Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.     

Production of anti-human thyroglobulin and anti-thyroid hormone antibodies in rabbits immunized with human thyroglobulin

Two rabbits (TG-1, TG-2) were immunized with human thyroglobulin (HTg) and bled serially. Antisera were obtained at different times after the first immunization and kept separately and studied. In both rabbits production of anti-HTg, and anti-thyroid hormone antibodies such as anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies was observed. Binding parameters of anti-HTg antibodies with HTg, T4, and T3 were calculated in two selected antisera (70-day and 249-day). The Scatchard's plots of these antibodies were all curve-linear and were analyzed in two components: one, higher binding constant (Ka1) and smaller binding capacity (Cap1) and the other, lower binding constant (Ka2) and larger binding capacity (Cap2). Ka1 values of anti-HTg, anti-T4, and anti-T3 antibodies in sera from TG-1 obtained from 70-day and 249-day bleeding were 1.1 X 10(10) M-1, 6.0 X 10(9) M-1. 7.9 X 10(8) M-1 and 1.7 X 10(10) M-1, 6.5 X 10(9) M-1, 1.0 X 10(9) M-1, respectively. Those from TG-2 were 1.7 X 10(10) M-1, 1.8 X 10(9) M-1, 6.4 X 10(8) M-1 and 2.0 X 10(10) M-1, 3.1 X 10(9) M-1, 1.6 X 10(9) M-1, respectively. The significance of the production of anti-HTg and anti-thyroid hormone antibodies in rabbits immunized with HTg in relation to the antigenic structure of HTg molecule was discussed.     

Noncompetitive antibody neutralization of IL-10 revealed by protein engineering and x-ray crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.     

Functional study of immature dendritic cells co-transfected with IL-10 and TGF-beta 1 genes in vitro

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previous studies suggest that immature dendritic cells (imDCs) might be involved in the induction of peripheral T cell tolerance. While interleukin-10 (IL-10) functions at different levels of the immune response, transforming growth factor-beta 1 (TGF-beta 1) is considered to be a key factor in immune tolerance. In this study, we investigated the effects of immature DC (imDC) co-transfected with IL-10 and TGF-beta 1 genes (IL-10-TGF-beta 1-imDC) on inducing immune tolerance. Moreover, we compared the effects of IL-10-TGF-beta 1-imDC with IL-10 transfected imDC (IL-10-imDC) and TGF-beta 1-transfected imDC (TGF-beta 1-imDC), respectively. IL-10-TGF-beta 1-imDC resulted in the down-regulation of MHC class II, CD80 and CD86. IL-10-TGF-beta 1-imDC could induce T cell hyporesponsiveness, and was reluctant to proliferate. IL-10-TGF-beta 1-imDC was more effective than IL-10-imDC and TGF-beta 1-imDC, respectively. In summary, co-expression of IL-10 and TGF-beta 1 affected the immunity of imDCs and enhanced their tolerogenicity. It might be a promising therapy for donor-specific tolerance after organ transplantation.     

Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.

Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor.     

Comparative role of retinol, retinoic acid and beta-carotene on progesterone secretion by pig corpus luteum in vitro

Quadruplicate wells of pig luteal cells were incubated for 24 h in the presence of different concentrations of retinol, beta-carotene (0, 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M) or retinoic acid (0, 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M). In addition, the responsiveness of luteal cells to LH challenge was also evaluated. Progesterone was assayed in the media. Cell viability was estimated using trypan blue exclusion and showed over 95% viability. In the presence of LH, progesterone content in the medium was increased by 7-fold. As compared to their respective controls, all concentrations of retinoic acid and beta-carotene increased progesterone content in the media. The highest level of stimulation was observed with 1 x 10(-6) M-retinoic acid (5-fold increase) and 1 x 10(-7) M-beta-carotene (10-fold increase). Only 1 x 10(-5) M-retinol stimulated progesterone secretion (over 3-fold). Therefore, retinol, retinoic acid and beta-carotene stimulate progesterone secretion by pig luteal cells in vitro.     

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.     

Interleukin-10 activation of the interleukin-10E1 pathway and tissue inhibitor of metalloproteinase-1 expression is enhanced by proteasome inhibitors in primary prostate tumor lines

The interleukin-10 (IL-10) activation of Janus kinase (JAK) family members (JAK1/TYK2) and IL-10E1 is subsequently inactivated by approximately 3-4 h in primary prostate tumor lines. We examined the effect of proteasome inhibition on IL-10 activation of the IL-10E1 pathway following stimulation of HPCA-10a cells. Treatment of HPCA-10a cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-10 receptor and IL-10E1 following stimulation. Further investigation showed that these stable phosphorylation events were the result of prolonged activation of JAK1 and TYK2 plus IL-10E1. IL-10E1 signaling normally induced the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and LLnL treatment of the HPCA-10a and HPCA-10c cells significantly enhanced IL-10 induction of TIMP-1 levels to block tumor cell invasion in modified Boyden chamber invasion assays. These observations were confirmed using pharmacologic inhibitors by Western blot and ELISAs. In the presence of LLnL, stable phosphorylation of IL-10E1 and induction of TIMP-1 was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on IL-10E1 phosphorylation and TIMP-1 could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting that phosphorylated IL-10E1 could be stabilized by phosphatase, but not by proteasome inhibition. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the IL-10E1 pathway and TIMP-1 induction by regulating the deactivation of JAK1/TYK2.     

Ligand binding by the chlorocruorin from Eudistylia vancouverii.

Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.     

Effect of histamine and antihistamines on interleukin-1 production by human monocytes

This study was carried out on the effect of histamine hydrochloride and its antagonists on the production of interleukin-1 (IL-1) by lipopolysaccharide (LPS)-stimulated adherent human monocytes (AHM) from normal healthy blood donors. IL-1 activity was evaluated by incorporation of [3H]-thymidine in mouse thymocytes in samples of 1:3 dilution. The result indicated that histamine hydrochloride significantly suppressed IL-1 production by AHM at 10(-3) M and 10(-10) M in 14 donors with maximal suppression observed at 10(-3) M. A 1-hr incubation with histamine hydrochloride (10(-3) M) before addition of LPS was found to be appropriate. Cimetidine, an H2-antagonist at 10(-3) M, 10(-5) M, and 10(-7) M significantly inhibited the effect of histamine hydrochloride (10(-3) M) and gave maximum inhibition at 10(-5) M, whereas chlorpheniramine maleate, and H1-antagonist had no significant inhibitory effect at the concentrations studied (10(-4) M, 10(-5) M, and 10(-7) M). Histamine hydrochloride (10(-3) M) added alone had no significant suppressive effect, while cimetidine (10(-5) M) alone had a significant stimulatory effect on IL-1 production by AHM.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Influence of the human blood serum on contractility and beta-adrenoreactyvity of the isolated human myocardium

On strips of the isolated myocardium of right hearts auriculum of the 43 patients with ischemic illness of heart and 9 patients with heart diseases of various ethyology at statement venous canule during aorto-coronary shunting, estimated influence of adrenaline (10(-9)-10(-4) g/ml) on amplitude caused by electrostimulus (1H, 5ms, 25-30 V) contractions, and also inotropic and adrenomodulation activity of serum blood (in dilution 1 : 10000, 1: 1000, 1 : 500, 1: 100, 1 : 50, 1: 10 and 1 : 5) nonpregnant women. Direct dependence of amplitude of contraction on size of fraction of of blood emission on Teyholts is revealed. It means, that strips of right auriculum myocardium reflect contractility of a left ventriculum myocardium. Adrenaline in concentration 10(-7)-10(-6) g/ml dependent of dose raised amplitude of the caused contraction not influencing it in concentration of 10(-9) and 10(-8) g/ml (the constant of dissotiation has 2 x 10(-7) g/ml), that as a whole, speaks about decrease in efficiency of activation beta-AP. Blood Serum in dissolutions 1 : 10000-1 : 50 did not influence on amplitude of contraction, and in dissolutions 1 : 10 and 1 : 5 strengthened it, that speaks presence in blood the endogenous activator of myocyte contractility (EAMC). Serum showed beta-adrenomodulation activity that speaks presence in it endogenous sensitizer of beta-adrenoreceptors (ESBAR) and endogenous blocker of beta-adrenoreceptors (EBBAR). In particular, in experiences with adrenaline in subthreshold concentration (10(-8) g/ml) serum showed ESBAR-activity (in dissolutions 1 : 1000, 1 : 500, 1 : 100 and 1 : 50), and in experiences with adrenaline in as much as possible effective concentration (10(-6) g/ml) serum showed ESBAR-activity (in dissolutions 1 : 50 and 1 : 10) and EBBAR-activity (in dissolutions 1:500) Hence, containing in blood serum endogenous modulators of beta-adrenoreactivity - ESBAR and EBBAR can modulate efficiency of beta-adrenoreceptors activation of human cardiomyocytes. It speaks about perspectivity of application of ES BAR analogues in cardiology.     

The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E2 (PGE2) production, were concentration-dependently inhibited by propolis (3 x 10(-3) - 3 x 10(2) microgml(-1)) with an IC50 of 2.7 microgml(-1) and 4.8 x 10(-2) microgml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4) - 28 microgml(-1); 10(-9) - 10(-4) M) and galangin (2.7 x 10(-4) - 27 microgml(-1); 10(-9) - 10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) microgml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) microgml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 microgml(-1) (15 x 10(-6) M) and 3 x 10(-2) microgml(-1) (120 x 10(-9) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 microgml(-1) and 5.3 x 10(-1) microgml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001).These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective.     

Design and analysis of an engineered human interleukin-10 monomer

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nm with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.     

Susceptibility of common voles to experimental toxoplasmosis

Common voles (Microtus arvalis) in groups of nine to 10 animals were inoculated per os with a dose of 1, 10, 1x10(2), 1x10(3), and of the K1 strain of Toxoplasma gondii. All the common voles inoculated with 1 to 1 x 10(3) oocysts remained subclinical and survived. Three of the 10 voles inoculated with 1 x 10(4) oocysts died between days 7 and 12 post inoculation (p.i.). Antibodies were demonstrated in all the infected voles killed on day 60 p.i. The highest antibody titres in voles detected by the dye test (DT) and latex agglutination test (LAT) were 1,024 and 1,280, respectively.     

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.     

Heat Shock Protein 90-α Mediates Aldo-Keto Reductase 1B10 (AKR1B10) Protein Secretion through Secretory Lysosomes

Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Our previous studies have shown that AKR1B10 is secreted through a lysosome-mediated nonclassical pathway, leading to an increase in the serum of breast cancer patients. This study illuminates the regulatory mechanism of AKR1B10 secretion. The cytosolic AKR1B10 associates with and is translocated to lysosomes by heat shock protein 90α (HSP90α), a chaperone molecule. Ectopic expression of HSP90α significantly increased the secretion of endogenous AKR1B10 and exogenous GFP-AKR1B10 fusion protein when cotransfected. Geldanamycin, a HSP90α inhibitor, dissociated AKR1B10-HSP90α complexes and significantly reduced AKR1B10 secretion in a dose-dependent manner. We characterized the functional domain in AKR1B10 and found that helix 10 (amino acids 233–240), located at the C terminus, regulates AKR1B10 secretion. Targeted point mutations recognized that amino acids Lys-233, Glu-236, and Lys-240 in helix 10 mediate the interaction of AKR1B10 with HSP90α. Together, our data suggest that HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. This finding is significant in exploiting the use of AKR1B10 in cancer clinics.     

The combination of the interleukin-1alpha (IL-1alpha-889) genotype and the interleukin-10 (IL-10 ATA) haplotype is associated with increased interleukin-10 (IL-10) plasma levels in healthy individuals

Family studies have demonstrated striking differences between individuals in their ability to produce IL-10 following lipopolysaccharide (LPS) stimulation of whole blood cultures in vitro, suggesting that differences in IL-10 production involve a considerable hereditary component. The first aim of this study was to analyse the possible effect of IL-10 genotypes and haplotypes on IL-10 plasma levels in a healthy Finnish population. As previous reports have demonstrated that endogenously produced IL-1 induces LPS-stimulated IL-10 production and that IL-10 inhibits synthesis of IL-1 in human monocytes, it is apparent that these two cytokines form an autoregulatory feedback loop. Secondly, we were interested whether any relationship could be found between IL-10 and IL-1beta in vivo. To examine this, the influence of IL-1alpha -889, IL-1beta -511 and IL-1Ra VNTR genotypes and IL-10 genotypes/haplotypes (ACC, GCC and ATA) on IL-10 plasma levels, and a putative correlation between IL-10 and IL-1alpha plasma levels were analysed. Four hundred adult blood samples were obtained from the Finnish Red Cross Blood Transfusion Centre, Tampere. The IL-10, IL-1alpha, IL-1beta and IL-1Ra gene polymorphisms were analysed using PCR. IL-1beta and IL-10 plasma levels were measured using an ELISA method. Our results indicated that increased IL-10 plasma levels were associated with the ATA haplotype (p = 0.03) and, surprisingly, with the IL-1alpha allele 2 carrier status (p = 0.02) in healthy individuals. This IL-1alpha 2+/ATA+ combination was found in 93 subjects out of 400 analysed (23%) and was associated with significantly high IL-10 plasma levels (p = 0.002). When individuals were classified into three groups, with no detectable IL-10 plasma levels (n = 145), with moderate levels (n = 152) and with high levels (n = 100) of IL-10, the IL-1alpha2+/ATA+ combination was more likely present among those with high levels than among those with undetectable levels of IL-10 (OR = 3.3, 95% CI 1.8 - 6.0, p < 0.001) or those with moderate levels of IL-10 (OR = 2.0, 95% CI 1.2 - 3.6, p = 0.012). Besides the observed association between IL-1alpha genotype and IL-10 levels, a moderate correlation was found between IL-10 and IL-1beta levels (r = 0.6, p = 0.01) among IL-10 producers (n = 252). The present findings suggest that the genotype combination of IL-1alpha 2+/ATA+ has a regulatory effect on basal IL-10 levels and that among individuals with measurable IL-10 plasma levels, IL-1beta and IL-10 basal levels correlate. Until now, data on the feedback loop between IL-1 and IL-10 cytokines have been based on studies in vitro, but now our results suggest that this relationship may also exist in vivo.     

Effects of veratrine and paeoniflorin on isolated mouse vas deferens

In this study, we attempted to identify the interactions and mechanisms between veratrine and paeoniflorin on isolated mouse vas deferens. Paeoniflorin had no effect on isolated mouse vas deferens. Veratrine (1 x 10(-5) approximately 1 x 10(-3) g/ml) could directly induce contraction of isolated rat and mouse vas deferens. The concentration induced by veratrine (1 x 10(-5) g/ml) was completely inhibited by Ca2+-free solution and verapamil (1 x 10(-5) M), in both the epididymal and the prostatic portions of isolated mouse vas deferens. Naloxone (1 x 10(-5) M) did not alter the contraction induced by veratrine (1 x 10(-5) g/ml) in either the epididymal or the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) inhibited the contraction induced by veratrine (1 x 10(-5) g/ml) in both the epididymal and the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) potentiated norepinephrine (1 x 10(-5) M)-induced phasic contraction in the epididymal portion, but decreased contractions in the prostatic portion. Paeoniflorin (4.8 x 10(-5) g/ml) increased KCI (56 mM)-induced phasic contraction in the epididymal portion, but decreased the tonic contraction in either the epididymal or the prostatic portion. Veratrine (1 x 10(-5) g/ml)-induced contractions could be decreased by pretreatment with ryanodine (1 x 10(-5) M) in both the epididymal and the prostatic portions. Pretreatment with the combination of paeoniflorin (4.8 x 10(-5) g/ml) and ryanodine (1 x 10(-5) M) did not potentiate the inhibition of paeoniflorin in the veratrine-induced contraction in both the epididymal and the prostatic portions of isolated mouse vas deferens.     

Anti-thyroid hormonal activity of tetrabromobisphenol A, a flame retardant, and related compounds: Affinity to the mammalian thyroid hormone receptor, and effect on tadpole metamorphosis

The thyroid hormone-disrupting activity of tetrabromobisphenol A (TBBPA), a flame retardant, and related compounds was examined. TBBPA, tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA) and 3,3'-dimethylbisphenol A (DMBPA) markedly inhibited the binding of triiodothyronine (T3; 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-7)-1 x 10(-4) M, while bisphenol A and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA and DMBPA did not exhibit thyroid hormonal activity in a thyroid hormone-responsive reporter assay using a Chinese hamster ovary cell line (CHO-K1) transfected with thyroid hormone receptor alpha1 or beta1, but TBBPA and TCBPA showed significant anti-thyroid hormone effects on the activity of T3 (1 x 10(-8) M) in the concentration range of 3 x 10(-6) - 5 x 10(-5) M. The thyroid hormone-disrupting activity of TBBPA was also examined in terms of the effect on amphibian metamorphosis stimulated by thyroid hormone. TBBPA in the concentration range of 1 x 10(-8) to 1 x 10(-6) M showed suppressive action on T3 (5 x 10(-8) M)-enhancement of Rana rugosa tadpole tail shortening. These facts suggest that TBBPA, TCBPA, TMBPA and DMBPA can act as thyroid hormone-disrupting agents.