K-ATPase and the large increase in the immunostaining by these cells after parasympathetic stimulation. It is inferred that there must be an inaccessibility to immunobinding sites of Na,K-ATPase in resting glands, and that the increase in binding which occurs after parasympathetic stimulation represents a conformational change that did not affect its enzyme catalytic reaction with this substrate.


Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus. Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.


Migration of erythrosin-labelled saliva during unilateral chewing in man. The aim was to monitor the distribution of erythrosin-labelled saliva around the mouth during normal, unilateral chewing on gum. Thirty-one subjects chewed chewing-gum containing erythrosin on the preferred side for 5 min. The mouth was examined and the distribution of the dye recorded on standardized charts. The dye was present mainly ipsilateral to the chewing side. Although the dye did cross the mid-line, it was rarely present in the contralateral premolar and molar regions.


Progression rates of cytomegalovirus retinopathy in ganciclovir-treated and untreated patients. Cytomegalovirus retinopathy lesions in patients with the acquired immunodeficiency syndrome may continue to enlarge despite ganciclovir sodium treatment. In an historical cohort study, we used serial, masked retinal photographs to calculate progression rates for 14 ganciclovir-treated patients known to have disease progression and for 17 untreated patients. The median period of evaluation was 23.5 days for ganciclovir-treated patients and 28 days for untreated patients (P = .89). In both groups, the rate at which borders of preexisting lesions advanced into uninfected retina varied in different directions. Anterior progression rates were usually faster than posterior progression rates. The median progression rate with which disease approached the fovea in ganciclovir-treated patients was 11.5 microns/d (range, 0 to 25.0 microns/d) and the median progression rate for untreated patients was 24.0 microns/d (range, 0 to 164.0 microns/d) (P = .01). These results suggest that ganciclovir provides a therapeutic benefit for patients with progressive disease by slowing the rate with which infection spreads. The size and appearance of lesions may also be related to their response to treatment and to progression rates of untreated disease.


Immunohistochemical study of pleomorphic adenoma of the nasal septum. A rare case of pleomorphic adenoma of the nasal septum is reported. A 48-year-old man complaining of nasal obstruction and nasal bleeding was referred to our hospital for treatment of a left nasal tumor. The tumor, including the nasal septum, was resected by the modified Denker operation and lateral rhinotomy. The tumor was firm, parenchymatous, pedunculated, and 24 x 22 x 14 mm in size. Pathological examination revealed pleomorphic adenoma with slight cellular atypism. Immunohistochemical observations suggested that characterization of this tumor might be similar to pleomorphic adenoma of the parotid gland.


Assessment of retrovirus-expressed nucleoprotein as a vaccine against lethal influenza virus infections of chickens. Hemagglutinin-based influenza vaccines stimulate protection in chickens that is limited to the serotype of the expressed hemagglutinin. To evaluate whether a more highly conserved influenza virus protein might stimulate a broader protective response, the influenza virus nucleoprotein (NP) was introduced into a retroviral vector (mRCAS/NP). NP is an internal influenza virus protein that has been shown to stimulate cytotoxic T-cell responses in influenza-virus-infected mice. Cells infected with mRCAS/NP expressed approximately 10% of the level of NP observed in influenza-virus-infected chicken embryo fibroblasts. Immunocompetent chicks were vaccinated intramuscularly with approximately 1 x 10(5) NP-expressing units of mRCAS/NP. Four weeks later, chicks were bled and challenged with a highly pathogenic avian influenza virus (A/Chicken/Victoria/1/85). The NP-expressing vector stimulated an influenza-virus-specific response, as indicated by the presence of antibody to NP, but failed to protect against the lethal challenge.


Differentiation of pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV genome. There are no simple, direct methods to reliably distinguish oncogenic serotype 1 Marek's disease viruses (MDVs) from their attenuated variants. The present study was an attempt to apply polymerase chain reaction (PCR) to develop a rapid and sensitive assay for the presence of the MDV genome. PCR oligos were chosen to flank the 132-base-pair tandem direct repeats in the serotype 1 MDV genome. The PCR reaction was specific for serotype 1 MDVs, amplifying fragments corresponding to one to three copies of the tandem repeats present in Md11/8, JM/102W, and GA viruses. A high-molecular-weight DNA smear was observed when the DNA from an attenuated Md11/100 was PCR-amplified. Use of the PCR technique allowed the detection of two copies of the 132-base-pair repeat in the DNA extracted from MDV-induced lymphomas removed from two chickens. No DNA was amplified from the DNA extracted from lymphomas induced by either an avian leukosis virus (RAV-1) or reticuloendotheliosis virus (chick syncytial virus).


Demonstration of the genetic stability of a Mycoplasma gallisepticum strain following in vivo passage. A Mycoplasma gallisepticum strain designated 6/85 (MGI) exhibiting reduced virulence for both chickens and turkeys was sequentially passaged 10 times in each species. DNA extracted from organisms before passage and those isolated after the third, sixth, and 10th passages was studied by restriction endonuclease DNA analysis using BamHI, BglII, EcoRI, HindIII, and PstI endonucleases. The virulent-type strain designated S6 was used as a comparison. Comparison of DNA fragment patterns of MGI and S6 strains showed distinct differences, although some similarities were evident. Passage of the strain in vivo did not affect DNA fragment patterns of the MGI strain. Electrophoretic protein patterns produced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed very similar band patterns in both the MGI and S6 strains. The most notable differences were seen in bands located in the molecular-mass regions of approximately 46.5, 50-54, 58-64, and 105-140 kilodaltons. Alteration of band pattern profiles following in vivo passage of the MGI strain was apparent in a single band at approximately 86 kilodaltons that appeared to stain more intensely following passage.


Differences among restriction endonuclease DNA fingerprints of Pennsylvania field isolates, vaccine strains, and challenge strains of infectious laryngotracheitis virus. Restriction endonuclease fingerprints of infectious laryngotracheitis virus (ILTV) DNA from 13 Pennsylvania field isolates, embryo-propagated and tissue-culture-propagated vaccine strains, and three reference strains were compared. These comparisons were made to evaluate the possible contribution of mutation of ILTV vaccine strains to recent outbreaks of infectious laryngotracheitis (ILT) in Pennsylvania. Six different restriction enzymes were used to generate the fingerprints. Differences in DNA banding patterns were revealed between the currently used ILTV vaccine strains and six of the 13 field isolates. Even greater DNA banding pattern differences were found between the older ILTV reference strains and the vaccine strains. The ILTV DNA fingerprints generated in the present study suggest that at least five different strains of ILTV have contributed to the outbreaks of ILT that have occurred since 1987 in Pennsylvania.


Occurrence of acute infectious bursal disease with high mortality in Japan and pathogenicity of field isolates in specific-pathogen-free chickens. Highly virulent infectious bursal disease virus (IBDV) was isolated from field cases, and the pathogenicity of the isolates was examined in specific-pathogen-free chickens. Chickens inoculated with the isolates developed severe clinical disease with a high mortality rate. Histopathologically, infectious bursal disease was characterized by bursal and thymic necrosis, aplastic anemia, acute hepatitis with fatty change, and systemic inflammatory response. In addition to functional abnormalities in the liver, a hypoxic state was induced by aplastic anemia and severe inflammation in the pulmonary air capillary walls. These pathological changes appeared to be closely related to the cause of death.


Experimental factors affecting mortality following inoculation of chickens with avian nephritis virus (G-4260). Groups of approximately 20 one-day-old chickens were inoculated with G-4260, the reference strain of avian nephritis virus (ANV), or saline. Based on mortality rates from severe nephritis in comparable experiments, light Sussex chickens generally were more susceptible than Rhode Island red (RIR) chickens. Mortality was greater in those given broiler starter than those given other feeds, and was greater when light Sussex chickens were given broiler starter feed and cold-stressed at 15 +/- 1 C for 2 hr daily during the first week rather than brooded normally. Inoculation with G-4260 either orally or by intraperitoneal injection produced similar results in RIR chickens. Thirty-three inoculated chickens died of severe nephritis between 4 and 12 days postinoculation, and 24 (73%) of them had visceral urate deposits. Inoculated inbred white leghorn Line 15 chickens with maternal antibody to ANV were brooded normally and given broiler feed: they were susceptible to infection as evidenced by subsequent histological lesions in the kidneys and serology, but mortality was not a feature. There were no deaths from nephritis in inoculated non-inbred white leghorn chickens free of maternal antibody to ANV that were given broiler feed and brooded normally. These results have implications in standardizing experimental conditions for the study of mortality induced by G-4260 and similar viruses.


Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification. Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.


Plant polyphenolic complex inhibits the reproduction of influenza and herpes simplex viruses. A polyphenolic complex (PC), isolated from the Bulgarian medicinal plant Germanium sanguineum L., inhibited the reproduction of influenza virus types A and B in vitro and in ovo. It protected white mice in an experimental lethal influenza infection. When influenza viruses were treated with a high dose (1 mg/mL), their hemagglutination, neuraminidase and infections activities were reduced completely. It was assumed that the PC-specific inhibitory effect on influenza virus reproduction in tissue cultures (50 micrograms/mL) was mediated by partial inactivation of viral glycoproteins which prevented the initial stages of reproduction. The PC also affected the intracellular stages of viral replication. The effect was dependent on the mode of application, the viral strain, and the biological test system. The PC also inhibited the reproduction of herpes simplex virus in vitro. Infectious titers were reduced and newly synthesized virion with damaged protein envelopes were observed by electron microscopy.


Bone histomorphometry in 1,25(OH)2D3- and vitamin D3-treated aged laying hens. Aged laying hens at oviposition were given four levels of dietary vitamin D3 and three levels of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) to determine the effects of the additives on the medullary and endosteal bone. Oxytetracycline hydrochloride labeling and histomorphometry of undecalcified sections were used in the determination of changes in medullary bone and endosteal surface of mid-diaphyseal tibiotarsal bones. Treatment with 1,25(OH)2D3 increased mineralized medullary bone area, endosteal surface length, amount of tetracycline labeling in medullary and endosteal bone, total number of osteoclasts on endosteal and medullary bone, and appositional rate of mineralizing bone. Vitamin D3 increased mineralized medullary bone area and total tetracycline labeling in endosteal bone but decreased endosteal mineral apposition rate. Medullary bone matrix with no mineral was observed only in the group receiving neither supplement. These areas were much larger than typical osteoid seams usually seen in trabecular bone. Both 1,25(OH)2D3 and vitamin D3 were effective in stimulating bone formation and retention of mineral within medullary bone matrix at oviposition.


An outbreak of Mycoplasma synoviae infection in North Carolina turkeys: comparison of isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis. Mycoplasma synoviae (MS) isolates made in 1988-89 from turkey flocks in North Carolina, Missouri, and Ontario, Canada, were compared with each other and MS reference strains (WVU-1853, F10-2AS, Neb-3S, and K1968) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell proteins and restriction endonuclease analysis (REA) of DNA. SDS-PAGE and REA indicated considerable homology among MS reference strains and recent field isolates. However, sufficient differences were resolved to identify the MS reference strains as different from each other and the field isolates, and to classify seven of nine recent field isolates as a cluster of nearly identical strains. The results suggest that flocks infected with members of the cluster were epizootiologically associated, possibly by a common or point source of infection.


Use of polymerase chain reaction for efficient cloning of dsRNA segments of infectious bursal disease virus. A method is described for efficient cloning of the large RNA segment of infectious bursal disease virus (IBDV) after reverse transcription and cDNA amplification. Complementary DNA segments of IBDV were prepared using reverse transcriptase and specific primers homologous to the conserved region at the 3' end of the IBDV sequence. The resulting cDNA segments were amplified using Taq DNA polymerase and a pair of specific primers. Three separate primer pairs were used for amplification, each yielding a cDNA fragment of the predicted size. The amplified products were directly used for cloning into a cloning vector, pCR1000. Three overlapping cDNA clones, containing the entire coding region of the large RNA segment of IBDV, were obtained. The identity of these clones was confirmed by hybridization with IBDV-specific probe, as well as by sequence analysis. By this method, approximately 3.2 kilobases of the large genome segments of two different strains of IBDV were cloned, and their complete nucleotide sequence was determined.


Effect of retinoid status on alpha, beta and gamma retinoic acid receptor mRNA levels in various rat tissues. We have investigated the effects of retinoids, vitamin D and thyroid hormone on the levels of retinoic acid receptor (RAR)alpha, RAR beta and RAR gamma mRNAs in intact animals. Although vitamin A deficiency caused no significant changes in the levels of RAR alpha and RAR gamma mRNAs, the level of RAR beta transcripts was greatly decreased in various tissues of vitamin A-deficient rats, but was restored rapidly to a normal level after administration of retinoic acid. Retinol also restored the RAR beta mRNA level, but the magnitude and kinetics of the induction differed from those by retinoic acid. The use of specific inhibitors demonstrated that this autoregulation of RAR beta gene expression in vivo occurred at the transcriptional level. In addition, from these results it was postulated that the maintenance of the normal RAR beta mRNA levels seemed to require a threshold serum retinol concentration (about 25 micrograms/dl). Moreover, we found that administration of retinol and retinoic acid to normal rats caused the overexpression of RAR beta transcripts (2-15-fold) when compared with the control levels of RAR beta mRNA, although the levels of RAR alpha and RAR gamma mRNAs were not affected. Vitamin D and thyroid hormone did not modulate the levels of RAR transcripts. These findings clearly indicate the specific ligand regulation of RAR beta gene expression in intact animals. The altered levels of RAR beta according to retinoid status may affect retinoid-inducible gene expression.


Economic impact of a documented case of reovirus infection in broiler breeders. The following parameters were used to calculate the economic impact to a commercial broiler company of a pathogenic reovirus infection in broiler breeders during egg production: hatchability, selling of eggs, vaccination, mortality, medication, slowing of line speed at processing, and downgrading and trimming at processing. The cost due to reovirus infection in one flock of broiler breeders was approximately $48,000.


Functional inhibition of protein kinase C-mediated effects in myocardial tissue is due to the phosphatase 2A. An endogenous protein which inhibits protein kinase C (PKC)-mediated effects has been detected in rat heart ventricular tissue. This functional PKC-inhibitory activity was completely abolished by okadaic acid, making it possible to measure PKC activity in non-purified cell fractions. This suggests that the PKC-inhibitory activity is a type 1 or 2A serine/threonine phosphatase. Confirming this, membrane and cytosolic PKC-inhibitory preparations were found to contain phosphatase activity which was suppressed by okadaic acid, exhibiting an IC50 (concn. required for 50% inhibition) of 1.5-2 nM. Furthermore, okadaic acid stimulated prostacyclin production in rat cardiomyocytes and aortic smooth-muscle cells and, like the PKC activator phorbol 12-myristate 13-acetate, it augmented the prostacyclin formation induced by the Ca2+ ionophore A23187. Our results strongly suggest that the endogenous PKC 'inhibitor' is the cellular phosphatase 2A, which plays an important role in regulating the phosphorylation level of PKC target proteins.


Localization of methyltransferase activities throughout the endomembrane system of flax (Linum usitatissimum L) hypocotyls. A microsomal fraction from flax hypocotyls (Linum usitatissimum L) showed a methylation ability from S-adenosyl-methionine on to the cell wall polysaccharides. Two kinds of methylation were found: (i) a methyl esterification of uronic acids in the oxalate extracts and (ii) an O-methylation of the hydroxyl groups in the NaOH extracts. The methyltransferase study showed a rapid decrease of the methyl esterification abilities, whereas the O-methylation on to the hydroxyl groups was maintained throughout the culture duration. The localization of such activities in the flax endomembrane system was performed using isopycnic centrifugation. Enzymic marker tests allowed us to identify the different membrane types. Methyltransferase activities in the different enriched fractions appeared to be associated with the Golgi apparatus for the O-methylation, and with the plasma membrane, Golgi apparatus and endoplasmic reticulum compartments for the carboxymethyl esterification.


Modulation of cytosolic-[Ca2+] oscillations in hepatocytes results from cross-talk among second messengers. The synergism between the alpha 1-adrenergic response, glucagon and cyclic AMP, and their antagonism by insulin and diacylglycerol manifest themselves in the control of the cytosolic-[Ca2+] oscillations. Hepatocytes respond to stimulation by glycogenolytic agonists acting via phosphoinositide (PI) breakdown through oscillations of the free cytosolic concentration of Ca2+ ([Ca2+]cyt.). Since the second-messenger repertoire of hepatocytes includes many other factors besides Ca2+, we investigated to what degree the regulation of [Ca2+]cyt. oscillations is integrated into these other signalling systems. [Ca2+]cyt. was recorded in single rat hepatocytes by using the Ca(2+)-indicator fura-2. Parallel stimulation with phenylephrine (an alpha 1-adrenergic agonist of PI breakdown) and glucagon resulted in a synergistic stimulation of [Ca2+]cyt. oscillations. Direct activation of the cyclic-AMP-dependent pathway with several stimuli (forskolin, 8-bromo cyclic AMP, 8-CPT cyclic AMP) mimicked the response to glucagon. In contrast, [Ca2+]cyt. oscillations induced by various combinations of these agonists could be antagonized by the glycogenic hormone insulin. As one of the options in the insulin-signalling network, we tested a diacylglycerol activator of protein kinase C, DiC8. It also acted as an inhibitor of [Ca2+]cyt. oscillations. We investigated how these observations could be reconciled with our previously introduced model of [Ca2+]cyt. oscillations in hepatocytes [Somogyi and Stucki (1991) J. Biol. Chem. 266, 11068-11077]. First of all, the effect of calmodulin inhibitors (calmidazolium and CGS 9343 B), acting at the core of our model on the feedback of Ca2+ on Ins(1,4,5)P3-induced Ca2+ release, was not altered by the new modulators. In addition, all agonists and antagonists could be used interchangeably in combination and introduced no significant change in the oscillatory pattern or spike shape. Since the response was solely limited to frequency modulation, over- or understimulation of the oscillatory system, there is no need to create a new oscillator or to introduce further reaction steps into the core of the model. We conclude that the regulation of [Ca2+]cyt. via the explored second-messenger pathways can be embedded into the oscillatory system as modulation of rate constants already present in this model.


Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions. Lipid peroxidation within human arterial lesions is thought to play an important role in the development of atherosclerosis. Peroxidation can be accelerated by the presence of 'catalytic' iron or copper ions. Gruel samples from advanced atherosclerotic lesions in the abdominal aortae of human cadavers were tested for pro-oxidant properties. All samples contained bleomycin-detectable iron and phenanthroline-detectable copper. Almost all gruel samples stimulated peroxidation of rat liver microsomes, and this was usually inhibited by the iron-ion chelator desferrioxamine. Some samples stimulated formation of hydroxyl radicals from H2O2 in the presence of ascorbate, a reaction again inhibited by desferrioxamine. We conclude that the interior of human advanced atherosclerotic lesions is a highly pro-oxidant environment, and that the use of copper or iron ions to promote peroxidation of low-density lipoproteins in vitro may be a valid model for events in the arterial wall.


Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases. A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.


Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis. As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.


Gene expression of regulatory enzymes of glycolysis/gluconeogenesis in regenerating rat liver. Levels of mRNA for glucokinase, L-pyruvate kinase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase were analysed during liver regeneration. Levels of mRNA for glycolytic enzymes (glucokinase and L-pyruvate kinase) decreased rapidly after partial hepatectomy. Glucokinase mRNA increased at 16-24 h, returning to normal values after this time. L-pyruvate kinase mRNA recovered control levels at 168 h. In contrast, phosphoenolpyruvate carboxykinase mRNA increased rapidly after liver resection and remained high during the regenerative process. However, the levels of fructose-1,6-bisphosphatase mRNA were not modified significantly. These results correlate with the reported increased rate of gluconeogenesis and changes in enzyme levels after partial hepatectomy. The effect of stress on the mRNA levels was also studied. All enzymes showed variations in their mRNA levels after the surgical stress. In general, the differences were more pronounced in regenerating liver than in sham-operated animals, being practically normalized at 24 h.


Cell shrinkage follows, rather than mediates, the short-term effects of glucagon on carbohydrate metabolism. The initial effects of glucagon on glycogen breakdown in isolated hepatocytes were found to be independent of cell volume and, when it occurred, cell shrinkage followed rather than mediated the glycogenolytic effect of glucagon. Similar conclusions could be drawn for the effect of glucagon on glycolysis/gluconeogenesis and for the antagonistic effect of insulin on glucagon action.


Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis. The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.


Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations. The activation of insulin-stimulated protein-serine/threonine kinases has been investigated in CHO cell lines transfected with cDNAs encoding either wild-type or mutant human insulin receptors. (1) Insulin treatment of CHO cells over-expressing wild-type insulin receptors resulted in the rapid and substantial (5-10-fold) activation of cytosolic protein kinases which phosphorylated myelin basic protein, Kemptide and two peptide substrates based on sites phosphorylated on ribosomal protein S6 in vivo. (2) Further fractionation of cytosolic extracts by MonoQ chromatography revealed two peaks of insulin-stimulated myelin basic protein kinase activity which were highly related to the previously described mitogen-activated protein (MAP) kinases ERK1 and ERK2. In addition, at least two major peaks of S6 kinase activity were resolved, which exhibited properties similar to the 70 kDa and 90 kDa S6 kinases described by others; the predominant effect of insulin was on the activity of the 90 kDa enzyme and was in excess of 10-fold. (3) MonoQ fractionation of extracts from parental CHO cells, or cells expressing kinase-deficient receptors, showed all insulin-stimulated peaks of activity to be almost completely absent. (4) Further studies demonstrated that substitution of tyrosine residues 1162 and 1163 (or 1162 alone) with phenylalanine led to a substantial reduction in the ability of insulin to stimulate these protein kinase activities when assayed in cytosolic extracts. In contrast, deletion of 69 amino acids from the C-terminus of the insulin receptor beta-subunit caused a leftward shift in the insulin dose-response curve of the MAP kinase activity, but apparently not in that of the 90 kDa S6 kinase activity.


Liver cell volume and protein synthesis. Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentration the rate of [3H]leucine incorporation was 5.8 +/- 0.2 nmol/h per mg of cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60 and 74% respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosM), but stimulated protein synthesis in hyperosmotically (440 mosM) pre-shrunken cells almost to rates found in normo-osmotic (320 mosM) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8% respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypo-osmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the above-mentioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.


Characterization of retroendocytosis in rat liver parenchymal cells and sinusoidal endothelial cells. After receptor-mediated endocytosis, internalized ligands may be recycled to the cell surface instead of being routed to lysosomes for degradation, a process termed retroendocytosis. We have investigated the kinetics and extent of retroendocytosis of neoglycoproteins after internalization via two carbohydrate-specific receptors in rat liver cells: galactose receptors in parenchymal cells (PC) and mannose receptors in sinusoidal endothelial cells (EC). Retroendocytosis in both cell types occurred with first-order kinetics, and the rate of recycling of internalized ligands was about 4 times higher in EC than in PC. As the length of the internalization pulse was increased, the extent of subsequent retroendocytosis decreased, indicating that retroendocytosis takes place from a relatively early stage in the endocytic pathway. Furthermore, as the degree of carbohydrate substitution of the neoglycoprotein ligands increased, the affinities of the receptors for the ligands and the extent of ligand retroendocytosis increased. In the EC, the relationship between degree of substitution and extent of retroendocytosis was not immediately apparent, as some of the neoglycoprotein ligands used may also bind to and be internalized by scavenger receptors on the EC, causing a decreased apparent retroendocytosis. However, when this interaction was inhibited, this relationship was restored. We conclude that retroendocytosis mainly occurs because of incomplete dissociation of ligands from receptors before receptor recycling to the cell surface and that the affinities of a receptor for its ligand at the cell surface and in the endosomal environment are major factors in determining the extent of retroendocytosis.