Differential Inhibitory Effects Research Articles

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri- o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-( o-cresyl)-4 H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di- o-tolyl- o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

Enhanced TARC production by dust-mite allergens and its modulation by immunosuppressive drugs in PBMCs from patients with atopic dermatitis

Background : Thymus and activation regulated chemokine (TARC) is a CC chemokine that attracts CCR4 + T cells. We reported previously that TARC is an important chemokine that defines Th2 imbalance in the pathogenesis of atopic dermatitis (AD). Objectives : This study was undertaken to clarify TARC producing cells in peripheral blood mononuclear cells (PBMCs), the regulation of dust mite-allergen clude extract (DME) and different immunosuppressive drugs (Tacrolimus (FK506), cyclosporine (CsA), dexamethasone (Dex)) on TARC production by peripheral PBMCs from AD patients in vitro. Methods : Monocyte derived dendritic cells (MoDCs) were generated from and TARC mRNA levels were examined and comapared with those from T cells in PBMCs from AD patients. PBMCs were cultured with or without DME and/or immunosuppressive drugs (Tacrolimus, CsA, Dex) for 7 days and TARC levels were measured. Results : PBMCs from AD patients which were cultured with DME stimulation for 7 days showed significantly higher levels of TARC production than those from healthy controls. RT–PCR demonstrated that TARC mRNA was expressed in CD4 + T cells, CD8 + T cells and MoDCs. Tacrolimus, CsA and Dex individually suppressed TARC production by PBMCs from AD patients which were co-cultured with DME for 7 days. Gel shift analysis revealed differential inhibitory effects of these immunosuppressive drugs on NFκB activity in PBMCs from AD patients. Conclusion : Our data demonstrate that TARC producing cells are MoDCs, T cells as well as epidermal keratinocytes in AD. We suggest that MoDCs might regulate the immune responses by attracting T cells and CD25 + T cells in the pathogenesis of AD. We also showed the important role of DME on TARC production and the inhibitory effect of the immunosuppressive drugs on TARC production by PBMCs from AD patients, that can regulate ongoing immune responses in the pathogenesis of AD.

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

A comparison of the immunosuppressive effects of salivary gland extracts from two laboratory strains of Boophilus microplus

This study addresses three questions related to the immune response of cattle to tick salivary gland extracts. Firstly, is there a difference in the inhibition of proliferation of Concanavalin A (ConA) stimulated bovine lymphocytes induced by salivary gland extracts of the N and Y strains of Boophilus microplus? Second, is there a difference in the development rate of the Y and N tick strains? Third, does the host affect the inhibitory effect of salivary gland extract on the proliferation of ConA stimulated lymphocytes from the two tick strains? Salivary gland extract of the Y strain inhibited in vitro proliferation of lymphocytes stimulated by ConA significantly more than that of the N strain, when each strain was raised on different animals. A difference in the development rate was observed between the tick strains when raised on the same animal, with female ticks of the Y strain developing faster and reaching a greater fully engorged weight than ticks of the N strain. The difference in their rate of development did not appear to contribute to a difference in inhibitory effects of the salivary gland extracts and there was no difference between the inhibitory effects of salivary gland extracts from both strains. However, when Y strain ticks were raised on different animals, there was a significant difference in the inhibition of lymphocyte proliferation between the two salivary gland extracts. Therefore, it was concluded that there is no difference between the inhibitory effects of the two tick strains and that the host has an influence on salivary gland extract composition of B. microplus and its inhibitive properties.

Expressions of inhibitory Smads, Smad6 and Smad7, are differentially regulated by TPA in human lung fibroblast cells

Smad6 and Smad7 are inhibitory Smads (I-Smads) with differential inhibitory effects on the regulation of the cellular signalings induced by TGF-β superfamily. Here, we show that phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) down-regulates Smad6 mRNA expression and up-regulates Smad7 mRNA expression in IMR-90, a human lung fibroblast cell line. These regulations of I-Smads by TPA were suppressed by one PKC inhibitor (Gö6983), but not by another (Gö6976). TPA treatment had little effect on the phosphorylation of novel PKCs (PKCδ and PKCε), but specifically induced PKCμ phosphorylation, and this effect was inhibited by Gö6983, but not by Gö6976. Additionally, Gö6983 but not Gö6976 inhibited ERK- and JNK-phosphorylation as well as Smad7 promoter activity induced by TPA. MEK inhibitor U0126 inhibited the down-regulation of Smad6 mRNA expression but not the up-regulation of Smad7 mRNA expression. In contrast, JNK inhibitor SP600125 had no such effects. Luciferase reporter analysis revealed that TPA did not induce NF-κB activation. In addition, TPA up-regulated Smad7 expression in the presence of NF-κB inhibitor TLCK. These findings indicate that TPA down-regulates Smad6 expression presumably via PKCμ-ERK-dependent pathway and up-regulates Smad7 expression via PKCμ-dependent mechanism(s) which need no MAPK and NF-κB activation.

A 71-kD heat shock protein (hsp) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis.

The effects of a mycobacterial 71-kD hsp antigen have been investigated for its ability to modulate arthritis in rats. Subcutaneous injection (base of tail) of increasing amounts of hsp71 from Mycobacterium tuberculosis (MTB) produced dose-dependent differential inhibitory effects on induction of arthritis by MTB and CP20961 in rats. As little as 1 microgram of the hsp71 produced a reduction in MTB arthritis, whereas complete protection was observed when 50 micrograms were administered. When 71-kD-treated rats were challenged with CP20961, all developed reduced symptoms of arthritis compared with control rats, but in this model no complete protection was observed over the dose range studied. The effects of 71-kD pretreatment on collagen II arthritis were not significant, but in general symptoms of arthritis were milder than in the control group. The same pattern of results was observed previously when hsp65 was used in the different models. These results show that the modulatory effects of hsp on adjuvant arthritis are not restricted to the hsp65 series, but are also mediated by a member of the hsp70 family.

Effect of taurine deficiency on adenosine receptor-mediated relaxation of the rat aorta

We recently demonstrated that chronic taurine supplementation or deficiency causes alterations in reactivity of the rat aorta to several vasoactive agents. In the present investigation, we examined the effects β-alanine-induced endogenous taurine deficiency on the mechanical responsiveness of the isolated rat aorta to adenosine receptor stimulation with 2-chloroadenosine (CAD), 5′- N-ethylcarboxyamidoadenosine (NECA), and N 6-cyclopentyladenosine (CPA). The adenosine analogs produced concentration-dependent (1×10 −9−3×10 −3 M) relaxations of aortas from both control and β-alanine-treated rats with the rank order of potencies NECA>CAD>CPA, which was consistent with A 2 receptor identification. CAD and NECA induced both endothelium-dependent and -independent relaxations of the aortas. The endothelium-dependent responses to both agents and the independent responses to CAD were significantly attenuated by β-alanine treatment. The relaxation responses of the aortas from control and taurine-deficient rats to CAD and NECA were markedly antagonized by ZM241385 (10 −5 M), suggesting the involvement of A 2A adenosine receptors. Further, N -nitro- l-arginine methyl ester ( l-NAME; 10 −5 M) significantly attenuated the endothelium-mediated relaxation produced by CAD and NECA in both groups. However, the inhibitory effect of l-NAME was less on the β-alanine-treated tissues, providing evidence that the effect of taurine deficiency was linked to a reduction in nitric oxide generation. As in the aorta, CAD produced both endothelium-dependent and -independent relaxation responses in the rat superior mesenteric artery, and both responses were inhibited by chronic β-alanine treatment, suggesting that not only similar responses can be generated by a given adenosine agonist in different vascular beds, but also β-alanine treatment modulates these responses. On the other hand, while CPA elicited only endothelium-independent aortic relaxation, this response was not altered by taurine deficiency. The results indicate that endogenous taurine deficiency causes differential inhibitory effects on adenosine receptor-mediated vasorelaxation, depending upon the agonists used. Given the recognized role of adenosine in the vasculature, these alterations suggest taurine-mediated modulation of blood flow regulation.

Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes.

Beneficial effects attributed to green tea, such as its anticancer and antioxidant properties, may be mediated by (-)-epigallocatechin-3-gallate (EGCG). In this study, the effects of EGCG on cell proliferation and UV-induced apoptosis were investigated in normal epidermal keratinocytes. When topically applied to aged human skin, EGCG stimulated the proliferation of epidermal keratinocytes, which increased the epidermal thickness. In addition, this topical application also inhibited the UV-induced apoptosis of epidermal keratinocytes. EGCG was found to increase the phosphorylation of Bad protein at the Ser112 and Ser136. Moreover, EGCG-induced Erk phosphorylation was found to be critical for the phosphorylation of Ser112 in Bad protein, and the EGCG-induced activation of the Akt pathway was found to be involved in the phosphorylation of Ser136. Furthermore, EGCG increased Bcl-2 expression but decreased Bax expression, causing an increase in the Bcl-2-to-Bax ratio. In addition, we demonstrate the differential growth inhibitory effects of EGCG on cancer cells. In conclusion, this study demonstrates that EGCG promotes keratinocyte survival and inhibits the UV-induced apoptosis via two mechanisms: by phosphorylating Ser112 and Ser136 of Bad protein through Erk and Akt pathways, respectively, and by increasing the Bcl-2-to-Bax ratio. Moreover, these two proposed mechanisms of EGCG-induced cell proliferation may differ kinetically to promote keratinocyte survival.

Effects of glycyrrhetinic acid derivatives on hepatic and renal 11beta-hydroxysteroid dehydrogenase activities in rats.

The purpose of this study was to examine the structure and activity relationships of glycyrrhetinic acid derivatives on the inhibition of hepatic and renal 11beta-hydroxysteroid dehydrogenases (HSDs) in rats. Furthermore, we explored whether inflammatory effect of the derivatives is involved in the inhibition of 11beta-HSD activity. 18beta-Glycyrrhetinic acid (Ia) potently inhibited 11beta-HSD activity of hepatic (IC50 (concentration giving 50% inhibition of cortisone production) = 0.09 microM) and renal (IC50 = 0.36 microM) homogenate. The inhibitory effect of 18beta-glycyrrhetol (Id) modified at the 30-position of glycyrrhetinic acid was weaker than that of glycyrrhetinic acid itself. 18beta-24-Hydroxyglycyrrhetinic acid (Ie), oxidized at the 24-position, remarkably reduced the inhibitory activity for both enzymes. 18beta-11-Deoxoglycyrrhetinic acid (IIc) showed the same inhibitory effect as glycyrrhetinic acid on hepatic 11beta-HSD activity, but less effect on renal 11beta-HSD activity. Furthermore, the inhibitory activity of 18beta-deoxoglycyrrhetol (IIa), modified at the 11- and 30-position, was markedly decreased. Dihemiphthalate derivatives (IIb, IIIb and IVb) of deoxoglycyrrhetol (IIa), 18beta-olean-9(11), 12-diene-3beta, 30-diol (IIIa) and olean-11, 13(18)-diene-3beta, 30-diol (IVa), which are anti-inflammatory agents, also showed weak inhibition against both hepatic and renal 11beta-HSDs. While glycyrrhetinic acid (200 mg kg(-1), p.o.) significantly inhibited 11beta-HSD activity in rat liver and kidney at 3 h after administration, compound IVb (100 mg kg(-1), p.o.) had no effect on either enzyme activity. In addition, the circulating corticosterone level was slightly increased by glycyrrhetinic acid but not by compound IVb. These results suggest that the anti-inflammatory effects of compound IVb, derived from glycyrrhetinic acid, are not due to accumulation of steroids induced by the inhibition of 11beta-HSD activity. Our data also showed that the 11-, 24- and 30-positions of glycyrrhetinic acid may play important roles in the differential inhibitory effects on 11beta-HSD isozyme activity.

Long term depression of human nociceptive skin senses induced by thin fibre stimulation

We have recently shown that stimulation, through a multi-electrode array, of thin nerve fibres close to the dermo-epidermal junction in the skin, produces powerful inhibition of itch and, to a lesser degree, cutaneous pain in humans. Here, we have studied the induction time and frequency dependency (range 1–10 Hz) of the inhibitory effects of such stimulation on itch, mechanical, and thermal pain, in 20 healthy subjects. Sixteen electrodes applied on the skin were consecutively stimulated using a method termed cutaneous field stimulation (CFS). The results show that different treatment periods with CFS were required for the induction of significant inhibitory effects on different nociceptive qualities: 1st heat pain (1 min), itch (3 min), 2nd heat pain (6 min), pinch evoked pain (8 min). Six to ten minutes stimulation sufficed to induce peak inhibitory effects on all these sensory qualities while longer stimulation (up to 40 min) did not cause significantly stronger inhibition. The effects on itch, 1st and 2nd heat pain lasted over 55 min after termination of CFS. There was no effect on prickle. No significant difference in inhibitory effects of different stimulation frequencies (1, 4 and 10 Hz/electrode) was found. The induction time and effective stimulation frequencies may suggest that the underlying mechanisms are similar to those of long term depression (LTD) previously described in the spinal cord in animal experiments.

Differential SERM Effects on Corepressor Binding Dictate ERα Activity in Vivo

Selective estrogen receptor modulators (SERMs) show differential effects upon ERalpha activation function 1 (AF-1). Tamoxifen allows strong ERalpha AF-1 activity, whereas raloxifene allows less and ICI 182,780 (ICI) allows none. Here, we show that blockade of corepressor histone de-acetylase (HDAC) activity reverses the differential inhibitory effect of SERMs upon AF-1 activity in MCF-7 cells. This suggests that differential SERM repression of AF-1 involves HDAC-dependent corepressors. Consistent with this, ICI and raloxifene are more potent than tamoxifen in promoting ERalpha-dependent sequestration of progesterone receptor-associated corepressors. Moreover, ICI and raloxifene are more efficient than tamoxifen in promoting ERalpha binding to the corepressor N-CoR in vivo and in vitro. An ERalpha mutation (537X) that increases N-CoR binding in the presence of all SERMs blocks AF-1 activity. An ERalpha mutation (L379R) that decreases N-CoR binding increases AF-1 activity in the presence of ICI and raloxifene and reverses the effect of the 537X mutation. The 537X and L379R mutations also alter the ligand preference of ERalpha action at AP-1 sites and C3 complement, an action that also involves AF-1. Together, our results suggest that differential SERM effects on corepressor binding can explain differences in SERM effects on ERalpha activity. We propose a model for differential effects of SERMs on N-CoR binding.

α1-Adrenergic Receptor Subtypes Differentially Control the Cell Cycle of Transfected CHO Cells through a cAMP-dependent Mechanism Involving p27 Kip1

Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed.

Quaternary benzo[c]phenanthridine alkaloids as inhibitors of dipeptidyl peptidase IV-like activity bearing enzymes in human blood plasma and glioma cell lines

Quaternary benzo[c]phenanthridine alkaloids (QBA), fagaronine (FA), sanguinarine (SA), chelerythrine (CHE) and the QBA extract from Macleya cordata (EX) exerted differential inhibitory effect on the hydrolytic activity of particular dipeptidyl peptidase (DPP)-like enzyme isolated from human blood plasma and from human and rat glioma cell lines. The low-MW form of DPP-IV-like enzyme activity, corresponding most probably to DPP-8, observed only in glioma cells but not in human plasma, was inhibited preferentially by SA, CHE and EX, and only slightly by FA. The alkaloid inhibitory effect was concentration-dependent in the range 25-150 M and directly pH-related. In addition, a subtle but consistent inhibition of the intermediate-MW form of DPP-IV-like enzyme activity, ascribed to DPP-IV/CD26, observed only in human plasma and of the attractin (high-MW form of DPP-IV-like enzyme activity, expressed in U87 glioma cells) by the studied alkaloids was observed. We conclude that some of the QBA biological effects could be determined by tissue and cell type specific dipeptidyl peptidase IV-like molecules expression pattern.

Dual innervation of the lamprey retina by GABAergic and glutamatergic retinopetal fibers: A quantitative EM immunogold study

A quantitative electron microscopic analysis of glutamate and GABA immunocytochemistry, using the postembedding immunogold technique, was undertaken in the lamprey retina to determine the proportion of glutamate-immunoreactive (GLU-ir) centrifugal visual terminals which were identified by anterograde axonal transport after an iontophoretic deposit of HRP in the sectioned optic nerve. Single immunogold labeling carried out with two different GABA and GLU antibodies showed that about 45% of the retinopetal axon terminals were GABAergic, and that two types of GLU-ir terminals (GLU-ir/1 and GLU-ir/2) were observed in the inner plexiform layer. The former type showed a high density of gold particles (Neosystem: 19.38±0.74; Sigma: 106.26±5.70) which statistically differed from the GLU-ir/2 (Neosystem: 3.23±0.31; Sigma: 31.73±5.61). Subcellular estimates showed that gold particles were concentrated over the vesicular pool in the GLU-ir/1 terminals and over mitochondria in the second terminal type. Consecutive sections alternately processed for GABA and GLU revealed that the GABA-immunonegative terminals corresponded to the GLU-ir/1, whereas the GABA-ir terminals only contained few GLU-ir particles, mostly concentrated in mitochondria. The occurrence of GABAergic and glutamatergic retinopetal terminals in the adult lamprey retina is discussed in functional terms of their differential inhibitory and excitatory effects on ganglion cell activity and the possible role of the centrifugal visual system in visually-guided behavior.

Differential Inhibitory Effect of alpha‐, beta‐, gamma‐, and delta‐Tocopherols on the Metal‐Induced Oxidation of Cholesterol in Unilamellar Phospholipid‐Cholesterol Liposomes

ABSTRACT: Cholesterol oxidation products (COPs) are present in biological tissues and in foods. The inhibitory effect of antioxidants, such as tocopherols, on COPs formation has been only partially investigated. The antioxidant effect of dl alpha‐, dl beta‐, dl gamma‐, and dl‐delta tocopherol on the metal‐induced oxidation of phosphatidylcholine (PC): cholesterol liposomes was assayed. Formation during liposome oxidation of six different COPs was monitored by gas chromatography. dl alpha‐, and dl gamma‐tocopherol show good inhibitory effect against PC‐fatty acid oxidation and also on COPs formation. dl delta‐Tocopherol is less effective than the alpha‐and gamma‐homologous, beta‐tocopherol being unable to prevent PC and cholesterol oxidation. dl alpha‐, and dl gamma‐Tocopherol are more effective to prevent the oxidation of the lateral chain of cholesterol molecule. At the highest tocopherol concentration assayed, dl alpha‐tocopherol shows prooxidant effect, enhancing liposomal oxidation and COPs formation. It is concluded that the tocopherols assayed can inhibit cholesterol oxidation but to a different degree.

Differential inhibitory effects of indomethacin, dexamethasone, and interferon-gamma (IFN-gamma) on IL-11 production by rheumatoid synovial cells.

IL-11, a member of the IL-6 type cytokines, has some biological activity related to the joint destruction in rheumatoid arthritis (RA), such as induction of osteoclast differentiation. However, its expression and regulation in rheumatoid inflamed joints has not been clarified. In the present study we examined the capacity of fresh rheumatoid synovial cells (fresh RSC) to produce IL-11, and the effect of indomethacin, dexamethasone and IFN-gamma on IL-11 production. Fresh RSC obtained from eight patients with RA produced large amounts of IL-11, measured by ELISA, and showed strong expression of IL-11 mRNA, determined by Northern blotting. Indomethacin inhibited the production of IL-11 by about 55%. Prostaglandin E2 (PGE2) completely prevented the inhibition, suggesting that IL-11 production by fresh RSC was in part mediated by PGE2. Dexamethasone inhibited the production of IL-11 by more than 80%. Interestingly, the inhibition was not abolished by PGE2. IFN-gamma inhibited the production of IL-11 from IL-1alpha-stimulated cultured rheumatoid synovial fibroblasts, although IFN-gamma did not inhibit the production of IL-11 by fresh RSC. These results suggest that the production of IL-11 by rheumatoid synovia was differentially regulated by PGE2 and IFN-gamma, and that treatment with indomethacin or dexamethasone decreased the level of IL-11 at inflammatory joints in patients with RA.

Simple shifts in redox/thiol balance that perturb blood coagulation

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.

Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3β-Hydroxysteroid Dehydrogenase

Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.

Electrophysiological studies on the role of the NMDA receptor in nociception in the developing rat spinal cord

The present study investigated the effects of spinally applied N-methyl- d-aspartate (NMDA) antagonists 2-amino-5-phosphonovaleric acid (AP5) and ketamine on convergent neurones in the deep dorsal horn of rats, in vivo at different postnatal ages (P) 14, 21, 28, and 56 days. AP5 inhibited the primary afferent fibre input, the C fibre, post-discharge and windup evoked responses in a dose-dependent manner at each age, and was significantly more effective in the pups than adult rats ( P<0.03 at 100-μg dose). AP5 100 μg abolished windup almost completely in the pups, whilst the adults required 10-fold higher doses. In contrast there was no difference in ketamine potency between age groups. Windup in the ketamine groups was reduced in a dose-dependent manner equally across all the age groups. The differential inhibitory effects of AP5 and ketamine may be due to postnatal changes in density, localisation and receptor subunit composition, altering receptor affinity and kinetics.

Differential inhibitory effects of sophoricoside analogs on bioactivity of several cytokines

Effects of sophoricoside and its analogs on proinflammatory cytokines have been investigated. Sophoricoside, genistein and orobol exhibited inhibitory effects on IL-5, IL-3, GM-CSF and IL-6 bioactivities. Genistin showed inhibitory effects on IL-5 and IL-3 bioactivities, but did not inhibit GM-CSF and IL-6 bioactivities. None of the sophoricoside analogs showed inhibitory effects on both IL-1β and TNF-α bioactivities. Among the compounds, sophoricoside exhibited the highest inhibitory effects on IL-5, IL-3 and IL-6 bioactivities with IC 50 values of 1.9 μM, 6.9 μM and 6.0 μM, respectively and orobol did show on GM-CSF bioactivity with an IC 50 value of 18.0 μM. The result would provide an additional mechanism by which the compounds exert immunosuppressive and anti-inflammatory effects.