Differences In Inhibitory Activity Research Articles

Effects of Escherichia coli RraA Orthologs of Vibrio vulnificus on the Ribonucleolytic Activity of RNase E In Vivo

RraA is a recently discovered protein inhibitor of RNase E that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. In the genome of Vibrio vulnificus, two open reading frames that potentially encode proteins homologous to E. coli, RraA-designated RraAV1 and RraAV2, have respectively 80.1% and 59.0% amino acid identity to RraA. The authors report that coexpression of RraAV1 protein in E. coli cells overproducing RNase E rescued these cells from growth arrest and restored their normal growth, whereas coexpression of RraAV2 protein further inhibited the growth of E. coli cells, whose growth was already impaired by overproduction of RNase E. Analyses of the steady-state level of various RNase E substrates indicated that the coexpression of RraAV1 more efficiently inhibited RNase E action than coexpression of RraA, and consequently resulted in the more increased abundance of each RNA species tested in vivo. The inhibitory effect by RraAV2 coexpression on RNase E was observed only in the case of trpA mRNA, indicating the possibility of RNA substrate-dependent inhibition of RraAV2 on RNase E. The findings suggest that these regulators of ribonuclease activity have both a conserved inhibitory function and a differential inhibitory activity on RNase E-like enzymes across the species.

Cytostatic and Antiviral Activity Evaluations of Hydroxamic Derivatives of Some Non‐steroidal Anti‐inflammatory Drugs

Non-steroidal anti-inflammatory drugs (NSAID) pharmacophores are interesting in designing potential anticancer drugs. Indeed, numerous experimental, epidemiologic and clinical studies suggest that NSAIDs are promising anticancer drugs. Herein, NSAID hydroxamic acids 3a-i were prepared by a new synthetic procedure and evaluated for their antiviral and cytostatic activity against malignant tumor cell lines and normal human fibroblasts (WI38). Antiviral activity evaluation results indicated that 3f had only a minor activity against the influenza virus A/H1N1 subtype with a selectivity index of 7-10. On the other hand, the results of the in vitro cytostatic activity evaluations revealed that the majority of NSAID hydroxamic acid derivatives 3a-i exhibited a strong non-specific antiproliferative effect at the highest concentration (100 microM) on the tested cell line panel. Only compounds 3b, 3e and 3i exerted a differential dose-dependent inhibitory activity against the growth of HeLa cells (p < 0.05) at concentration 10 microM. Among those three compounds, only compound 3b showed a selective cytostatic effect on HeLa in comparison with normal fibroblasts.

The immunosuppressive activities of newly synthesized azaphenothiazines in human and mouse models

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 μg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.

Differential Inhibitory Activities and Stabilisation of DNA Aptamers against the SARS Coronavirus Helicase

The helicase from severe acute respiratory syndrome coronavirus (SARS‐CoV) possesses NTPase, duplex RNA/DNA‐unwinding and RNA‐capping activities that are essential for viral replication and proliferation. Here, we have isolated DNA aptamers against the SARS‐CoV helicase from a combinatorial DNA library. These aptamers show two distinct classes of secondary structure, G‐quadruplex and non‐G‐quadruplex, as shown by circular dichroism and gel electrophoresis. All of the aptamers that were selected stimulated ATPase activity of the SARS‐CoV helicase with low‐nanomolar apparent K m values. Intriguingly, only the non‐G‐quadruplex aptamers showed specific inhibition of helicase activities, whereas the G‐quadruplex aptamers did not inhibit helicase activities. The non‐G‐quadruplex aptamer with the strongest inhibitory potency was modified at the 3′‐end with biotin or inverted thymidine, and the modification increased its stability in serum, particularly for the inverted thymidine modification. Structural diversity in selection coupled to post‐selection stabilisation has provided new insights into the aptamers that were selected for a helicase target. These aptamers are being further developed to inhibit SARS‐CoV replication.

Stereoelectronic properties of spiroquinazolinones in differential PDE7 inhibitory activity.

A detailed computational study on a series of spiroquinazolinones showing phosphodiesterase 7 (PDE7) inhibitory activity was performed to understand the binding mode and the role of stereoelectronic properties in binding. Our docking studies reproduced the essential hydrogen bonding and hydrophobic interactions for inhibitors of this class of enzymes. The N1 proton of the quinazolinone scaffold was involved in H-bonding to an amide side chain of the conserved glutamine residue in the active site. The central bicyclic ring of the molecules showed hydrophobic and pi-stacking interactions with hydrophobic and aromatic amino acid residues, respectively, present in the PDE7 active site. The docked conformations were optimized with density functional theory (DFT) and DFT electronic properties were calculated. Comparison of molecular electrostatic potential (MEP) plots of inhibitors with the active site of PDE7 suggested that the electronic distribution in the molecules is as important as steric factors for binding of the molecules to the receptor. The hydrogen bonding ability and nucleophilic nature of N1 appeared to be important for governing the interaction with PDE7. For less active inhibitors (pIC(50) < 6.5), the MEP maximum at N1 of the spiroquinazolinone ring was high or low based on the electronic properties of the substituents. All the more active molecules (pIC(50) > 6.5) had MEP highest at N3, not N1. Efficient binding of these inhibitors may need some rearrangement of side chains of active-site residues, especially Asn365. This computational modeling study should aid in design of new molecules in this class with improved PDE7 inhibition.

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

BackgroundComplementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal FindingsCDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/SignificanceThe high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Differential Inhibition of Long Interspersed Element 1 by APOBEC3 Does Not Correlate with High-Molecular-Mass-Complex Formation or P-Body Association

The human cytidine deaminase APOBEC3G (A3G) and other APOBEC3 proteins exhibit differential inhibitory activities against diverse endogenous retroelements and retroviruses, including Vif-deficient human immunodeficiency virus type 1. The potential inhibitory activity of human APOBEC proteins against long interspersed element 1 (LINE-1) has not been fully evaluated. Here, we demonstrate inhibition of LINE-1 by multiple human APOBEC3 cytidine deaminases, including previously unreported activity for A3DE and A3G. More ancient members of APOBEC, cytidine deaminases AID and APOBEC2, had no detectable activity against LINE-1. A3A, which did not form high-molecular-mass (HMM) complexes and interacted poorly with P bodies, was the most potent inhibitor of LINE-1. A3A specifically recognizes LINE-1 RNA but not the other cellular RNAs tested. However, in the presence of LINE-1, A3A became associated with HMM complexes containing LINE-1 RNA. The ability of A3A to recognize LINE-1 RNA required its catalytic domain and was important for its LINE-1 suppression. Although the mechanism of LINE-1 restriction did not seem to involve DNA editing, A3A inhibited the accumulation of nascent LINE-1 DNA, suggesting interference with LINE-1 reverse transcription and/or integration or intracellular movement of LINE-1 ribonucleoprotein. Thus, association with P bodies or cellular HMM complexes could not predict the potency of APOBEC3 anti-LINE-1 activities. The catalytic domain of APOBEC3 proteins may be important for proper folding and target factors such as RNA or protein interaction in addition to cytidine deamination.

Inhibitory Effect of Select Nitrocompounds on Growth and Survivability of Listeria monocytogenes In Vitro

We report the effects of 2-nitro-1-propanol (2NPOH), 2-nitroethanol (2NEOH), and nitroethane (NE) on growth and survivability of Listeria monocytogenes. In all cases, inhibition was greatest with 2NPOH and least with NE. For example, specific growth rates of L. monocytogenes strain 18 declined (P < 0.05) 76, 60, and 29% from controls during aerobic culture at 37°C in brain heart infusion broth containing 10mM 2NPOH, 2NEOH, or NE, respectively. Mean specific growth rate for the controls incubated likewise without added nitrocompound was 0.62±0.02 h−1. Specific growth rates of L. monocytogenes Scott A decreased (P < 0.05) 67, 45, and 11%, respectively, from controls (0.67±0.02 h−1) when cultured similarly. Specific growth rates for L. monocytogenes strain 18 incubated similarly except at 30°C were reduced (P < 0.05) 76, 60, and 30%, respectively, and were reduced (P < 0.05) 78, 23, and 23% during anaerobic culture at 30°C in brain heart infusion broth containing 15mM 2NPOH, 2NEOH, or NE (control rates ranged from 0.37±0.07 to 0.74±0.05 h−1). Survivability of L. monocytogenes strain 18 was reduced (P < 0.05) during aerobic storage (4 months at 4°C) in brain heart infusion broth containing 2NPOH or 2NEOH (by 7.8 and 1.9 log units, respectively) but not NE. The inhibitory effect of 2NPOH was approximately 20% greater during growth at pH 7.0 than at pH 5.6 or 8.0. These results demonstrate the differential inhibitory activity of 2NPOH, 2NEOH, and NE against L. monocytogenes in vitro.

α-Tocopheryl Succinate Induces Apoptosis in Prostate Cancer Cells in Part through Inhibition of Bcl-xL/Bcl-2 Function

Although the antitumor effect of alpha-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby alpha-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that alpha-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 microm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of alpha-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of alpha-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.

The ascaphins: a family of antimicrobial peptides from the skin secretions of the most primitive extant frog, Ascaphus truei

The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1–8, were isolated from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus. Ascaphins 2–7 may be represented by the consensus amino acid sequence GX 2DX 2KGAAKX 3KTVAX 2IANX·COOH whereas ascaphin-1 (GFRDVLKGAAKAFVKTVAGHIAN·NH 2) and ascaphin-8 (GFKDLLKGAAKALVKTVLF·NH 2) contain a C-terminally α-amidated residue. The ascaphins show no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the cationic, amphipathic α-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.

Expression Patterns of α2,3-Sialyltransferases and α1,3-Fucosyltransferases Determine the Mode of Sialyl Lewis X Inhibition by Disaccharide Decoys

A variety of human adenocarcinomas express sialylated, fucosylated Lewis blood group antigens on cell surface and secreted mucins. Binding of these antigens to P-selectin on platelets is thought to facilitate formation of platelet-tumor emboli in the circulation, which in turn allows sequestration of the tumor cells in the microvasculature. Here we report a pharmacologic approach for blocking these interactions through metabolic inhibition of sialylation. Peracetylated forms of Galbeta1,4GlcNAcbeta-O-naphthalenemethanol and GlcNAcbeta1,3Galbeta-O-naphthalenemethanol were taken up by LS180 human colon carcinoma cells, O-deacetylated, and utilized as biosynthetic intermediates, resulting in heterogeneous oligosaccharides. The primed oligosaccharides included sialylated, sulfated, and fucosylated products based on mass spectrometry. Assembly of free oligosaccharides on the glycosides decoyed glycosylation of cellular glycoproteins, as assessed by altered binding of lectins and carbohydrate-specific antibodies. Expression of alpha2,3-sialylated oligosaccharides on the cell surface was diminished specifically, whereas alpha2,6-sialylation and fucosylation were not. In U937 lymphoma cells, the glycosides decreased fucosylation without affecting sialylation. The differential inhibitory activities correlated inversely with fucosyltransferase and sialyltransferase activity based on enzyme assays and microarray analysis. Regardless of the mechanism, the disaccharides blocked the cells from forming selectin ligands and inhibited adhesion to immobilized selectins, suggesting that the glycosides might prove useful for interfering with tumor cell adhesion and metastasis.

Antagonism by IL-4 and IL-10 of Endotoxin-Induced Tissue Factor Activation in Monocytic THP-1 Cells: Activating Role of CD14 Ligation

Background.Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation.Results.TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 μg/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells.Conclusions.Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.

Chickpea Defensive Proteinase Inhibitors Can Be Inactivated by Podborer Gut Proteinases1

Abstract Developing chickpea (Cicer arietinum L.) seeds 12 to 60 d after flowering (DAF) were analyzed for proteinase inhibitor (Pi) activity. In addition, the electrophoretic profiles of trypsin inhibitor (Ti) accumulation were determined using a gel-radiographic film-contact print method. There was a progressive increase in Pi activity throughout seed development, whereas the synthesis of other proteins was low from 12 to 36 DAF and increased from 36 to 60 DAF. Seven different Ti bands were present in seeds at 36 DAF, the time of maximum podborer (Helicoverpa armigera) attack. Chickpea Pis showed differential inhibitory activity against trypsin, chymotrypsin, H. armigera gut proteinases, and bacterial proteinase(s). In vitro proteolysis of chickpea Ti-1 with various proteinases generated Ti-5 as the major fragment, whereas Ti-6 and -7 were not produced. The amount of Pi activity increased severalfold when seeds were injured by H. armigera feeding. In vitro and in vivo proteolysis of the early- and late-stage-specific Tis indicated that the chickpea Pis were prone to proteolytic digestion by H. armigera gut proteinases. These data suggest that survival of H. armigera on chickpea may result from the production of inhibitor-insensitive proteinases and by secretion of proteinases that digest chickpea Pis.

Differential Interaction of the Human Cholesteryl Ester Transfer Protein with Plasma High Density Lipoproteins (HDLs) from Humans, Control Mice, and Transgenic Mice to Human HDL Apolipoproteins

Plasma high density lipoproteins (HDLs) from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoprotein A-II (HuAIITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with the human cholesteryl ester transfer protein (CETP) was studied. Whereas cholesteryl ester transfer rates were gradually enhanced by the addition of moderate amounts of HDL from the different sources, striking differences appeared when HDL levels kept increasing beyond a maximal transfer value. Indeed, while a plateau value corresponding to maximal CETP activity was maintained when raising the concentration of HuAITg HDL and HuAIAIITg HDL, inhibitions could be observed with the highest levels of human, control mouse, and HuAIITg mouse HDL. The concentration-dependent inhibition of CETP activity could be reproduced by the addition of delipidated HDL apolipoproteins from control mice, but it was abolished by a 1-h preheating treatment at 56 degrees C. In contrast, no significant inhibition of CETP activity was observed with the delipidated protein moiety of HuAITg HDL, and cholesteryl ester transfer rates remained unchanged before and after a 1-h, 56 degrees C preheating step. Finally, the CETP-mediated transfer of radiolabeled cholesteryl esters from human low density lipoprotein to human HDL was significantly higher in the presence of lipoprotein-deficient plasma from HuAITg mice than in the presence of lipoprotein-deficient plasma from control mice. Interestingly, cholesteryl ester transfer rates measured with both control and HuAITg lipoprotein-deficient plasmas became remarkably similar following a 1-h, 56 degrees C preheating treatment. It is concluded that human, control mouse, and HuAIITg mouse HDL contain a heat-labile lipid transfer inhibitory activity that is absent from HDL of HuAITg and HuAIAIITg mice. Alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitory activities.

In vitro suppression of HIV-1 replication by ajoene [(e)-(z)-4,5,9-trithiadodeca-1,6,11-triene-9 oxide

Studies were performed to establish whether synthetic ajoene exhibited differential inhibitory activity against human immunodeficiency virus (HIV)-1 (IIIB) and to clarify the mechanism of its antiviral effects. Our results demonstrate that ajoene protected acutely infected Molt-4 cells against HIV-1 and blocked further destruction of CD4 T-cells in vitro. Ajoene showed dose-dependent inhibition, with 50% cytotoxic concentration (CTC50%) and 50% effective inhibitory concentration (EIC50%) values of 1.88 microM and about 0.35 microM, respectively, when the test compound was added before or after HIV-1 infection and incubation carried out at 37 degrees C for 4 days. Ajoene proved relatively more active than dextran sulfate in blocking HIV-1 virus-cell attachment. The mode of anti-HIV action of ajoene can be ascribed to the inhibition of early events of viral replication, particularly virus adsorption.

In vitro Activity of Extracts fromPolyporaceae against Human Immunodeficiency Virus Type 1

Aqueous extracts of Fomitella supina, Phellinus rhabarbarinus, Trichaptum perrottettii, and Trametes cubensis (Polyporaceae) from the Amazonian rain forest of Venezuela exhibited differential cytotoxicity and inhibitory activity against the human immunodeficiency virus type 1 (HIV-1). These extracts inactivated extracellular virions and suppressed HIV-1 replication by blocking HIV-1-induced cell–cell fusion in an in vitro culture system. They also stimulated interferon (IFN) production, active against HIV-1 and vesicular stomatitis virus (VSV) cytopathic effects. The results suggest that these extracts contain novel anti-HIV active compounds which, individually or in combination, can exert several distinct suppressive effects. The test extracts appear to act specifically at the level of CD4-gp120 binding.

Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Apple Fruit

A polygalacturonase-inhibiting protein (PGIP) was purified from mature 'Golden Delicious' apple fruit. The protein was cell wall bound and had a molecular mass of 44 to 54 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chemical deglycosylation of purified apple PGIP released a 34-kDa polypeptide, suggesting that differential glycosylation accounted for the heterogeneity in molecular mass. Apple PGIP showed differential inhibitory activity against five polygalacturonase isozymes purified from Botrytis cinerea grown in liquid culture. However, inhibition was not detected against polygalacturonase extracted from apple fruit inoculated with the same fungus. Kinetic studies suggested a mixed-type inhibition. N-terminal amino acid sequence of apple PGIP shared 96%, 68%, and 60% identity with those from pear, tomato, and bean, respectively.

C-C chemokines induce the chemotaxis of NK and IL-2-activated NK cells. Role for G proteins.

The C-C chemokines MIP-1 alpha, MCP-1, and RANTES, but not MIP-1 beta, induce the chemotaxis of NK and IL-2-activated NK (IANK) cells, as determined in microchemotaxis assay. Only RANTES and MCP-1, but not MIP-1 alpha were able to induce the chemokinesis of NK cells. In contrast, none of the C-C chemokines tested was able to induce the chemokinesis of IANK cells. IANK cell chemotaxis in response to MCP-1 or RANTES but not MIP-1 alpha, was inhibited by pertussis toxin (PT). In contrast, cholera toxin (CT) inhibited the ability of all three chemokines to induce the chemotaxis of IANK cells. IANK cells intoxicated with PT lost their ability to migrate in response to RANTES and MCP-1 but not MIP-1 alpha, whereas those intoxicated with CT lost their ability to migrate in response to the three C-C chemokines tested. These results suggest that guanine nucleotide binding (G) proteins are coupled to C-C chemokine receptors in IANK cells. Subsequently, we observed that MIP-1 alpha, MCP-1, and RANTES, but not MIP-1 beta, enhance the binding of guanosine 5'-O-(thiotriphosphate), and increase the hydrolysis of [32P]GTP in IANK cell membranes. Further analysis showed that MIP-1 alpha, RANTES, or MCP-1 did not enhance GTP binding in membranes prepared from IANK cells intoxicated with CT, whereas only RANTES and MCP-1 but not MIP-1 alpha lost their ability to enhance GTP binding to IANK cell membranes prepared from PT-intoxicated cells. The differential inhibitory activity of CT and PT suggests that C-C chemokine receptors are coupled to different G proteins in IANK cells.

Differential effects of calyculin A and okadaic acid on the glucose-induced regulation of glycogen synthease and phosphorylase activities in cultured hepatocytes

The effects of the phosphatase inhibitors calyculin A and okadaic acid were investigated to determine the roles of protein phosphatases type 1 and 2A in the regulation of the activities of glycogen synthase and phosphorylase by glucose in a primary culture of hepatocytes. Glycogen synthesis, as measured by the incorporation of labelled glucose into glycogen, was inhibited in a dose-dependent manner by calyculin A (IC 50 = 2.2 nM) and okadaic acid (IC 50 = 14 nM). Glucose-induced activation of glycogen synthase was inhibited by calyculin A and okadaic acid with IC 50 values of 3.7 nM and 90 nM, respectively. Phosphorylase was simultaneously activated by these inhibitors with calyculin A again being more active ( P < 0.001) than okadaic acid. The differing potencies ( P < 0.001) of these inhibitors on the activities of glycogen synthase and phosphorylase were also observed with varying concentrations of glucose (5.6–60 mM) in the medium and at different incubation periods upto 120 min. It has been previously shown that both inhibitors inhibit protein phosphatase-2A with equal potency and calyculin A is a more potent inhibitor of protein phosphatase-1 than okadaic acid. Heat- and proteinase-treated cytosolic fractions from hepatocytes incubated with calyculin A and okadaic acid showed similar differential inhibitory activities towards purified types 1 and 2-A protein phosphatases. Hence, these data provide further evidence that protein phosphatase type-1 plays a major role in the control of glycogen synthesis by regulating the activities of glycogen synthase and phosphorylase.

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets.

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic beta-cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of 3H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.