Plasma clearance and net uptake of alpha-tocopherol and low-density lipoprotein by tissues in WHHL and control rabbits. The mechanism(s) of uptake of vitamin E (alpha-tocopherol) by tissues is poorly understood. It has, however, been suggested from studies in vitro that the apolipoprotein B/E (apo B/E) receptor pathway for low-density lipoprotein (LDL) may be involved. To investigate the role of the apo B/E receptor pathway in vivo, we have studied the transport and uptake of alpha-tocopherol by tissues in Watanabe Heritable Hyperlipidaemic (WHHL) rabbits, which lack functional LDL (apo B/E) receptors, and controls. [3H]alpha-Tocopherol incorporated within LDL labelled with [14C]sucrose was used in these studies, as this enabled the uptake of both alpha-tocopherol and LDL to be studied independently. The principal findings were as follows. (1) Concentrations of the circulating lipids (including alpha-tocopherol) and LDL were increased and the plasma fractional disappearance rates of alpha-tocopherol and LDL decreased in the WHHL rabbits. (2) The WHHL rabbits clear more LDL and alpha-tocopherol from the circulation than controls do, because of their increased pool sizes of alpha-tocopherol and LDL. (3) The lipoprotein composition of the WHHL rabbits differed from that of the controls, and there was exchange of alpha-tocopherol between the lipoprotein fractions in vivo and in vitro. (4) High-affinity apo B/E receptors were not essential for the uptake of alpha-tocopherol by tissues. (5) Evidence from the plasma-clearance and tissue data suggest that alpha-tocopherol can be taken up by tissues in association with, and also independent of, LDL. We conclude that there are several different mechanisms for the uptake of alpha-tocopherol by tissues, which include receptor-dependent and receptor-independent pathways, independent transport and co-transport of alpha-tocopherol and LDL, and uptake from a number of different lipoproteins.


The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding. Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.


Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers. Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.


Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene. The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.


Steric and electronic requirements for muscarinic receptor-stimulated phosphoinositide turnover in the CNS in a series of arecoline bioisosteres. A series of arecoline derivatives was utilized to assess steric and electronic effects important for activating muscarinic receptors in the CNS. Arecoline derivatives in which the methyl ester moiety was replaced by hexyloxy-1,2,5-oxadiazole (2b), hexyloxythiophene (3b) or hexyloxypyrazine (4b) were compared with the hexyloxy-1,2,5-thiadiazole compound (1b) (Hexyloxy-TZTP), known from previous work to be active as an M1/M3 partial agonist. MNDO calculations showed that the N-S bonds of the alkoxythiadiazole ring were highly polarized with the ability to form H-bonds to the N's. On the other hand, the smaller oxadiazole had lower polarities in the N-O bonds and reduced ability to form H-bonds, the thiophene was of comparable size to the thiadiazole and had large C-S bond polarities without the H-bond capability and the pyrazine had limited ability to form H-bonds. The compounds were compared with respect to their abilities to stimulate phosphoinositide (Pl) turnover in the hippocampus of the rat brain. 1b was more active than 2b-4b for stimulating the Pl turnover response. The data suggest that the ability to form H-bonds is an important factor for the ability of 1 to stimulate M1 muscarinic receptors in the CNS.


Cell-cycle dependent phosphorylation of HSP28 by TGF beta 1 and H2O2 in normal mouse osteoblastic cells (MC3T3-E1), but not in their ras-transformants. Transforming growth factor (TGF) beta 1 increased phosphorylation of a specific protein of approximately M(r) = 30,000 (p30) in mouse osteoblastic MC3T3-E1 cells. This protein, p30, was identified as one of the small heat shock proteins (HSP) 28 from the electrophoretic pattern on two-dimensional gels, and its peptide map compared with that of heat shock-inducible p28. The increase in phosphorylation of HSP 28 seemed to correlate with growth inhibition in this cell line, since it was increased by growth inhibitory agents, such as TGF beta 1, H2O2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), but not by the growth stimulating agent, epidermal growth factor (EGF), and this phosphorylation was observed only when the cells were sensitive to growth inhibition by these agents, in the late G1 phase of the cell cycle. Furthermore, in ras-transformants, whose DNA synthesis was not inhibited by these agents, this phosphorylation was not increased by these stimuli. These results indicate that phosphorylation of HSP 28 may be coupled to inhibition of DNA synthesis in this cell line.


Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury. The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.


Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase. Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A. cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer. The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra. These results indicate that an earlier report that the A. cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO2- is reduced. Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.


Two different signal transductions of neuropeptide Y1 receptor in SK-N-MC cells. The signal transduction systems of the neuropeptide Y (NPY) Y1 receptor were studied in SK-N-MC human neuroblastoma cells. NPY induced an increase in intracellular calcium ion concentration ([Ca2+]i) and inhibition of forskolin-stimulated cyclic AMP accumulation, which were mediated through Y1 receptors. One-min preincubation of cells with phorbol 12-myristate 13-acetate (PMA) inhibited both signal transductions dose-dependently, but its effect on [Ca2+]i was about 100-fold more potent than that on cyclic AMP. PMA had no effect on [125I]BH-NPY binding in SK-N-MC cells and hardly inhibited the endothelin-1-induced increase in [Ca2+]i. Pertussis toxin also inhibited the NPY-induced [Ca2+]i increase 30-fold more effectively than the NPY-mediated inhibition of cyclic AMP accumulation. These results indicate that Y1 receptors in SK-N-MC cells couple to two signal transduction systems that have different sensitivities to phorbol ester and pertussis toxin treatments.


Expression of recombinant myeloperoxidase using a baculovirus expression system. Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.


Organ distribution and characterization of porcine peptides (VIP, CGRP and PHI) that increase cAMP in rat platelets. Using an assay for rat platelet cAMP, we investigated the organ distribution of peptides that increase cAMP in rat platelets in porcine tissues. Marked activity was observed in the duodenum, pancreas and brain. By analysis with reverse phase high performance liquid chromatography (HPLC), three major peaks of activity were observed in porcine tissues. The first peak was vasoactive intestinal polypeptide (VIP), and the second peak was calcitonin gene-related peptide (CGRP). The third peak of activity was isolated from porcine duodenum. By analysis with a gas phase sequencer and with an amino acid analyzer, this peptide was identified as peptide histidine isoleucine (PHI). In a glucagon-secretin family of neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) significantly increased platelet cAMP levels in a dose-dependent manner; however, glucagon did not. These results suggest that not only VIP and CGRP but also PHI and PACAP act upon platelets, as well as vascular tissues.


Respiratory chain enzymes in muscle of endurance athletes: effect of L-carnitine. The effects of L-carnitine on respiratory chain enzymes in muscle of long distance runners were studied in 14 athletes. These subjects received placebo or L-carnitine (2 g orally b.i.d.) during a 4-week period of training. Athletes receiving L-carnitine showed a significant increase (p < 0.01) in the activities of rotenone-sensitive NADH cytochrome c reductase, succinate cytochrome c reductase and cytochrome oxidase. In contrast, succinate dehydrogenase and citrate synthase were unchanged. No significant changes were observed after placebo administration. The levels of both total and free carnitine from athletes receiving placebo were significantly decreased (p < 0.01) after treatment. By contrast, total and free carnitine levels were markedly increased (p < 0.01) after supplementation with L-carnitine. Our results suggest that L-carnitine induces an increase of the respiratory chain enzyme activities in muscle, probably by mechanisms involving mitochondrial DNA.


Activation of transforming G protein-coupled receptors induces rapid tyrosine phosphorylation of cellular proteins, including p125FAK and the p130 v-src substrate. We have used the family of human muscarinic receptors (mAChRs) as a model for receptors coupled to G proteins and have shown that genes for certain mAChR subtypes can behave as potent agonist-dependent oncogenes. Furthermore, transforming mAChRs can transduce mitogenic signals in transfected NIH 3T3 cells. In this study, we show that in cells expressing ml mAChRs, the cholinergic agonist carbachol, induces a rapid and dose-dependent increase in tyrosine phosphorylation of cellular proteins which are different from those induced by PDGF. Interestingly, carbachol, but not PDGF, induces an increase in tyrosine phosphorylation of the p125FAK and p130 v-src substrates. Thus, growth promoting pathways activated by receptors coupled to G proteins might involve tyrosine phosphorylation of a small set of cellular proteins previously identified as substrates for oncogene-encoded tyrosine kinases.


Electron transport between cytochrome c and alpha tocopherol. Using liposomes we have demonstrated an electron transfer between tocopherol (vitamin E) and cytochrome c. Reduced cytochrome c protects vitamin E from oxidation induced either directly by ultraviolet light or indirectly by soybean lipoxygenase-catalyzed oxidation of arachidonic acid. Oxidized cytochrome c is reduced by tocopherol and tocopherol homologues (chromanols) resulting in accumulation of tocopheroxyl radicals which we detected by ESR. The peak height of the ESR spectrum of tocopheroxyl radicals (which is proportional to the amount of radical present) is proportional to the ratio of reduced to oxidized cytochrome c. In mitochondrial membranes succinate-cytochrome c reduction is inhibited by antimycin A. Addition of exogenous chromanols facilitates a by-pass of the antimycin A blocked electron pathway, and succinate-dependent cytochrome c reductase activity is restored. Cytochrome c may act as a water-soluble complement to the lipid-soluble ubiquinol in regenerating mitochondrial tocopherol from tocopheroxyl radical.


Diacylglycerol rather than Ca2+ mediates GnRH inhibition of FSH induced steroidogenesis in ovarian granulosa cells. Treatment of cultured granulosa cells with PLC or GnRH stimulated the rapid generation of DAG and phosphoinositide turnover. The PKC activators PLC (3 mU/ml) and TPA (10(-7)M) or the decapeptide GnRH (10(-6)M) elicited similar inhibitory responses on FSH or cAMP stimulated granulosa cell steroidogenesis. Mobilization of intracellular Ca2+ with A23187 (10(-8)M) was followed by a slight increase in the steroidogenic activity of cultured granulosa cells, whereas elevation of extracellular K+ (50 mM) largely augmented the steroid biosynthetic activity of the granulosa cells. These results suggest that the inhibitory effect of GnRH on granulosa cell steroidogenesis is mediated by generation of DAG, rather than by increases in intracellular Ca2+ concentrations.


Binding of mutant insulins to a mutated insulin receptor. We studied the binding of mutant insulins to both the normal human insulin receptor and an insulin receptor in which the sequence 240-250 of the receptor alpha subunit was mutated to provide an additional net positive charge. One mutant insulin (AspB10), which has an additional negative charge, bound to both types of receptors with a higher affinity than native insulin. Moreover, this mutant insulin was more effective in activating the tyrosine kinase activity of both types of receptors. This study suggests, therefore, that charge interactions between insulin and its receptor may play a role in insulin receptor binding and action.


Hepatitis B virus DNA integration and expression of an erb B-like gene in human hepatocellular carcinoma. Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.


Camptothecin analog (CPT-11)-sensitive human pancreatic tumor cell line QGP-1N shows resistance to SN-38, an active metabolite of CPT-11. In the course of our study to determine the cross-sensitivity between CPT-11 and its active metabolite, SN-38, we found a SN-38-resistant human pancreatic tumor cell line, QGP-1N, which shows sensitivity to CPT-11. The IC50 of SN-38 was 152 times greater for QGP-1N than for SUIT-2, also a human pancreatic tumor cell line, whose IC50 of CPT-11 was similar to that for QGP-1N. The uptakes of CPT-11 and SN-38 and the intracellular conversion of CPT-11 to SN-38 could not explain the difference in sensitivity. DNA synthesis of QGP-1N cells was inhibited by CPT-11 which did not affect that of SUIT-2, while SN-38 inhibited the DNA synthesis of SUIT-2 at lower concentrations than that of QGP-1N. The inhibition test of topoisomerase I catalytic activity by CPT-11 or SN-38 revealed no difference in the biochemical properties of the topoisomerase I enzymes to the compounds between these two cell lines. These results indicate that CPT-11 should have its own inhibitory effect on DNA synthesis through a yet unknown mechanism in QGP-1N cells, although SN-38 plays an essential role in the antitumor activity of CPT-11 in SUIT-2 cells. In some cases, the antitumor effect of CPT-11 might be consequent not only on SN-38 but also on CPT-11 itself.


An assessment of human insulin receptor phosphorylation and exogenous kinase activity following deletion of 69 residues from the carboxyl-terminus of the receptor beta-subunit. A mutant human insulin receptor with a carboxyl-terminal deletion of 69 amino acids (proreceptor residues 1287-1355) is expressed as a stable protein in transiently transfected COS cells. We find that in intact cells this mutant is phosphorylated in an insulin-dependent manner on core tyrosines 1158, 1163 and 1163. As expected, the carboxyl-terminal beta-subunit phosphorylation sites (serines 1305/6, tyrosines 1328/34 and threonine 1348) are absent from this mutant. However, the two major insulin-stimulated serine phosphopeptides remain. In intact cells, insulin stimulates exogenous substrate phosphorylation by the truncated receptor only approximately 1.9-fold (cf. approximately 9-fold for the wild-type receptor in these cells), a consequence of a approximately 4.8-fold elevation in basal insulin-independent kinase activity.


Neurotoxin-binding activity in the supernatant fraction of the electric organ from Torpedo californica. We found neurotoxin-binding activities in the supernatant fraction obtained by ultracentrifugation of a homogenate of the electric organ dissected from the electric ray, Torpedo californica. While about half of the activity was estimated as due to acetylcholine receptors in dispersed microparticles, the remainder was unassigned. A part of the latter, detected with alpha-bungarotoxin, eluted ahead of the alpha-bungarotoxin-acetylcholine receptor complex on a Sepharose CL-6B column in the presence of 1% Triton X-100. Another component eluted after this complex. Although these activities were immunologically related to AChR, they were different from AChR in their size and reactivity with Concanavalin A. We are currently seeking to characterize these toxin-binding components at present. The existence of such activities is interesting since they may possibly function in regulating signal transduction at the neuromuscular junction.


Antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from bovine brain inhibit the holoenzyme formation. Mouse monoclonal antibodies to the bovine brain regulatory subunit type II (RII) were produced and antibodies of 11 clones were characterized. We determined their subclass, affinity constants, competivity and influence on two RII functions, namely cAMP binding and inhibition of the catalytic subunit (C) activity. 4 clones produced antibodies that increased RII-cAMP binding 1,5-2-fold. Antibodies of other 3 clones prevented R-C association and inhibition of C phosphotransferase activity. Some antibodies affected neither of these RII functions.


Effects of calcium on Na+,K(+)-ATPase isolated from human placenta. It has been reported that Ca2+ can be either stimulatory or inhibitory of the activity of Na+,K(+)-ATPase obtained from different sources. The aim of the present work was to study the effect of increasing concentrations of Ca2+ on Na+,K(+)-ATPase activity and of placenta. The temperature dependence of Na+,K(+)-ATPase activity and of spectral parameters of a spin label paramagnetic maleimide) were also (MSL, 2,2,6,6-tetramethylpiperidin-1-oxyl-4-maleimide) were also investigated in absence and in presence of 2 mM Ca2+. Ca2+ affects positively the enzymatic activity and this effect is evident at all temperature tested. Maleimide spectral parameters are not affected by the presence of 2 mM Ca2+, showing that the microenvironments of SH groups are not modified following ion interactions.


Rat serum fructose 1,6 bisphosphatase: modifications in different experimental conditions. Fructose 1,6 bisphosphatase activity was measured in rat serum. The activity of the enzyme presented the following characteristics: pH optimum alkaline, between 8-8.5 and inhibited by AMP. The activity was measured in different experimental situations, such as streptozotocin diabetes, hepatocellular injury produced by carbon tetrachloride (CCl4), and bile-duct ligation. In diabetic rat, serum activity increased 2-fold with respect to the control values. In animals treated with CCl4 the activity increased 10-14 fold. On the contrary bile duct ligation decreased activity according to the cholestasis time. The results obtained in this study show that fructose 1,6 bisphosphatase is an enzyme measurable in serum, which changes according to different situations of liver cell injury.


Role of the adenylate cyclase, phosphoinositidase C and receptor tyrosyl kinase systems in the control of hepatocyte proliferation by hepatocyte growth factor. Hepatocyte growth factor (HGF) is the most potent known mitogen for hepatocytes in primary culture. However, the mechanisms through which HGF induces hepatocyte proliferation have not been defined. Here we have investigated the role of the adenylate cyclase, phosphoinositidase C and tyrosine kinase signalling systems in the control of hepatocyte proliferation by HGF using freshly isolated or cultured adult rat hepatocytes. We show that human recombinant HGF caused a dose-dependent increase in hepatocyte DNA synthesis with a maximal effect at 10 ng/mL and an EC50 of 5.9 ng/mL. HGF had no effect on hepatocyte adenylate cyclase activity or intracellular cAMP levels. Elevation of hepatocyte cAMP levels resulted in inhibition of HGF-stimulated DNA synthesis. HGF stimulated inositol phospholipid hydrolysis with a maximal effect at 25 ng/mL and potentiated the effect of vasopressin (10(-8) and 10(-9)M). HGF (100 ng/mL) caused an increase in the phosphorylation on tyrosine of an unknown hepatocyte protein with a molecular mass of 36 kDa. Thus, we have shown that HGF, like epidermal growth factor (EGF), can activate the phosphoinositidase C and tyrosine kinase systems in rat hepatocytes. As with EGF, these intracellular signalling systems may underlie HGF-induced hepatocyte proliferation.


Central effects of the preservative, methylparaben. In vivo activation of cAMP-specific phosphodiesterase and reduction of cortical cAMP. The phenolic preservative, methylparaben (MPB), has in the past been demonstrated to harbour definite pharmacological effects. In an attempt to examine the possible central effects of MPB, notably on cyclic nucleotides and cyclic nucleotide phosphodiesterase (PDE; EC 3.1.4.17), rats were orally treated with the drug (0.4% in rat food) for 3 weeks with cortex extracts being used for the various determinations. Three isozymes were identified by DEAE-cellulose anion exchange chromatography, namely the calmodulin/calcium-stimulated form or PDE I (peak I), the cGMP-stimulated form or PDE II (peak II), and an independent form not affected by either calmodulin or cGMP also known as PDE IV (peak III). The presence of MPB induced a significant decrease in cortical cAMP, as well as strongly stimulating the activity of PDE IV (peak III). In addition, a small, yet significant, increase in cGMP levels was observed. Since no increase in cGMP hydrolysis was observed, we conclude that chronic ingestion of MPB induces a preference for cAMP hydrolysis, which was confirmed by the increase in PDE IV (peak III) activity. PDE IV is a membrane-bound, low Km PDE exhibiting high selectivity for cAMP hydrolysis. While there was an increase in cGMP, we failed to observe an increase in the activity of the cGMP-stimulated PDE (PDE II). These data are discussed with reference to the possible membrane effects of MPB allowing it to alter both the kinetic properties of PDE IV with the resultant effects on cAMP, as well as a means whereby it may activate guanyl cyclase and increase cGMP.


Evaluation of central adrenergic receptor signal transmissions after an antidepressant administration to the rat. The effects of several antidepressants, amitriptyline, citalopram, desipramine, fluoxetine, maprotiline, mianserin, nialamide, nomifensine, tranylcypromine and viloxazine, on the accumulation of cyclic AMP and inositol monophosphates were studied in rat cerebral cortical slices. The two enzymatic systems were stimulated either by adrenergic agonists or by forskolin. Cyclic AMP and inositol monophosphates (IPs) formed were determined by a double label method. In vitro all drugs, except inhibitors of monoamine oxidase, nialamide and tranylcypromine, inhibited alpha 1-agonist-mediated production but did not modify the cyclic AMP accumulation. Otherwise, chronic desipramine but not citalopram administration decreased the accumulation of cyclic AMP (-39%) elicited by beta-adrenoceptor agonists; no change was observed in inositol phosphate metabolism after administration of these two drugs. These data support previous investigations showing a decrease in cyclic AMP production after chronic treatment with norepinephrine uptake blockers but do not confirm the hypothesis of a modification of alpha 1-adrenoceptor-stimulated inositol phosphate metabolism.


The effect of gammalinolenic acid on the subfractions of plasma high density lipoprotein of the rabbit. The effect of dietary supplementation with evening primrose oil (containing 70% gammalinolenic acid) on the concentration of plasma lipids and lipoproteins of the New Zealand White rabbit was investigated. No significant changes were observed in the concentrations of plasma cholesterol or triglycerides during the treatment, although an increase in high density lipoprotein (HDL) cholesterol (P < 0.01) was observed at 4 weeks of evening primrose oil intake and 2 weeks after withdrawal. However, when HDL subpopulations were resolved by gradient gel electrophoresis, major alterations were observed in the distribution of HDL subfractions. These included an increase in HDL2b (P < 0.001) and HDL3c (P < 0.001) and the appearance of very large particles of HDL. These findings suggest that supplementation of diets with n-6 fatty acids may be effective in the long-term prevention of atherosclerosis.


Arylamine N-acetyltransferase in human red blood cells. N-Acetyltransferase activities associated with erythrocytes from 20 individuals have been determined with p-aminobenzoic acid as substrate. A three-fold variation in Vmax is found. The N-acetyltransferase genotype of the individuals has been determined and there is no correlation between the extent of acetylation measured in the individuals' erythrocytes and the inheritance of alleles at the polymorphic NAT locus. Folate is confirmed to be an inhibitor of arylamine N-acetyltransferase activity measured in erythrocytes. The content of folate in erythrocytes of individuals also varies. The individual with the maximum folate content has the minimum N-acetyltransferase activity. The monomorphic N-acetyltransferase gene from individuals spanning the range of N-acetyltransferase activity have been amplified, using the polymerase chain reaction. The pattern of restriction enzyme digestion of the monomorphic N-acetyltransferase gene with a series of eight restriction enzymes is the same for individuals spanning the activity range of arylamine N-acetyltransferase in their erythrocytes.


Histamine-induced differentiation of HL-60 cells. The role of cAMP and protein kinase A. When HL-60 cells were stimulated with histamine, a significant differentiation of the cells toward neutrophils was elicited. Histamine increased phagocytic activity, but it reduced myeloperoxidase activity of HL-60 cells. Histamine-induced differentiation in HL-60 cells was inhibited not only by H2 antagonists, such as cimetidine, ranitidine and famotidine, but also by an inhibitor of protein kinase A (A kinase), KT-5720. Histamine increased the cAMP level and A kinase activity in HL-60 cells; both increases preceded the cell differentiation. Histamine also enhanced phosphorylation of a 160 kD protein in HL-60 cells, while H2 antagonists and KT-5720 inhibited this phosphorylation. The results of the present study indicate that an activation of A kinase via H2 receptor stimulation may cause the phosphorylation of a 160 kD protein and that this phosphorylation is probably involved in the process leading to differentiation of HL-60 cells.


Hepatic protoporphyria is associated with a decrease in ligand binding for the mitochondrial benzodiazepine receptors in the liver. Protoporphyrin IX (PP) and N-methylprotoporphyrin IX (N-MePP) added in vitro to liver membranes reduced dose-dependently the affinity of [3H]PK 11195 for the mitochondrial benzodiazepine receptors (MBRs), the latter being about 20 times more potent (Ki 4.5 and 0.25 microM). Preincubation of these two porphyrins with liver homogenates for 120 min at 4 degrees resulted in significant inhibition of [3H]PK 11195 binding even after repeated washings of the membranes due to the residual presence in the membranes of about 35 and 5% of PP and N-MePP, respectively. Thus, the hypothesis that an in vivo increase in the hepatic porphyrin content modifies the binding of the isoquinoline PK 11195 to the MBRs was investigated in an experimental model of protoporphyria. PP and N-MePP were allowed to accumulate in vivo through treatment with 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) (100 mg/kg i.p., once), and rats were killed 5 h after treatment when hepatic porphyrin accumulation was marked (10-fold increase), PP predominating. In the liver, treatment reduced the affinity (Kd) of [3H]PK 11195 for MBRs (from 3.56 to 15.37 nM, P < 0.01) and the maximum number of binding sites (Bmax) (55% decrease, P < 0.05); the affinity (Ki) of RO 5-4864 for [3H]PK 11195 binding sites was also reduced (from 23.9 to 72.99 nM, P < 0.05). No significant differences were found in the brain cortex. Liver and brain diazepam binding inhibitor levels and plasma corticosterone levels were unchanged. The reduction in [3H]PK 11195 binding to MBRs in the liver of DDC-treated rats thus appears to be attributable to a specific effect of the DDC-induced formation of the two protoporphyrins; this conclusion suggests that in hepatic protoporphyria processes modulated by MBRs may be altered.


Different 1,4-dihydropyridines exhibit discriminating effects on passive calcium uptake in rat liver plasma membrane vesicles. The effects of a number of calcium channel effectors on Ca2+ uptake by rat liver plasma membrane vesicles was examined. Nifedipine, verapamil and diltiazem had to be present at 1 mM in order to produce > 50% inhibition of Ca2+ uptake. The two structurally similar 1,4-dihydropyridines, nicardipine and nisoldipine exhibited opposite effects; nicardipine inhibited while nisoldipine stimulated Ca2+ uptake. The results show that low concentrations (microM) of calcium channel blockers of excitable cells have little effect on Ca2+ uptake by liver plasma membrane vesicles consistent with earlier findings of others that voltage-gated calcium channels are absent in hepatocytes. However, the opposite effects of higher concentrations (ca. 1 mM) of nicardipine and nisoldipine on Ca2+ uptake suggest a discriminatory action that might be useful in studying further the mechanism of passive Ca2+ uptake by these membrane vesicles.


In vivo occupancy of histamine H3 receptors by thioperamide and (R)-alpha-methylhistamine measured using histamine turnover and an ex vivo labeling technique. In the brain, the H3 type of histamine receptor has a pre-synaptic autoreceptor inhibitory role which regulates neuronal release and synthesis of histamine. To examine the interaction of the selective H3 receptor antagonist thioperamide with H3 receptors in the brain in vivo, we have used a functional and non-functional measurement of H3 receptor occupancy. In three species (rat, guinea-pig and mouse) peripheral administration of thioperamide caused dose-related increases in histamine turnover in the cerebral cortex (whole brain was examined in the mouse) and, in the same tissues, inhibited the ex vivo binding of the selective H3 receptor agonist [3H](R)-alpha-methylhistamine ([3H]-RAMH). The peak effect of thioperamide to inhibit ex vivo binding of [3H]RAMH was observed approximately 30 min after i.p. administration, whilst the maximum increase in histamine turnover did not occur until after at least 100 min. At a pretreatment time of 30 min, the ED50 of thioperamide to inhibit ex vivo binding of [3H]RAMH binding in the rat, guinea-pig and mouse brain was found to be 2.0 +/- 0.2, 4.8 +/- 0.6 and 2.6 +/- 0.3 mg/kg (mean +/- SEM, N = 4), respectively. We have also examined the effect of peripheral administration of RAMH on ex vivo binding of [3H]RAMH in rat cortex. Qualitatively and quantitatively similar results to those of thioperamide were observed following i.p. administration of RAMH to rats (ED50 = 3.9 +/- 0.4 mg/kg, mean +/- SEM, N = 4). An effect of RAMH on histamine turnover in rat cortex could not be determined as this compound displayed significant cross-reactivity with the antibodies used in the radioimmunoassay to measure histamine and telemethylhistamine. These data indicate that, following peripheral administration, both thioperamide and RAMH penetrate the brain where they can subsequently interact with H3 receptors. It would appear that binding of thioperamide to H3 receptors is linked with a concomitant increase in histamine turnover in the brain. In conclusion, the ex vivo binding technique, particularly when coupled with measurement of histamine turnover, should provide a valuable means for investigating the ability of any peripherally administered compound to cross the blood-brain barrier and subsequently interact with histamine H3 receptors.


Temperature and anion dependence of allosteric interactions at the gamma-aminobutyric acid-benzodiazepine receptor. The temperature dependence of [3H]flunitrazepam ([3H]FNZ) binding to rat brain membranes was examined in the presence of the anaesthetics, pentobarbitone, alphaxalone and propofol. Van't Hoff plots showed the binding of FNZ to be largely enthalpy driven. Alphaxalone and propofol increased the entropy of the binding reaction but not the enthalpy and therefore did not show temperature dependence in their efficacy. In contrast, pentobarbitone increased the enthalpy of FNZ binding and, therefore, is more efficacious at low temperatures. The EC50 values of all three modulators increased with temperature indicating that their interactions with the receptor may be enthalpy driven. The EC50 values of all three modulators were also anion dependent, showing a decrease in the presence of gamma-aminobutyric acid (GABAA)-channel permeant anions. The efficacies of alphaxalone and pentobarbitone, but not that of propofol, also increased with increasing chloride ion concentration. The results indicate that all three modulators interact with the GABAA receptor at distinct recognition sites.


Chronic ethanol consumption and withdrawal affects mitochondrial benzodiazepine receptors in rat brain and peripheral organs. Ethanol administration to rats for 30 days resulted in a significant decrease (-28%; P < 0.05) in the density of mitochondrial benzodiazepine receptors (MBR) in the olfactory bulb. The reduction in [3H]PK 11195 binding persisted 24 hr after cessation of alcohol and had returned to normal values when measured 4 days later. Alterations were confined to this brain region and were not detected in the cerebral cortex, cerebellum or hippocampus. [3H]PK 11195 binding was elevated in the liver (100%; P < 0.01), heart (43%; P < 0.01) and testis (27%; P < 0.05) 30 days following ethanol consumption and this persisted for 1 and 4 days after abrupt withdrawal. A transitory decrease (-20%; P < 0.05) in MBR density was observed in the adrenal gland following 30 days of alcohol ingestion, but disappeared during withdrawal. The alterations in these receptors may be relevant to the cellular damage or dysfunction induced by chronic exposure to ethanol.


Heterogeneity of binding sites for ICI 198,615 in human lung parenchyma. We have identified and characterized two different subclasses of binding site for the novel peptido-leukotriene (LT) antagonist, [3H]ICI 198,615, in membranes from human lung parenchyma using a receptor-ligand assay. This novel compound is representative of a new class of LT receptor antagonists and it has been demonstrated to be several orders of magnitude more potent and selective than most other LT antagonists described to date. The binding of [3H]ICI 198,615 is rapid, specific and saturable. Equilibrium was reached within 5-10 min. Non linear fitting of dissociation time courses has revealed the presence of two different components (K(off)1 = 8.3 +/- 6.8 x 10(-4) sec-1 and K(off)2 = 0.79 +/- 1.66 x 10(-3) sec-1) of the kinetic curves, suggesting heterogeneity of the binding sites. Computer analysis of equilibrium binding data obtained at 25 degrees results in a model with two classes of binding sites, a high affinity-low capacity class with Kd1 = 0.024 +/- 0.014 nM and Bmax1 = 0.015 +/- 0.004 pmol/mg protein and a low affinity-high capacity class with Kd2 = 6326 +/- 3859 nM and Bmax2 = 473 +/- 383 pmol/mg protein. In competition studies, LTD4 was also found to interact with two classes of binding site (Kd1 = 0.016 +/- 0.008 nM and Kd2 = 15195 +/- 8965 nM). On the contrary, LTE4 and LTC4 were found to interact with a homogeneous class of sites only with Kd = 7466 +/- 4629 nM and Kd = 428 +/- 73 nM, respectively. Furthermore, we have evaluated the effect of a number of LT antagonists on the binding of [3H]ICI 198,615. Ro 24-5913 (Kd = 3.0 +/- 2.1 nM), FPL55712 (Kd = 4945 +/- 2868 nM), LY171883 (Kd = 19628 +/- 12365 nM), SKF 104353 (Kd = 74.2 +/- 46 nM) and its enantiomer SKF 104373 (Kd = 13627 +/- 6813 nM) were found to interact with a single class of binding sites. The present studies indicate a heterogeneity of binding sites for ICI 198,615 in membranes from human lung parenchyma and that ICI 198,615 is a very potent and selective antagonist of LTD4 receptors in this tissue.


Inhibition of sodium pump by bepridil. An in vitro and microcalorimetric study. The effects of diltiazem, verapamil, bepridil, nicardipine and nifedipine were studied in vitro on Na+,K(+)-ATPase from dog kidney (EC 3.6.1.37). Except diltiazem, all the drugs tested showed an inhibitory effect on Na+,K(+)-ATPase activity in a dose-dependent manner. Among these drugs bepridil is far more effective than the others (IC50 approximately 10(-4) M). Competition studies showed that bepridil acted in a non-competitive manner with the ATP-Mg2+ complex and in a partially competitive manner with K+. Since ouabain acted similarly under the same experimental conditions, we tested the interaction of bepridil and ouabain on Na+,K(+)-ATPase. With low doses of ouabain, the enzyme inhibition corresponded to a potentiated synergy of the two drugs. We then studied the action of bepridil on the sodium pump activity of intact red blood cells by an ex vivo microcalorimetric technique. At 10(-5) M bepridil caused a significant decrease in sodium pump activity (33 +/- 8%).


Effect of sphingosine and its N-methyl derivatives on oxidative burst, phagokinetic activity, and trans-endothelial migration of human neutrophils. Neutrophils display three major functions: (i) oxidative burst, (ii) phagokinetic activity, and (iii) trans-endothelial migration. Sphingosine (SPN) is known to inhibit oxidative burst in human neutrophils via inhibition of protein kinase C (PKC). SPN is metabolically converted into N,N-dimethylsphingosine (DMS) in some tissues and cell lines. In previous studies, we have demonstrated that the PKC-inhibitory effect of DMS is stronger than that of SPN, and that of the synthetic analogue N,N,N-trimethylsphingosine (TMS) is even stronger. Therefore, in the present study, we compared the effects of SPN, DMS, and TMS on the neutrophil functions mentioned above. These three compounds, at 10-20 microM, showed equal inhibition of phorbol 12-myristate 13-acetate (PMA)-dependent superoxide (O2-) production and O2 consumption. They and other known PKC inhibitors (H-7, staurosporine, calphostin C), at 1-5 microM, showed equal inhibition of the phagokinetic activity of neutrophils. On the other hand, trans-endothelial migration of neutrophils was suppressed by SPN, DMS, and TMS at 5-10 microM, but was relatively unaffected by the other PKC inhibitors. All of these compounds inhibited PMA-induced phosphorylation of major neutrophil proteins with a M(r) of 60 and 47 kDa; this effect is ascribable to inhibition of PKC. Despite the similar effects of SPN, DMS, and TMS on neutrophil function, TMS was considerably less cytotoxic to neutrophils under the same experimental conditions. Furthermore, SPN and DMS at 10-20 microM caused obvious morphological changes of the endothelial cells, but TMS did not. SPN undergoes rapid metabolic conversion to various sphingolipid compounds, but TMS is stable. In view of all these findings, TMS appears to be a superior pharmacological agent, compared to SPN derivatives or other PKC inhibitors, for suppression of neutrophil overfunction associated with inflammatory processes and tissue injury.


Inhibition of glutathione reductase by flavonoids. A structure-activity study. A structure-activity study of fourteen chemically related flavonoids was conducted to evaluate their abilities to inhibit glutathione reductase (GR). By comparing the I50 values of flavonoids from different classes possessing an identical hydroxyl configuration, we determined the following order of potency for inhibition of GR: anthocyanidin > dihydroflavonol = chalcone > flavonol > catechin. Enzyme inhibition by delphinidin chloride and myricetin was partially prevented in a N2 atmosphere which implicates a role for oxygen in the mechanism of inhibition. To determine the role of oxygen species in enzyme inhibition, GR was preincubated with either mannitol, diethylenetriaminepenta-acetic acid (DETAPAC), superoxide dismutase (SOD), catalase (CAT), or SOD and CAT prior to assays for enzyme inhibition by flavonoids. Enzyme inhibition by delphinidin chloride and myricetin was suppressed by the addition of SOD, suggesting that superoxide (O2-.) is involved. However, inhibition by quercetin and morin was not sensitive to antioxidants. To further investigate the role of O2-. in GR inhibition, a superoxide generating system was utilized in the presence and absence of flavonoid. The O2-. generating system failed to inhibit GR in the absence of flavonoid but enhanced the inhibition by myricetin, indicating that the O2-. did not directly inhibit GR but reacted directly with certain flavonoids to form a reactive intermediate which, in turn, inhibited GR. These findings suggest that the mechanism of inhibition of GR by flavonoids is complex and may have oxygen-dependent and oxygen-independent components.


[Identification of a segment of the gene for the substance B receptor in the human DNA genome using the polymerase chain reaction]. Polymerase chain reaction was applied to human genomic DNA using primers corresponding to the rat substance P receptor cDNA. As a result, a fragment of 94 b.p. was isolated identical to the fragment 771-864 of the above-mentioned cDNA, with the exception of the G796----A substitution (Val----Ile in the amino acid sequence). A comparison of the established sequence with the published structures of tachykinin receptors of NK-1, NK-2 and NK-3 types allows its assignment to the substance P receptor (NK-1 tachykinin receptor) gene detected in the human genome.


[The structure of carbohydrate chains of human IgG4-paraproteins differing in the ability to bind cell receptors]. Comparative oligosaccharide analysis by HPLC revealed structural differences in the carbohydrate chains of human IgG4 paraproteins, varying in ability to induce the rhesus monkey's passive skin anaphylaxis. An atypical IgG4 paraprotein, which is inactive in this reaction and also does not bind the IgG4-subclass specific monoclonal antibody IH2, has a much higher proportion of the carbohydrate chains lacking terminal galactose residues than two typical IgG4 paraproteins. This structural feature may be one of the reasons for the atypical IgG4 not to bind by the mast cell Fc gamma receptor.


Reduction of the severity of canine osteoarthritis by prophylactic treatment with oral doxycycline. In vitro studies have indicated that levels of neutral metalloproteinases in osteoarthritic (OA) cartilage are elevated and that doxycycline (doxy) inhibits collagenolytic and gelatinolytic activity in extracts of OA cartilage. The purpose of the present study was to test the effect of oral doxy administration on the severity of cartilage degeneration in OA. OA was induced in 12 adult mongrel dogs by transection of the anterior cruciate ligament (ACL) 2 weeks after dorsal root ganglionectomy. Six dogs received doxy orally from the day after ACL transection until they were killed 8 weeks later; the other 6 served as untreated OA controls. The unstable knee of each untreated dog exhibited extensive full-thickness cartilage ulceration of the medial femoral condyle. In sharp contrast, cartilage on the distal aspect of the femoral condyle of the unstable knee was grossly normal in 2 doxy-treated dogs, and exhibited only thinning and/or surface irregularity in the others. Degenerative cartilage lesions on the medial trochlear ridge, superficial fibrillation of the medial tibial plateau, and osteophytosis were, however, unaffected by doxy treatment. Collagenolytic activity and gelatinolytic activity in cartilage extracts from OA knees of untreated dogs were 5-fold and 4-fold greater, respectively, than in extracts from dogs given doxy. Prophylactic administration of doxy markedly reduced the severity of OA in weight-bearing regions of the medial femoral condyle. It remains to be determined whether administration of doxy after OA changes have developed is also effective.


Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases. Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis. We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF) samples from patients with rheumatic diseases and compared their levels with levels of soluble interleukin-2 receptors (sIL-2R). Sensitive enzyme-linked immunosorbent assays (ELISA), specific for p55 and p75 sTNF-R and for sIL-2R, were used. Serum levels of p75 sTNF-R were 3-4-fold higher than levels of p55 sTNF-R, and both were significantly elevated in patients with osteoarthritis (OA) and rheumatoid arthritis (RA) compared with healthy controls. RA SF levels of sTNF-R were 4-5-fold higher than levels in serum, suggesting local production in the joint, and were significantly higher than levels in the SF of patients with seronegative arthropathy or OA. Furthermore, levels of p55 and p75 sTNF-R, but not sIL-2R or TNF alpha measured by ELISA, were increased in the SF of patients with clinically active RA. The soluble TNF-R in RA and OA SF were functional since they inhibited TNF activity in a cytotoxicity assay in proportion to the levels of inhibitor present. Evaluation of serially obtained serum samples suggested that sTNF-R may be a useful parameter for monitoring RA disease activity. Biologically active soluble TNF-R are up-regulated in patients with rheumatic disease and are produced locally in the joints. Measurement of serum levels of TNF-R may be useful for monitoring of disease, and determination of SF levels could be of diagnostic value.


Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor alpha. We have previously described the location of tumor necrosis factor alpha (TNF alpha)-producing cells in synovial tissue and cartilage-pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNF alpha, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase-conjugated mouse anti-alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage-pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R-expressing cells were in the vicinity of TNF alpha-containing cells, and some TNF alpha-containing cells also expressed TNF-R. TNF-R-expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNF alpha in this tissue. This study further supports the hypothesis that TNF alpha plays a major role in the pathogenesis of RA.


Light microscopic characterization of the fibroblast-like synovial intimal cell (synoviocyte). To reassess synovial intimal cell populations by light microscopy. Non-inflamed, rheumatoid and osteoarthritic synovia were analyzed as tissue sections and cytospin preparations by a series of combined immunohistochemical and cytochemical staining techniques. Two populations of intimal cells were identified. The first carried macrophage markers. The second showed high uridine diphosphoglucose dehydrogenase (UDPGD) activity, minimal cytoplasmic CD68, absent non-specific esterase (NSE) activity, and absent leukocyte and endothelial antigens. The majority of these cells showed a high content of prolyl hydroxylase. Combined cytochemical staining for NSE and UDPGD activity allows effective separation of intimal cell populations. We suggest that the cells of high UDPGD activity are the fibroblast-like or type B synovial intimal cells defined by electron microscopy. High UDPGD activity probably reflects a preferential ability to synthesize glycosaminoglycans, including hyaluronan.


Effects of converting enzyme inhibitors and the calcium antagonist nifedipine alone and in combination on precontracted isolated rabbit aortic rings. The calcium antagonist nifedipine markedly relaxed intact rabbit aortic rings precontracted with low level potassium chloride while converting enzyme (CE) inhibitors, ramiprilat, enalaprilat, and captopril were only marginally effective. Either CE inhibitor combined with nifedipine, however, significantly accentuated this relaxation. The CE inhibitors increased vascular cyclic GMP content while nifedipine was not active. Thus, inhibition of potassium chloride-induced calcium influx by nifedipine may unmask the vasorelaxant potential of CE inhibitors that in turn may result from local accumulation of endothelium-derived bradykinin.


Negative inotropic and chronotropic activity of calcium channel ligands possessing a xanthone 1,4-dihydropyridine backbone. A series of xanthone 1,4-dihydropyridine derivatives were prepared. The compounds were evaluated for inotropic, chronotropic, and calcium antagonist properties.


Pharmacological studies of the new antiinflammatory agent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4'-1-benzopyran-4-o ne. 2nd communication: effect on the arachidonic acid cascades. The in vitro and in vivo effects of 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-on e (T-614, CAS 123663-49-0), a new antiinflammatory agent, on arachidonic acid metabolism were investigated in comparison with those of reference drugs. Although the inhibitory effect of T-614 on the synthesis of prostaglandins by rabbit renal microsomes was very weak (IC50 of 58 micrograms/ml), T-614 effectively inhibited the prostaglandin E2 (PGE2) generation by mouse fibroblasts stimulated with bradykinin with an IC50 value of 0.47 micrograms/ml. The suppressive effect of T-614 on the PGE2 generation in fibroblasts was also found when cells were stimulated with Ca ionophore A23187, but not when induced with arachidonic acid. T-614 suppressed the A23187-induced PGE2 generation by rat macrophages, but not the leukotriene B4 production. In addition, 5-lipoxygenase activities in guinea-pig peritoneal exudated cells were not inhibited. In in vivo experiments, at doses of more than 1 mg/kg, T-614 reduced the PGE2 contents in inflammatory exudate of rat carrageenin-sponge type inflammation, but it was almost inactive in inhibiting gastric prostaglandins production up to 100 mg/kg. T-614 also did not affect the urinary PGE2 excretion in rats, and slightly inhibited the thromboxane synthesis in rat blood. Furthermore, the convulsion-induced increase of PGE2 in mouse brain was inhibited by T-614. These data suggest that T-614 inhibits the production of cyclooxgenase-mediated products with apparently different mode from classical non-steroidal antiinflammatory drugs, and may partly explain the discrepancy in pharmacological properties between this compound and other drugs.


Pharmacokinetics of silybin in bile following administration of silipide and silymarin in cholecystectomy patients. The biliary excretion of silybin, the main active component of silymarin, was evaluated by using a specific HPLC method in 9 cholecystectomy patients with T-tube drainage following single oral doses of silipide (CAS 134499-06-2), a lipophilic silybin-phosphatidylcholine complex (IdB 1016), and of silymarin (120 mg, expressed as silybin equivalents). After intake of silipide, the concentration of silybin in bile reached a peak within 4 h and declined thereafter with a mean time of about 10 h. After administration of silymarin, biliary silybin concentrations were several-fold lower than those observed after intake of silipide. The bile collected after silymarin intake also contained considerable amounts of isosilybin (a silybin isomer) and very low levels of silydianin and silycristin. The amount of silybin recovered in bile in free and conjugated form within 48 h accounted for 11% of the dose after silipide and for 3% of the dose after silymarin. Plasma silybin concentrations, determined in 3 subjects, were several-fold lower than those in bile after intake of silipide and mostly undetectable after intake of silymarin. These data indicate that the bioavailability of silybin is much greater after administration of silipide than after administration of silymarin. This results in increased delivery of the compound to the liver, which represents the target organ for pharmacological action.


Effect of aciclovir on the replication of herpes simplex virus type 1 in MA-104 cell line resistant to aciclovir. A culture of monkey kidney cells (MA-104) resistant to aciclovir (CAS 59277-89-3) was established. The resistant cells were continuously grown in medium containing 200 mumol/l aciclovir whereas normal cells died in such medium. The doses of aciclovir required for 50 and 90% reduction in virus yield were for several Herpes simplex virus Type 1 clinical isolates up to 5-fold higher in resistant than in normal MA-104 cells; in contrast, doses required for 99% reduction in virus yield could be as much as 50-fold higher in the resistant cells. Virus yields developed in the presence of 30 mumol aciclovir in culture medium were at least 10(3)-fold higher in resistant than in normal MA-104 cells. The results showed that the antiviral effect of aciclovir may be significantly decreased in the cell line which developed resistance to aciclovir.


Survey on spontaneous peripheral neuropathy in aging rats. The incidence of peripheral neuropathy in rats (Charles River bred, caesarean derived, Sprague-Dawley origin) obtained from long term studies over a 10 year period is reported. The survey included peripheral nerve samples from 14,237 males and 13,668 females. The incidence of peripheral neuropathy is 23% in untreated males and 18% in untreated females at 80-104 weeks of age. The total incidence in all rats in the survey was somewhat higher in males than females. The light and electron microscopic features of age-related peripheral neuropathy in rats is described. The peripheral neuropathy was spontaneous in origin and was age-related.


The effect of 25-hydroxycholesterol on the regulation of apolipoprotein E mRNA levels and secretion in the human hepatoma HepG2. The human hepatoma cell line, HepG2, was cultured with 25 OH cholesterol, a potent inhibitor of HMG-CoA reductase, in order to examine the effect of the oxysterol on apo E synthesis and secretion. Treatment of cells with oxysterol (2.5 microM) resulted in a greater than 90% inhibition of HMG-CoA reductase activity and a 3-fold reduction in its cognate mRNA level. However, apo E mRNA level and secretion were not affected after 24 h of drug treatment. This drug treatment was associated with a reduction in both cellular free and esterified cholesterol levels by 50% and 40%, respectively. Exposure of HepG2 cells to an ACAT inhibitor, the Sandoz compound (58-035) for 24 h, at a concentration of 5 micrograms/ml, resulted in a 30% increase and 70% decrease in the intracellular levels of free and esterified cholesterol, respectively. Under this regimen of drug treatment, the level of apo E mRNA was increased by approximately 70%, while HMG-CoA reductase mRNA level was decreased by 35%. When the cells were exposed to the combination of the ACAT inhibitor and 25 OH cholesterol, the cellular levels of free and esterified cholesterol were reduced by 30% and 80%, respectively. This combination of drugs had no effect on apo E mRNA; however, the level of HMG-CoA reductase mRNA was decreased by 3.5-fold. Taken together, the data suggested that reduction in the intracellular levels of either free or esterified cholesterol had no effect on apo E mRNA level. By contrast, a small increment in cellular free cholesterol content was associated with a significant induction in apo E mRNA level. Furthermore, 25 OH cholesterol caused a significant redistribution (50%) of apo E from the HDL fraction to the d greater than 1.21 g/ml infranatant. By using high performance liquid chromatography and molecular sieve columns, it was found that the appearance of a lipid-poor apo E particle was not an artifact of ultracentrifugation. This particle contained 85 wt% protein and 15 wt% of free cholesterol and phospholipid. The results suggested that a lipid-poor apo E particle was secreted by the HepG2 cells under certain circumstances.


Lack of tolerance to ethanol-induced stimulation of mesolimbic dopamine system. Rats maintained for 10 days on a 10% ethanol solution as the sole source of fluid developed marked tolerance to ethanol-induced loss of righting reflex, but no tolerance to the stimulatory effects on mesolimbic dopaminergic system. An ethanol challenge stimulated both the electrical activity of A10 dopaminergic cells and dopamine output in the ventral striatum of behaviourally tolerant animals and of controls to the same extent. These results are compatible with the hypothesis that an increased dopamine neurotransmission in the limbic system participates in the reinforcing effect of ethanol.


Functional alterations in cerebral GABAA receptor complex associated with formation of alcohol dependence: analysis using GABA-dependent 36Cl- influx into neuronal membrane vesicles. The effect of alcohol dependence induced by ethanol inhalation on GABA-dependent 36Cl- influx into membrane vesicles prepared from the mouse brain has been examined. Ethanol, flunitrazepam and salsolinol induced a significant facilitation of the GABA-dependent 36Cl- influx into membrane vesicles obtained from the normal mouse brain. Ethanol inhalation induced the facilitation of GABA-dependent 36Cl- influx at 3-12 hr after the initiation of inhalation, but this facilitation returned to a normal level within 12 hr. In membrane vesicles obtained from the brain of an alcohol-dependent mouse at 7 days after the initiation of ethanol inhalation, not only was there a significant decrease of the GABA-dependent 36Cl- influx but there occurred also the disappearance of the activating effects of ethanol, flunitrazepam and salsolinol on the influx. This decrease in GABA-dependent 36Cl- influx was found to be recovered within 8 hr after the withdrawal of ethanol inhalation. On the other hand, behavioural withdrawal signs such as tonic-clonic convulsions with grimaces and heads thrown back appeared at 8 hr after the withdrawal of ethanol inhalation and continued for 8-16 hr. These results suggest that the observed functional deteriorations at cerebral GABAA receptors such as the decrease of GABA-dependent 36Cl- influx and the disappearance of the activating effects of ethanol, flunitrazepam and salsolinol on the influx may contribute to the preparation of the exhibition of ethanol withdrawal signs and/or the establishment of functional tolerance to alcohol, but are not directly related to the exhibition of alcohol withdrawal signs.


Biphasic protein kinase C translocation in PC12 cells in response to short-term and long-term ethanol exposure. Short-term and long-term effects of ethanol on protein kinase C (PKC) activity and PKC translocation from cytosol to membrane were examined in PC12 cells, a clonal cell line of neural crest origin. Treatment of PC12 cells with ethanol (30-100 mM) for 2 hr had no effect on PKC activity and PKC translocation. When PC12 cells were treated with 100 mM ethanol for 18, 44 and 74 hr, there was a biphasic effect on PKC translocation. At 18 and 44 hr ethanol treatment, PKC translocation was significantly (P < 0.001) increased, at 74 hr ethanol treatment, there was a significant decrease (P < 0.05). Less than 100 mM of ethanol had no effect on PKC activity and PKC translocation. Cyclic AMP and cyclic GMP-dependent protein kinase had no effect on PKC translocation. These findings indicate that biphasic PKC translocation from cytosol to membrane forms the basis of acute and chronic effects of ethanol on neurotransmission.


Endocrine profile during acquisition of free-choice alcohol drinking in rhesus monkeys; treatment with desglycinamide-(Arg8)-vasopressin. This study concerns the effect of spontaneous acquisition of alcohol drinking in rhesus monkeys on plasma levels of beta-endorphin, ACTH, prolactin, cortisol and testosterone. Twelve monkeys had free-choice access to water and two ethanol/water solutions (1%, 2%, v/v) for 4 weeks. During the first 2 weeks, six monkeys were injected (i.m.) twice daily with 0.50 microgram/kg desglycinamide-(Arg8)-vasopressin (DGAVP), a neuropeptide, that has been postulated to interfere with central positive reinforcement processes. The other six were treated with a placebo. Hormonal plasma levels after the first 2 weeks and after another 2 weeks of alcohol drinking were compared to pre-alcohol hormonal levels (baseline). The placebo-treated subjects significantly increased, but the DGAVP-treated subjects significantly decreased ethanol intake over time. After 2 weeks of alcohol, significant increases were found in beta-endorphin and ACTH levels. After 4 weeks, prolactin was increased, cortisol decreased and particularly beta-endorphin remained significantly increased. No relationship was found between baseline hormonal levels and subsequent ethanol intake. No significant differences in plasma hormonal changes were observed between DGAVP- and placebo-treated subjects. Two placebo-treated subjects that showed the highest increase in ethanol intake over time, reacted differently, by reducing beta-endorphin and ACTH levels over time, showing the largest decreases in cortisol and hardly any prolactin reaction. It is concluded that spontaneous alcohol drinking by naïve subjects disturbs hormonal processes and that two animals deviated with respect to the acquisition in alcohol drinking and endocrine functioning.


In-patient treatment of alcohol problems--predicting and preventing relapse. Seventy-five alcohol-dependent patients (35 males, 40 females) treated by a Minnesota Model in-patient programme were followed up for 1 year. A variety of outcome measures were included, and patients' GPs were also questioned with regard to various aspects of their involvement in treatment for alcohol problems. Males admitted to the programme had a longer history of drinking, consumed more alcohol and showed greater expenditure on alcohol. At 6 months, 66% of males and 45% of females were abstinent; at 1 year, the proportions abstinent were 53% and 39%, respectively. Good outcome for both sexes was associated with attendance of Alcoholics Anonymous meetings. Poor outcome at 1 year was associated with a lack of GP involvement in aftercare and failure to provide alcohol counselling in the community. Females appeared to be particularly disadvantaged by depressive comorbidity. Males showed poorer outcome if they belonged to social class IIIM or lower, were unemployed, or had a family history of alcoholism. It is suggested that closer attention should be paid to monitoring patients' mood state, with appropriate treatment of depression, and that GPs need on-going support and education for helping patients with alcohol problems.


[Hepatitis C virus].

[Hepatitis C virus]. Hepatitis C Virus, major causative agent of parenterally transmitted non-A non-B hepatitis was identified by Choo et al. in 1988 using molecular biology technologies. This virus contains a positive stranded RNA genome, it has been shown to have a small diameter. These structure and properties suggest that HCV shares many features in common with Pestiviruses and Flaviviruses. After many years of research, two major objectives were reached: -the identification of viral genome and the main purified viral polypeptide derived from recombinant yeast. Major information were recently appearing about the nucleotide sequences of different isolates coming from US, Europe and Japan; -the preparation of specific tool (ELISA anti-HCV) for detection of circulating HCV antibodies.


[Serological diagnosis of anti-HCV antibodies. Tests for screening and confirmation: analytical study. Le groupe "Hépatites virales" de la Société Française de Transfusion Sanguine]. Four screening and five confirmatory ELISA 2nd generation tests for anti-HCV serological diagnostic purpose are detailed herein. All studied assays were indirect ELISA procedures. Solid phase are coated with viral antigens (synthetic peptides or recombinant proteins) corresponding to structural and non structural HCV genes. Solid phase antigenic adsorbent, assay procedures and interpretations of results are analysed.


[European Acute Transfusion Hepatitis Interferon Study (EATHIS)]. Since 1989, a prospective, multicentric and collaborative study, named European Acute Transfusion Hepatitis Interferon Study (EATHIS), involving 16 blood centres in France is coordinated by a task force in collaboration with the Viral Hepatitis and Recipients working groups of the French National Society of Blood Transfusion. A phase of recipient screening may be completed by a therapeutic interferon protocol. Among 1,476 transfused recipients receiving an average of 4.4 blood components, 1,011 were followed up at least four months. The incidence of non-A, non-B post-transfusion hepatitis (PTH) was varying from 0.1 to 6% at the different locations. Out of 15 hepatitis cases, 10 were diagnosed as post-transfusional. These preliminary data indicate a reduced incidence of PTH, with a mean of 1% for the period 1989-1991. It emphasises the importance of a transfusion vigilance program including follow up and testing for transaminases and specific viral markers.


[Hepatitis C virus and transfusion: policy regarding the donation and the donor. Groupe "Hépatites Virales" de la Société Nationale de Transfusion Sanguine]. Hepatitis C virus (HCV) discovery and introduction of anti-HCV antibodies screening in blood transfusion imply the necessity of a good blood donations and blood donors policy. Detection of a seropositivity during the screening must be completed with a confirmatory test. The results are directly used to inform donors and define the blood products policy. Donors with positive results on confirmatory test are discarded and have physical and biological examinations in hepatology. Individuals with indeterminate or negative results must be retested for the HCV serology. Furthermore, because of a rapid improvement in the fields of technology, diagnosis and therapy of HCV, an adaptation of the policy is necessary.


Leigh encephalopathy: histologic and biochemical analyses of muscle biopsies. To elucidate the pathogenesis of Leigh encephalopathy, histologic, biochemical, and mitochondrial DNA analyses were performed on biopsied muscles from 33 patients with the clinical characteristics of this disorder. On muscle histochemistry, cytochrome c oxidase activity was decreased or absent in 7 patients (21%), although none had ragged-red fibers. In 2 patients with cytochrome c oxidase deficiency, staining for this enzyme was poor in the muscle fibers and fibroblasts but was normal in the arterial wall, indicating tissue-specific involvement. Ten patients (30%) had biochemical defects, including 2 with pyruvate dehydrogenase complex, 4 with cytochrome c oxidase, 1 with NADH-cytochrome c reductase (complex I), and 3 with multiple complex deficiencies. None of the 28 patients in whom muscle mitochondrial (mt)DNA was analyzed had DNA deletions or point mutation at nucleotide positions 3,243 or 8,344. These results indicate that the underlying defect in Leigh encephalopathy is heterogeneous because only 30% of patients had enzyme defects demonstrable in muscle biopsy material.


[Importance of PCR in the diagnosis of hepatitis C]. The identification of hepatitis C virus, based on DNA amplification, gives a precise estimation of the prevalence of the most frequent agent of NANB hepatitis. The first ELISA allowing the detection of anti-HCV antibodies, had too many false positive results and required the development of more sensitive and specific assays to confirm its results. PCR, allowing the hepatitis C virus diagnosis by showing directly HCV RNA sequences, offers a complementary approach to immunoserological tests. In blood donors with anti-HCV antibodies and with indeterminate or negative confirmatory tests, the finding of HCV RNA sequences reveals serum infectivity. During acute hepatitis, the delay in the appearance of anti HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on-going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness and a third one distinct from the previous two. However, its limits and constraints imply that PCR must not be considered as a routine assay. This emphasizes the need for more simple and rapid diagnostic tests, allowing the detection of HCV antigens and, as in hepatitis B, the progressive unravelling of the life cycle of HCV.


Characterization of a cell line derived from a human oligodendroglioma. A novel clonal cell line derived from a human glioma (HOG) was found to express some oligodendrocyte-specific proteins including a 15-kDa form of myelin basic protein (MBP) and high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Expression of the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide) was low. HOG cells did not express the characteristic astrocyte markers glial fibrillary acidic protein (GFAP) or significant glutamine synthetase (GS) activity. After initial plating, HOG cells were flat and epitheloid and thus showed a limited oligodendrocyte-like morphology. However, after cells became more confluent, some cells were phase-bright and elaborated short processes. Receptor types expressed by HOG cells included A2-adenosine, prostaglandin E1 (PGE1), and beta 2-adrenergic receptors (beta-ARs) linked to stimulation of adenylate cyclase, and muscarinic cholinergic and H1-histamine coupled to phosphatidyinositol turnover (Post and Dawson, 1991). HOG cells should therefore provide a useful model for studying the extracellular regulation and phosphorylation of oligodendrocyte-specific proteins.


An overview of principles for classifying brain tumors. The purpose of this review is to clarify for the nonneuropathologist some of the confusing issues concerning the classification of brain tumors. Following a short discussion of the commonly used methods to diagnose brain tumors clinically (frozen section, light and electron microscopy, immunohistochemistry), the general principles of classifying neural tumors are presented. Grading of tumors on the basis of histological anaplasia, and the concept that tumor cells can be related to specific cytological stages of normal cellular development (cytogenetic classification) are presented. The World Health Organization system of classifying neural tumors is an attempt to develop a standardized classification scheme with as few interpretative controversies as possible, but it has required revision as new information has been gained. The major clinical and biological features of the commonest tumor groups are then discussed. It is unlikely that improvements in the classification of brain tumors will be based solely on histological information. Definitions of tumor entities that will provide more accurate prognoses and bases for effective therapy will require considerably more information at the molecular level than is currently available.


Medulloblastomas and related primitive neuroectodermal brain tumors of childhood recapitulate molecular milestones in the maturation of neuroblasts. We review here recent data that have brought into sharper focus a number of important biological properties of the neoplastic cells in childhood primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS). Studies of this group of tumors, as exemplified by posterior fossa medulloblastomas (MBs), suggest that neoplastic cells in PNETs partially recapitulate stages in the maturation of normal human neuroblasts. These findings may contribute to the elucidation of the mechanisms involved in tumor initiation and progression because oncogenes and antioncogenes appear to exert their effects in a cell type-specific manner that also depends on the maturational state of a given cell. Currently, a large body of data suggests that populations of cells in PNETs (e.g., MBs) exhibit one or more molecular defects in the sequence of maturational events leading to the exit of stem cells or partially committed neuron-like precursors from the cell cycle, followed by their terminal differentiation into neurons. This, together with the orchestrated interactions of as yet unidentified oncogenes and antioncogenes in these PNET cells, may represent a cluster of molecular abnormalities that underly the emergence of the highly malignant phenotype that characterizes childhood PNETs.


[Type 6/11 and 16/18 squamous epithelial cancers of the upper respiratory tract and digestive system. An in situ hybridization study]. 61 squamous cell cancers (27 laryngeal, 12 hypopharyngeal, 14 tonsillary, 8 tongue) with different keratinization and grading and seven lymph node metastases of HPV 16/18 positive carcinomas were analysed for the presence of HPV-DNA by in situ hybridisation. 65.5% of them were found to be positive. Twelve laryngeal carcinomas (44%), five tonsillary tumours (35.7%), eight tumours of the hypopharynx (66.6%) and three tongue carcinomas (37.5%) were shown to contain HPV 16/18 DNA. The detection rates of HPV 6/11 were lower. 44 of the analysed tumours (72.1%) had a grading G2. 29 of these tumours (65.9%) were HPV positive. Only eight of the patients were no heavy smokers or alcoholic drinkers. One of the lymph node metastases was positive for HPV 16/18. The results indicate that HPV may be involved in the pathogenesis of squamous cell carcinomas of head and neck tumours.


Effect of ultraviolet irradiation on selected host cell proteins including Ro/SS-A and Epstein-Barr virus in cultured lymphoblastoid cell lines. Although systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) are distinct collagen vascular illnesses, they share certain features. Both have clinical manifestations involving skin and mucous membranes and characteristically have high titers of circulating autoantibodies to the cellular components Ro/SS-A, calreticulin/Ro, 52 kDa Ro and La/SS-B. Viruses have been postulated to be involved in the pathogenesis of both diseases. Sensitivity to sun is a cardinal feature of SLE, and UV light may be involved in its pathogenesis. Using human B-lymphoblastoid cell lines, the effect of the resident Epstein-Barr virus on the expression of the above cellular components was investigated by flow cytometry. Sublethal irradiation with ultraviolet B light appeared to diminish EBV antigen expression (gp350/220) during the first 48 to 72 hours in culture, whereas there was no change in the expression of MHC class I or immunoglobulin host cell proteins, and an apparent increase in the expression of host cell autoantigens. The virus appeared to be more sensitive to UVB-induced damage yet did appear to be able to undergo repair. No direct correlation could be made between the presence of the virus and the increase in autoantigen expression. La/SS-B and/or 52 kDa Ro antigen(s) were found to be present in the cytoplasm of the B lymphoblastoid cells at a higher base level in EBV-infected cell lines than in the EBV-negative cell lines.


Buprenorphine attenuates the effects of cocaine on adrenocorticotropin (ACTH) secretion and mood states in man. Adrenocorticotropin (ACTH) levels in plasma increased rapidly to 105% above baseline within 5 minutes after intravenous injection of cocaine (30 mg) in cocaine-dependent men. The time course of ACTH stimulation paralleled increases in plasma cocaine levels and self-reports of salient drug effects on mood states and did not occur after placebo administration. An opioid mixed agonist-antagonist, buprenorphine (4 mg/day sublingually), suppressed the acute cocaine-induced stimulation of both ACTH and euphoria. Buprenorphine's suppression of postcocaine ACTH and euphoria were not related to differences in plasma cocaine levels or cocaine-induced alterations of cardiovascular function.


Age-related differences in the phosphoinositide system in rat neostriatum. The phosphoinositide cycle is an important second messenger system that may be involved in the therapeutic action of lithium. Elderly patients taking lithium often experience beneficial effects at lower doses and serum levels than their younger counterparts. To investigate age-related differences in lithium's effects on the phosphoinositide system, neostriatal slices from 3-, 10-, and 24- to 26-month-old Fischer 344 rats were labeled with [3H]-myoinositol and exposed for a 25-minute incubation period to oxotremorine-M and varying lithium chloride concentrations. Inositol phosphatase accumulation was significantly greater in 10- and 24- to 26-month-old than in 3-month-old rats. Further investigations may clarify the underlying mechanisms for such age-related differences.


Analysis of the basis for persistence of herpes simplex virus type 1 in undifferentiated U937 cells. Replication of herpes simplex type 1 (HSV-1) is inhibited in the human monocyte-like cell line, U937, when the cells are in the undifferentiated state, but when the cells are stimulated to differentiate by treatment with the phorbol ester, phorbol 12-myristate 13-acetate virus is replicated. Because HSV-1 has been shown to persist in these cells and in their in vitro counterparts freshly isolated human blood monocytes, we initiated an analysis of viral persistence in undifferentiated U937 cells. No appreciable HSV-1 DNA replication was observed in undifferentiated U937 cells compared with differentiated U937 cells and with fully permissive Vero cells. However, using in situ hybridization, we established that a significant percent of the undifferentiated U937 cells contained viral DNA sequences. Interestingly, when analyzed by Southern blot hybridization, this DNA was found to have assumed a nonlinear configuration similar to that found in latently infected neurons. Analysis of viral proteins in undifferentiated U937 cells revealed a marked absence of proteins of all three kinetic classes. However, in transient transfection assays, the major viral transactivating protein ICP4, functioned normally, whereas ICP0, a promiscuous transactivator of both viral and cellular genes, was unable to transactivate viral promoters in undifferentiated U937 cells. Thus, a subtle dysfunction in the activity of ICP0 may account, at least in part, for the inability of undifferentiated U937 cells to support replication of HSV-1.


Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells. The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in fresh-water (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.


Characterization of a rat adrenocortical inner zone-specific antigen and identification of its putative precursor. We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization. Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30,000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. In-vivo treatment with ACTH (100 micrograms/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro. In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60,000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 mumol/l) or dibutyryl-cAMP (100 mumol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2.(ABSTRACT TRUNCATED AT 250 WORDS)


Microvascular perturbations in human allografts: analogies in preeclamptic placentae. The thromboresistance of endothelium is maintained as long as natural anticoagulant pathways are functionally present on endothelial plasma membranes. The principal anticoagulant pathways in human hearts and kidneys are thrombomodulin (TM) and heparan sulfate proteoglycan-antithrombin III (HSPG-ATIII). The downregulation of TM or the loss of ATIII is associated with fibrin deposition. This sequence of events occurs when stable allografts of hearts or kidneys become unstable or rejected. Human placentae do not contain the HSPG-ATIII natural anticoagulant pathway, but the TM system is uniformly represented on endothelium of normal chorionic villi. However, many villi in placentae from preeclamptic pregnancies contain thrombomodulin-negative endothelium, and these vessels contain fibrin thrombi. These thrombi compromise blood flow through the placental microcirculation and are associated with ischemic changes either with or without the presence of cellular infiltrates.


Fetal stem vessel endothelial changes in placentae from normal and abnormal pregnancies. Areas of immunocytochemically defined immunopathology are common in placentae from secondary recurrent spontaneous aborters. Endothelial cells of affected villi were found to be negative for the thrombomodulin natural anticoagulant pathway, and these cells were reactive with monoclonal antibodies to tissue factor and fibrin. Endothelial cells of normal villi adjacent to affected villi also were negative for thrombomodulin and positive for tissue factor and fibrin, but endothelium of normal villi distant to affected villi were thrombomodulin-positive, tissue factor-negative, and did not contain deposits of fibrin. These findings indicate that a loss of endothelial anticoagulant activity precedes the onset of immunopathology. This observation, coupled with the findings that fetal stem vessels normally lack the heparan sulfate proteoglycan-antithrombin III natural anticoagulant pathway and that normal cord blood contains a heparin-like anticoagulant, suggest that a failure of endothelial anticoagulation may be a primary event in the development of placental immunopathology.


[Effect of concentration of nutrients solution on the growth of Salvia miltiorrhiza Bge]. The experiment on the effect of various concentrations of nutrient solution on sand culture of Salvia miltiorrhiza was carried out. The results show that Salvia miltiorrhiza is a fertilizer tolerant plant. It can grow well and give high yield when the total concentration of nutrient reaches 0.37%.


[Ecological investigation on Schisandra chinensis (Turcz.) Baill. of the Changbai Mountain]. This paper reports the distribution and ecological environment of Schisandra chinensis in the Changbai mountain, as well as the characteristics of bud, root, branch, annual biomass, development period, flowering-fruiting habits and cultivation of the plant.


The effects on lipid metabolism of 4-phenyl-5,5-dicarbethoxy-2-pyrrolidenone in Sprague Dawley rats. 4-Phenyl-5,5-dicarbethoxy-2-pyrrolidenone [XIV] treatment in Sprague Dawley rats caused significant reduction of serum cholesterol and triglyceride levels with reduction of VLDL and LDL cholesterol levels. The compound significantly reduced regulatory enzyme activities, e.g. ATP dependent citrate lyase, HMG CoA reductase, acyl CoA cholesterol acyl transferase, cholesterol-7-alpha-hydroxylase, sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase. In tissue cultured cells, the compound suppressed LDL receptor activity and degradation, and elevated HDL receptor activity and HDL degradation. Rat bile cholesterol and phospholipids were elevated; however, overall bile acids were reduced. In situ loop studies suggest that the agent interfered with interhepatic reabsorption of cholesterol and cholic acids. At the therapeutic dose of compound XIV, no deleterious effects were demonstrated in mice.


Presence and regulation of a truncated proopiomelanocortin gene transcript in rat pancreatic islets. The demonstration of proopiomelanocortin-(POMC-) derived peptides in the endocrine pancreas suggests a local biosynthesis of these peptides. Using Northern blot analysis we demonstrated a 800 nucleotide POMC-like mRNA species. The POMC-like mRNA was demonstrable in a part of the pancreatic islets by the in situ hybridization technique. A small number of single cells within the exocrine pancreas were also found to give positive hybridization signals. Isolated islets were incubated under normoglycemic conditions with dibutyryl-cAMP (3mM) or dexamethasone (0.1mM) for 4 h. Dibutyryl-cAMP significantly (p < 0.01) increased the POMC-like mRNA content to 177 +/- 39.1% of the control whereas dexamethasone decreased it to 66.9 +/- 11.4% of the control. In vivo treatment of adult rats with 80 micrograms dexamethasone over 3 days showed also a reduction of the POMC-like mRNA level in the islets. In conclusions, the POMC gene is expressed in adult rat islets and can be regulated by dexamethasone and dibutyryl-cAMP. Whether the truncated POMC-like mRNA reported here is the source of the biosynthetic POMC-like products detected in the islets is not yet known.


Diastereomers of neopterin and biopterin in human urine. A new pteridine compound, named umanopterin, was isolated from human urine both of cancer patients and non-cancer controls. The structure was confirmed to be 2-amino-4(3H)-oxo-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridine , a diastereomer of neopterin. The amount of umanopterin relative to neopterin was about 10%, which was practically the same among the non-cancer controls and the patients of various cancers. A small amount of a threo diastereomer of biopterin, named orinapterin, was isolated from human urine for the first time. Its structure was shown to be 2-amino-4(3H)-oxo-6-[(1'S,2'S)-1',2'-dihydroxypropyl]pteridine. A non-enzymatic transformation of 7,8-dihydroneopterin and 7,8-dihydrobiopterin by a mechanism analogous to keto-enol tautomerism is postulated for the formation of umanopterin and orinapterin in human body.


Protease inhibitor homologues of dendrotoxin do not bind to dendrotoxin acceptors on synaptosomal membranes or facilitate neuromuscular transmission. The dendrotoxins are a homologous group of potassium channel-blocking polypeptides found in mamba snake venom. They are similar in sequence and structure to Kunitz-type serine protease inhibitors. Modified and native protease inhibitors were assayed for dendrotoxin-like activity using radioligand-binding and twitch tension-recording methods. Despite the large number and high concentration of compounds tested, no protease inhibitor displayed dendrotoxin-like activity. The results indicate that the protease-inhibiting and potassium channel-blocking activities of these two groups of polypeptides are not linked.


Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta). Distinctions between tumor necrosis factor, TNF-alpha, and lymphotoxin, TNF-beta, have previously been based on the differences between their protein sequences, biological activity, and molecular regulation. In the past year, elucidation of the molecular nature of the two molecules and the interactions with common receptors has emphasized their similarities, although profound differences continue to emerge with regard to their mode of production, transcription rates, mRNA half-lives, and the importance of various DNA regulatory sequences. A role for both TNF-alpha and TNF-beta has recently been suggested with regard to disease, particularly multiple sclerosis. The past year has also seen the description of an extensive microsatellite polymorphic system which should provide a more definitive understanding of the association of the TNF locus with disease.