Acute and subchronic effects of Rimcazole (BW 234U), a potential antipsychotic drug, on A9 and A10 dopamine neurons in the rat

The effects of acute and subchronic Rimcazole administration on A9 and A10 dopamine (DA) neurons were examined using extracellular single cell recording techniques. Intravenous injections of Rimcazole did not prevent or reverse the inhibition of firing rates of DA cells produced by DA agonist apomorphine (APO). Single intraperitoneal injection of Rimcazole decreased the number of spontaneously active DA cells in A10, but not in A9; it had no effect on the firing rate of DA neurons in either A9 or A10. Following prolonged administration of Rimcazole, 25 mg/kg/day for 28 days, there was a significant increase in the number of spontaneously active A10 DA neurons, but not A9 DA cells. The firing rate of both A9 and A10 DA cells decreased significantly following prolonged Rimcazole administration; however, the firing pattern of these cells did not change. In addition, chronic Rimcazole did not affect the ID50 of APO for DA neurons. These results suggest that Rimcazole has an indirect effect on DA neurons with a relative selectivity for A10 DA cells; it does not exhibit pharmacological profiles of previously reported antipsychotic drugs.     

Synthesis of shock proteins in cultured fetal mouse myocardial cells

We examined the synthesis of shock proteins in cultured fetal mouse myocytes. The preparation is free from fibroblasts, and the cells are vital and morphologically intact with respect to beat frequency and electron microscopy. Cultured myocytes from fetal mouse heart respond to heat shock and cadmium chloride, H2O2, allylamine, cyclosporine, and azathioprine exposure with the synthesis of shock proteins. Heat shock induces the de novo synthesis of two proteins of 71 and 68 kDa; cadmium chloride induces, in addition, a protein of 30 kDa. The other substances tested provoke the synthesis only of the 30-kDa polypeptide. The formation of heat shock proteins is concentration-dependent: Cyclosporine provokes the de novo synthesis of the 30-kDa polypeptide at concentrations above 10 ng/ml, whereas azathioprine causes the same effect at concentrations above 50 micrograms/ml. Hence cyclosporine might be cardiotoxic already at concentrations below the pharmacological dosages while azathioprine influences the myocytes only at concentrations much higher than the therapeutic level. Our results indicate that heat shock protein expression in cultured myocytes may be a useful tool to monitor cardiotoxicity.     

Serotonin sensitive adenylate cyclase activity in monkey anterior limbic cortex: antagonism by molindone and other antipsychotic drugs.

Serotonin (5-HT) sensitive adenylate cyclase in monkey anterior limbic cortex homogenates was further characterized and the effects of antipsychotic drugs and 5-HT anatagonists investigated. Differences in time course for stimulation by agonists and in responsiveness to receptor anatagonists of 5-HT-and dopamine (DA)-stimulated activities, were observed. Also there was an additivity of 5-HT and DA at maximally effective concentrations. Classical 5-HT antagonists blocked the 5-HT sensitive adenylate cyclase with a rank order of potency: methiothepin > cyproheptadine > methysergide. These 5-HT antagonists also effectively inhibited DA sensitive adenylate cyclase. Most antipsychotic drugs tested antagonized 5-HT stimulated activity although these drugs exhibited greater efficacies in blocking DA stimulated activity. Exceptions were molindone which failed to antagonize DA sensitive adenylate cyclase but effectively blocked 5-HT sensitive cyclase and pipamperone which was inactive in both cyclase systems. Haloperidol was a more selective antagonist of the DA sensitive adenylate cyclase than were the other antipsychotic drugs tested.     

In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence.

Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kDa), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3H]tetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kDa, were specifically labeled. When extracts were acylated with [3H] tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42-kDa polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction.     

Differential effects after repeated treatment with haloperidol, clozapine, thioridazine and tefludazine on SNC and VTA dopamine neurones in rats

The effects of repeated treatment (21 days) with different antipsychotic compounds (haloperidol, clozapine, thioridazine and tefludazine) on dopamine (DA) neurones in substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) were studied in rats using single unit recording techniques. A dose-dependent decrease in the number of spontaneously active DA neurones in SNC and in VTA was observed with haloperidol. Clozapine showed no significant effect on the activity in SNC while a dose-dependent decrease in the number of active DA neurones in VTA was observed. Thioridazine showed no or weak effect in SNC while repeated treatment induced a marked inhibitory effect on the DA neurones in VTA. Tefludazine, a potential antipsychotic compound, induced a dose-dependent decrease in both SNC and VTA DA activity. However, the effect on the DA neurones in VTA was more pronounced at all doses. Since the classical neuroleptic haloperidol is equally effective in both regions, while the atypical neuroleptics clozapine and thioridazine have selective or predominant effect in the VTA area it has previously been thought that the inhibition of spontaneously active DA neurones in VTA should indicate an antipsychotic effect of a compound while the inhibition of DA neurones in SNC should account for the development of neurological side effects. The data suggests that the potential antipsychotic compound tefludazine should not induce neurological side effects at lower doses but still has an antipsychotic activity while repeated treatment with higher doses of tefludazine might cause extrapyramidal side effects.     

Blocking of the receptor-mediated invasion of erythrocytes by Plasmodium knowlesi malaria with sulfated polysaccharides and glycosaminoglycans

Invasion of human erythrocytes by Plasmodium knowlesi requires the Duffy blood group antigen. P. knowlesi merozoites synthesize a 135-kDa polypeptide which binds to the Duffy antigen with receptor-like specificity. In this study, we show that the sulfated polysaccharide fucoidan and the glycosaminoglycan dextran sulfate inhibit the binding of the 135-kDa polypeptide to human Duffy-positive and rhesus erythrocytes while the chondroitin sulfates do not. Fucoidan and dextran sulphate also blocked the in vitro invasion of human Duffy b and rhesus erythrocytes cells by P. knowlesi merozoites. These inhibitors were more effective at blocking the binding of the 135-kDa polypeptide to human Duffy b erythrocytes than to rhesus erythrocytes, which correlated with them having a greater inhibitory effect on invasion of merozoites into human than into rhesus erythrocytes. The blocking by these sulfated sugars is not related to charge density on the polysaccharides; fucoidan with a relatively low charge density blocks binding of the 135-kDa polypeptide at 4 micrograms/ml, while the highly negatively charged chondroitin sulfates do not block binding even at the concentration of 1 mg/ml. Furthermore, fucoidan-Sepharose bound and removed the 135-kDa polypeptide from parasite culture supernatants with a selectivity equal to that of the Duffy blood group antigen. The negatively charged sulfate groups on fucoidan and dextran sulfate and the conformation in which they are held possibly mimic similarly charged groups on the Duffy antigen which bind the 135-kDa P. knowlesi polypeptide.     

Binding of Host-Associated Treponeme Proteins to Collagens and Laminin: A Possible Mechanism of Spirochetal Adherence to Host Tissues

The polypeptides of seven strains of human treponemes were investigated by immunoblot analysis for their binding to the human placental collagens and laminin. Of the treponemal polypeptides, eleven polypeptides, 45-kDa, 49-kDa, and 62-kDa polypeptides from T. pallidum ATCC 27087, a 48-kDa polypeptide from T. phagedenis biotype Reiter, 51-kDa and 53-kDa polypeptides from T. vincentii ATCC 35580, 30-kDa, 53-kDa and 63-kDa polypeptides from T. socranskii subsp. buccale ATCC 35534, a 52-kDa polypeptide from T. denticola ATCC 35405, and a 53-kDa polypeptide from T. denticola ATCC 33520 possessed an ability to bind to the laminin, type I, III, IV, or V collagen. An intermediate-sized human oral isolate strain G7201 did not possess any laminin- or collagen-binding polypeptides. Immunoelectron microscopy using intact treponemal cells with a single collagen-binding polypeptide and the corresponding antisera demonstrated that the 51-kDa and 53-kDa polypeptides from T. vincentii, the 53-kDa polypeptide from T. socranskii subsp. buccale ATCC 35534 and the 52-kDa polypeptide from T. denticola ATCC 35405, were outer envelope proteins.     

Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities.

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.     

Dopaminergic 3H-agonist receptors in rat brain. New evidence on localization and pharmacology

Recent methodological advances have allowed the reliable assay of specific dopaminergic 3H-agonist binding sites in rat striatum. Successful assay depends on preincubation of tissue homogenates at 37 degrees C; this results in a guanyl nucleotide-sensitive and dopamine (DA)-dependent increase in the density (Bmax) of 3H-agonist binding. Lesions of DA terminals or drugs which deplete DA levels prevent the preincubation-induced increase in binding, and this effect is completely reversible by preincubation with added DA. In contrast, kainic acid lesions irreversibly reduce 3H-agonist binding. It is concluded that the evidence supporting the existence of presynaptic "D-3" sites is artefactual and that 3H-DA binding sites are more likely related to post-synaptic receptors. 3H-DA binding involves two sites, one of which has pharmacologic properties similar to D-1 receptors, whereas the other resembles D-2 receptors. The affinity of 15 antipsychotic drugs for 3H-haloperidol binding sites was highly correlated (R = 0.94) with their inhibitory potency at a subset of 3H-DA binding sites. However, the inhibition of 3H-DA binding by antipsychotic drugs was noncompetitive. These findings can be explained by an allosteric model, whereby antagonists bind to a site different from but allosterically linked to a high-affinity 3H-DA binding site.     

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.     

Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.     

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW < 10 kDa), such as salicylic acid, can enhance this activity.     

Repression of quiescence-specific polypeptides in chicken heart mesenchymal cells transformed by Rous sarcoma virus.

Chicken heart mesenchymal cells do not proliferate in medium of physiological composition containing plasma (S. Balk, Proc. Natl. Acad. Sci. USA 77:6606-6610, 1980). To understand the molecular events involved in cell quiescence and in the initiation of cell division under physiological conditions, we examined the differences in the patterns of protein synthesis of quiescent, hormone-stimulated, and Rous sarcoma virus-transformed chicken heart mesenchymal cells. We describe the expression of a 20,000-kilodalton (kDa) polypeptide actively synthesized by quiescent cells but not by their transformed counterparts. Normal chicken heart mesenchymal cells stimulated with epidermal growth factor and insulin also repressed the synthesis of the 20,000-kDa polypeptide while actively growing but synthesized increasing amounts of the protein at high cell density (confluence). The synthesis of the 20,000-kDa protein is not restricted to chicken heart mesenchymal cells, since confluent, density-arrested chicken embryo fibroblasts also expressed high levels of the protein. Transformed chicken heart mesenchymal cells and embryo fibroblasts did not synthesize the protein even at high cell density. The 20,000-kDa polypeptide accumulated in the culture medium.     

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

Comparison of the effects of chronic haloperidol treatment on A9 and A10 dopamine neurons in the rat

The effects of chronic haloperidol (CHAL) treatment on A9 and A10 dopamine (DA) neurons were compared using extracellular single cell recording techniques. CHAL caused a time-dependent reduction in the number of spontaneously active A9 and A10 DA cells and induced an irregular firing pattern in many of the DA cells that remained active. Both of these effects occurred earlier and to a greater extent in A10 than in A9. Intravenous injection of the DA agonist apomorphine reversed both the reduction of active DA neurons and the irregular discharge pattern, suggesting that both effects were due to the process of depolarization inactivation. Lesions of the nucleus accumbens (NAc) produced by ibotenic acid prevented the development of depolarization inactivation of A10 DA neurons, indicating that this process is mediated primarily by NAc — A10 feedback pathways. The results suggest that the slow development of depolarization inactivation of DA cells produced by CHAL may contribute to the delayed onset of the clinical effects of long-term treatment with antipsychotic drugs.     

Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source.

The effects of ammonium on the de novo synthesis of polypeptides in the soil-nitrifying bacterium Nitrosomonas europaea have been investigated. Cells were incubated in the presence of both acetylene and NH4+. Under these conditions, the cells were unable to utilize NH4+ as an energy source. Energy to support protein synthesis was supplied by the oxidation of hydroxylamine or other alternative substrates for hydroxylamine oxidoreductase. De novo protein synthesis was detected by 14C incorporation from 14CO2 into polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In the presence of NH4+, acetylene-treated cells synthesized the 27-kDa polypeptide of ammonia monoxygenase (AMO) and two other major polypeptides (with sizes of 55 and 65 kDa). The synthesis of these polypeptides was completely inhibited by chloramphenicol and attenuated by rifampin. The optimal concentration of hydroxylamine for the in vivo 14C-labeling reaction was found to be 2 mM. The effect of NH4+ concentration was also examined. It was shown to cause a saturable response with a Ks of approximately 2.0 mM NH4+. Labeling studies conducted at different pH values suggest cells respond to NH3 rather than NH4+. No other compounds tested were able to influence the synthesis of the 27-kDa component of AMO, although we have also demonstrated that this polypeptide can be synthesized under anaerobic conditions in cells utilizing pyruvate- or hydrazine-dependent nitrite reduction as an energy source. We conclude that ammonia has a regulatory effect on the synthesis of a subunit of AMO in addition to providing nitrogen for protein synthesis.