Protective effects of the nuclear factor kappa B inhibitor pyrrolidine dithiocarbamate in bladder ischemia–reperfusion injury in rats

The aim of the present study was to evaluate the protective effects of the NF-кB inhibition with pyrrolidine-dithiocarbamate (PDTC) in ischemia–reperfusion (I/R) injury in the rat bladder. Twenty-four Sprague-Dawley male rats were divided into three groups. Group I; (n = 8) control, group II; (n = 8) I/R group; group III (n = 8) I/R and PDTC treatment. Superoxide dismutase (SOD), catalase (CAT), and gluatathione-S-transferase (GST) enzymes was studied in bladder tissue. Lipid peroxidation (as TBARS) levels in tissue homogenate were measured with thiobarbituric acid reaction. All the slides were stained with NF-кB, p53 and HSP60 immunohistochemistry for detection genome destruction and tissue stress, respectively. Our results show that the mean TBARS levels were significantly higher in group II (p < 0.05). The TBARS levels were significantly decreased in group III compared with the group II (p < 0.05). CAT, SOD and GST activities were decreased in group II, but these enzymes levels were significantly increased in group III according to the group II (p < 0.05). Under microscopic evaluation NF-кB expression increased significantly in group II compared to the group I (p < 0.05) and then decreased in group III (p < 0.05). HSP60 and p53 expression in group II was increased significantly compared with group I. Under microscopic evaluation we detected that HSP60 and p53 expression was increased significantly in group II compared with group I. In group III PDTC administration was decreased the HSP60 and p53 expression, this difference was statistically significant (p < 0.05). The results of the present study have demonstrated that NF-кB inhibition with PDTC protects and provides beneficial effects on ischemia/reperfusion stress related bladder tissue destruction.     

Effect of adenosine 3′,5′-(cyclic)-monophosphate on the synthesis of progestational steroids by rabbit ovarian tissue in vitro

1. Adenosine 3',5'-(cyclic)-monophosphate (3',5'-AMP) stimulates the synthesis of progestational steroids by rabbit ovarian tissue in vitro. 2. Other adenosine phosphates fail to increase steroidogenesis. 3. The ratio of 20alpha-hydroxypregn-4-en-3-one to progesterone, the maximal response of the tissue, and the responses of separated corpora lutea and interstitial tissue produced by luteinizing hormone are closely paralleled by 3',5'-AMP. 4. In tissues maximally stimulated by luteinizing hormone, 3',5'-AMP fails to produce an additional response. 5. The addition of theophylline, an inhibitor of phosphodiesterase, potentiates the effects of 3',5'-AMP and also luteinizing hormone. 6. The results obtained suggest that 3',5'-AMP is a mediator of the action of luteinizing hormone on progestational steroid synthesis by rabbit ovarian tissue.     

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart

The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups (n = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.     

Protective effects of dipeptidyl peptidase-4 (DPP-4) inhibitor against increased β cell apoptosis induced by dietary sucrose and linoleic acid in mice with diabetes

Chronic exposure to high glucose and fatty acid levels caused by dietary sugar and fat intake induces β cell apoptosis, leading to the exacerbation of type 2 diabetes. Oleic acid and linoleic acid are two major dietary fatty acids, but their effects in diabetes are unclear. We challenged β cell-specific glucokinase haploinsufficient (Gck(+/-)) mice with a diet containing sucrose and oleic acid (SO) or sucrose and linoleic acid (SL) and analyzed β cell apoptosis. In Gck(+/-) but not wild-type mice, SL significantly decreased the β cell mass and β cell proportion in islet cells arising from increased apoptosis to a greater degree than did SO. The mRNA expression of SREBP-1c was significantly higher, and that of E-cadherin was significantly lower in the islets of Gck(+/-) mice fed SL compared with mice fed SO. We next evaluated monotherapy with desfluorositagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in these mouse groups. DPP-4 inhibitor protected against β cell apoptosis, restored the β cell mass, and normalized islet morphology in Gck(+/-) mice fed SL. DPP-4 inhibition normalized the changes in the islet expression of SREBP-1c and E-cadherin mRNA induced by the SL diet. Furthermore, linoleic acid induced β cell apoptosis to a greater degree in the presence of high glucose levels than in the presence of low glucose levels in vitro in islets and MIN6 cells, whereas a GLP-1 receptor agonist prevented apoptosis. In conclusion, SL exacerbated β cell apoptosis in diabetic Gck(+/-) mice but not in euglycemic wild-type mice, and DPP-4 inhibition protected against these effects.     

Calcium- and calmodulin-independent modulation of calmodulin-sensitive hypothalamic cyclic nucleotide phosphodiesterase activity by the (11–19) fragment of thymosin β4

A fragment (11–19) of thymosin 4 was found to stimulate phosphodiesterase activity even in the absence of calcium and calmodulin. Half-maximal enzyme activation occurred with 10 nM peptide, and was further increased by phospholipids such as phosphatidylserine. The mechanism of stimulation is an increase in the V_max of cAMP degradation without a substantial change in the K_m for the substrate. In the presence of calcium ions and calmodulin the peptide was also stimulatory.     

Protective effect of N-(p-amylcinnamoyl) anthranilic acid,phospholipase A2 enzyme inhibitor,and transient receptor potential melastatin-2 channel blocker against renal ischemia–reperfusion injury

The production of reactive oxygen species and inflammatory events are the underlying mechanisms of ischemia-reperfusion injury (IRI). It was determined that transient receptor potential melastatin-2 (TRPM2) channels and phospholipase A₂ (PLA ₂) enzymes were associated with inflammation and cell death. In this study, we investigated the effect of N-( p-amylcinnamoyl) anthranilic acid (ACA), a TRPM2 channel blocker, and PLA ₂ enzyme inhibitor on renal IRI. A total of 36 male Sprague-Dawley rats were divided into four groups: control, ischemia-reperfusion (I/R), I/R + ACA 5 mg, I/R + ACA 25 mg. In I/R applied groups, the ischemia for 45 minutes and reperfusion for 24 hours were applied bilaterally to the kidneys. In the I/R group, serum levels of the blood urea nitrogen (BUN), creatinine, cystatin C (CysC), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and interleukin-18 increased. On histopathological examination of renal tissue in the I/R group, the formation of glomerular and tubular damage was seen, and it was detected that there was an increase in the levels of malondialdehyde (MDA), caspase-3, total oxidant status (TOS), and oxidative stress index (OSI); and there was a decrease in total antioxidant capacity (TAC) and catalase enzyme activity. ACA administration reduced serum levels of BUN, creatinine, CysC, KIM-1, NGAL, interleukin-18. In the renal tissue, ACA administration reduced histopathological damage, levels of caspase-3, MDA, TOS, and OSI; and it increased the level of TAC and catalase enzyme activity. It has been shown with the histological and biochemical results in this study that ACA is protective against renal IRI.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure–activity analyses: dissociation of antihypertensive and bradycardic effects

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr¹-Asn²-Trp³-Pro⁴-His⁵-Pro⁶-Gln⁷-Ile⁸-Pro⁹-Pro¹⁰). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala³, Bj-PRO-10c Ala⁷, Bj-PRO-10c Ala⁸ were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala², Bj-PRO-10c Ala⁴, Bj-PRO-10c Ala⁵, Bj-PRO-10c Ala⁹, and Bj-PRO-10c Ala¹⁰ kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala¹ and Bj-PRO-10c Ala⁶ were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr¹ and Pro⁶ residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro⁶ → Ala⁶ mutant is probably due to the fact that in the parent peptide the His⁵-Pro⁶ bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.     

In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC₅₀ values of 4.2 × 10^?9 M for B16MEL4A5 and 7.7 × 10^?11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.     

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.     

Structure–activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor

Structure–activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund’s adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res. 1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain 2006, 10, 537].     

Protective effects of green tea polyphenols and their major component, (–)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells

SUMMARY

Oxidative modification of low density lipoprotein (LDL) appears to be important in the pathogenesis of atherosclerosis. Inhibiting the oxidation of LDL may retard or prevent the atherogenic process. However, susceptibility of LDL to oxidation in vitro and its atherogenicity in vivo may not always correlate. Subjects with familial hypercholesterolaemia (FH) develop severe, premature atherosclerosis despite having large, bouyant LDL particles which are less susceptible to oxidation. High dose, long-term vitamin E increases the resistance of LDL to oxidation but, unlike probucol, has no effect on xanthoma regression in homozygous FH. In FH, the quantity of LDL takes priority and the main aim of therapy is reduction of LDL bulk. Individuals with small, dense LDL particles are at increased risk for atherosclerosis despite desirable plasma LDL cholesterol levels. Small, dense LDL particles are more susceptible to oxidation and in these subjects antioxidant therapy may be of greater benefit. In subjects with atherosclerosis, current management should be aimed primarily at reducing the LDL cholesterol level. In the future antioxidant therapy may complement our management of hypercholesterolaemia.     

Enhancement of permethrin efficacy in acaricide–attractant mixtures for control of the fowl tick Argas persicus (Acari: Argasidae)

Three acaricides, permethrin, propoxur and diazinon, were tested against Argas persicus ticks in a test of susceptibility and in a multiple choice test in bioassay. A mixture of guanine hydrochloride and diatomaceous earth in saline was used as an attractant in bioassays, causing 53.1–95.7% assembly. The attractant was mixed with acaricides to reduce their repellency and enhance their efficiency in bioassays. Permethrin was the most toxic (LC95 at day 7=0.5–1.4mg m–2 depending on the developmental stage) and most repellent acaricide. The mortality of males in the bioassay was significantly higher (76.7–94.3%, p<0.01) when acaricide in amounts of 16 and 160 g of active ingredient per filter paper disc were mixed with attractant (0.5 mg per filter paper disc) instead of acaricide alone (20–45.7% mortality only). The mean permethrin residue on the tick body at the end of bioassay with the acaricide– attractant mixture was significantly higher (13.62±11. 64 ng) than in experiments without the attractant (less than 1 ng). Propoxur was less toxic (LC95 at day 7=0.9–1.9 mg –2) and diazinon the least toxic (LC95 at day 7=2– 9.4 mg m–2), both being not or only slightly repellent. Males and females also assembled on filter paper discs treated with propoxur without an attractant. Diazinon displayed significant mortality only in amounts of 0.1 and 1 mg of active ingredient per filter paper disc with or without the attractant. Therefore, the repellency of permethrin can be reduced and its effectiveness enhanced when used in a mixture with an attractant. No similar effect was observed with propoxur or diazinon.     

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.     

Changes in cellular levels of cyclic AMP in rat mast cells during secretion of histamine induced by immunoglobulin E decapeptide and ACTH(1–24) peptide: Comparison with immunological and ionophore triggers

The role of cyclic AMP in the secretory mechanism of mast cells has been investigated by comparing the time course of changes in cellular levels of this cyclic nucleotide with the kinetics of secretion induced by basic peptides, antigen, anti-IgE and calcium ionophore. ACTH(1–24) peptide and a synthetic decapeptide representative of the sequence 497–506 within the Cε4 domain of human IgE induced a transient rise in cyclic AMP which reached approx. 150% of the resting levels by 10 s. Peptide-induced secretion of histamine was also rapid, reaching a maximum after 5–10 s. Immunological triggering of mast cells with antigen and anti-IgE raised levels of cyclic AMP to 150% of resting levels within 15 s, accompanying secretion of histamine which reached a maximum after 30 s. A relatively slower release of histamine induced by the calcium ionophore A23187 was paralleled by a significant reduction in cyclic AMP to 50% of the resting levels after 300 s. These data suggest a relationship between the accumulation of cyclic AMP in mast cells and secretion of histamine mediated by the Cε4 decapeptide and the ACTH(1–24) peptide as well as by IgE-dependent mechanisms. However, the simultaneous increase in cyclic AMP and secretion of histamine suggests that the two events may not be causally related.