Matrix metalloproteinases in diabetic retinopathy: potential role of MMP-9

INTRODUCTION: Diabetic retinopathy remains one of the most feared complications of diabetes. Despite extensive research in the field, the molecular mechanism responsible for the development of this slow progressing disease remains unclear. In the pathogenesis of diabetic retinopathy, mitochondria are damaged and inflammatory mediators are elevated before the histopathology associated with the disease can be observed. Matrix metalloproteinases (MMPs) regulate a variety of cellular functions including apoptosis and angiogenesis. Diabetic environment stimulates the secretion of several MMPs that are considered to participate in complications, including retinopathy, nephropathy and cardiomyopathy. Patients with diabetic retinopathy and also animal models have shown increased MMP-9 and MMP-2 in their retina and vitreous. Recent research has shown that MMPs have dual role in the development of diabetic retinopathy; in the early stages of the disease (pre-neovascularization), MMP-2 and MMP-9 facilitate the apoptosis of retinal capillary cells, possibly via damaging the mitochondria, and in the later phase, they help in neovascularization. AREAS COVERED: This article reviews the literature to evaluate the role of MMPs, especially MMP-9, in the development of diabetic retinopathy, and presents existing evidence that the inhibitors targeted toward MMP-9, depending on the duration of diabetes at the times their administration could have potential to prevent the progression of this blinding disease, and protect the vision loss. EXPERT OPINION: Inhibitors of MMPs could have dual role: in the early stages of the diseases, inhibit capillary cell apoptosis, and if the disease has progressed to the angiogenic stage, inhibit the growth of new vessels.     

Upregulation of phagocyte-like NADPH oxidase by cytokines in pancreatic beta-cells: Attenuation of oxidative and nitrosative stress by 2-bromopalmitate

Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β + TNFα + IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.     

A farnesylated G-protein suppresses Akt phosphorylation in INS 832/13 cells and normal rat islets: regulation by pertussis toxin and PGE₂

Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E?, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous protein kinase-B inhibitory protein [Probin] in islet function are discussed.     

Penicillin Sulfone Inhibitors of Class D β-Lactamases

OXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K_i values for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant K_i improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K_is for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t_n] = 0) and t_ns ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.The complexity and diversity of β-lactamases (EC 3.2.5.6) are largely determined by the unique manner in which the various amino acids define the active sites of these enzymes. OXA β-lactamases are class D enzymes (Bush-Jacoby group 2d) that are structurally related to the class C penicillin binding proteins (12, 14, 42, 61). Of primary importance, OXA β-lactamases are found in some of the most difficult-to-treat nosocomial pathogens (Pseudomonas aeruginosa and Acinetobacter spp.) (11, 33, 60). In these pathogens, OXA β-lactamases may confer resistance to penicillins, cephalosporins, and carbapenems (2, 27-29, 45, 55). The bla_OXA genes encoding resistance to β-lactams are located in the chromosome, on plasmids, or in integrons and may be inducible (25, 27, 30, 45, 51, 54).Currently, there are more than 140 different OXA β-lactamases reported (www.lahey.org). OXA-1 is a penicillinase found in Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa and exists as a monomer (58). Among the most-studied OXA enzymes are OXA-10, a dimer, and its clinically important derivatives, OXA-14 and -17. The last three enzymes confer resistance to ceftazidime and are regarded as extended-spectrum β-lactamases (ESBLs) of the class D family (22-24). The common OXA enzymes that confer resistance to carbapenems include OXA-23, -24/40, -48, -51, and -58 (8, 26, 34, 40, 52, 55). In contrast to the situation in P. aeruginosa, OXA carbapenemases are mostly found in Acinetobacter baumannii (49).The growing number of OXA β-lactamases found in nature generates considerable interest in both understanding the mechanistic basis of resistance to inactivation and developing effective inhibitors (6). When they are found in clinical isolates, OXA β-lactamases are poorly inhibited by the currently available β-lactam-β-lactamase inhibitor combinations (ampicillin-sulbactam, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, and piperacillin-tazobactam) (9, 13, 14, 45, 53). For the OXA-1 β-lactamase, the affinity of tazobactam is reduced, and the turnover of the inhibitor is significantly elevated (6). Moreover, little is known about the inactivation kinetics of other OXA β-lactamases with experimental inhibitors (1, 41, 44, 50).In the quest for new inhibitors, Buynak and coworkers designed and synthesized C-2-substituted 6-alkylidene penicillin sulfones and C-3-substituted 7-alkylidene cephalosporin sulfones as mechanism-based inactivators of class A β-lactamases (3, 16, 17, 48). In general, these β-lactamase inhibitors derive their success from their high affinities for the active site and ability to form stable reaction intermediates (46, 48). The aim of this present work was to determine the relative efficacies of C-2- and C-3-substituted 6/7-alkylidene penicillin and cephalosporin sulfones as β-lactamase inhibitors of the OXA-1, -10, -14, -17, and -24/40 β-lactamases. In contrast to what has been determined in the inactivation of the class C CMY β-lactamase and the class A SHV and TEM β-lactamases by mechanism-based or “suicide” inhibitors (clavulanic acid, tazobactam, and sulbactam; Fig. ​Fig.1,1, compounds 1 to 3), we show that C-2- and C-3-substituted penicillin and cephalosporin sulfone inhibitors form a covalent adduct that undergoes a unique reaction chemistry and does not fragment (10, 57, 59). This behavior may prove to be an important characteristic of successful β-lactamase inhibitors of class D enzymes.Open in a separate windowFIG. 1.Chemical structures of commercially available inhibitors: clavulanic acid, compound 1; tazobactam, compound 2; and sulbactam, compound 3. Substrates used in this study: penicillin G, compound 4; ampicillin, compound 5; oxacillin, compound 6, cephaloridine, compound 7; and nitrocefin, compound 8. Novel compounds used in this study: C-2-substituted 6-alkylidene penicillin sulfones, compounds 9 to 11; C-3-substituted 7-alkylidene cephalosporin sulfone, compound 12. The accepted ring numbering system is shown for cephaloridine, and C-3 of the isoxazole substituent on oxacillin is labeled.     

Periodic deworming with pyrantel in an industrial township

Between June 1986 and September 1987 a population of 100 families was dewormed every 3 months (quarter) by using a single dose of pyrantel. Stool samples were examined by Kato's thick smear method; height (m), weight (kg), hemoglobin concentration (g/dl) and clinical morbidity were recorded before each deworming treatment. Clinical morbidity was recorded in another 100 control families who did not receive anthelmlntics. There were 477 and 490 individuals in the study and the control families respectively. In the study group the prevalence of roundworm was reduced from 10.3% to to 0% and that of hookworm infection was reduced from 2.9% to 0% after 2 quarters. At the end of the 4th quarter the mean hemoglobin rose by 0.1 g/dl (P less than 0.01) and the mean BMI increased by 0.37 (P less than 0.01). There was a significant reduction in the clinical morbidity in the study group compared with the control group (P less than 0.05). There were no side effects reported to pyrantel treatment. Thus quarterly treatment with pyrantel was found to be effective in keeping roundworm and hookworm prevalence at 0% in an industrial township.     

Possible involvement of cholinergic and opioid receptor mechanisms in fluoxetine mediated antinociception response in streptozotocin-induced diabetic mice

Clinical and experimental studies have been reported that antidepressant drugs can be used as co-analgesics in the management of neuropathic pain. However, the mechanism through which they alleviate pain still remains unclear. The aim of the present study was to investigate the possible mechanism of action of fluoxetine-induced antinociceptive effect in streptozotocin-induced diabetic mice, especially the involvement of non-serotonergic neurotransmitters and their receptors. Diabetes was induced in male Laka mice with a single intraperitoneal injection of streptozotocin (200 mg/kg). Four weeks after streptozotocin, diabetic mice were tested for pain responses in the tail-immersion and hot-plate assays. Diabetic mice exhibited significant hyperalgesia as compared with control mice. Fluoxetine (10 and 20 mg/kg, i.p) injected into diabetic mice produced an antinociceptive effect in both tail-immersion and hot-plate assays. The antinociceptive effect of fluoxetine in diabetic mice was significantly lower as compared with that in control mice. Pretreatment with a muscarinic receptor antagonist, atropine (2 and 5 mg/kg, i.p) and an opioid receptor antagonist, naloxone (2 and 5 mg/kg, i.p), but not the alpha(2)-adrenoreceptor antagonist, yohimbine (2 and 5 mg/kg, i.p) reversed the antinociceptive effect of fluoxetine (20 mg/kg). These results suggest that apart from serotonin pathway, muscarinic and opioid receptors also participate in fluoxetine-induced antinociception in diabetic neuropathic pain.     

Liquid chromatographic tandem mass spectrometry method for the quantification of miglitol in human plasma

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of miglitol (CAS 72432-03-2), an alpha-glucosidase inhibitor, in human plasma using gabapentin (CAS 60142-96-3) as internal standard (IS). Following protein precipitation, the analytes were separated using an isocratic mobile phase on a reversed phase phenyl column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 208/146 for miglitol and m/z 172/154 for the IS. The assay exhibited a linear dynamic range of 100-6000 ng/mL for miglitol in human plasma. The lower limit of quantification was 100 ng/mL with a relative standard deviation of less than 5 %. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. The average absolute recoveries of miglitol and the IS from spiked plasma samples were 40.5 +/- 2.7 and 47.1 +/- 2.9 %, respectively. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The miglitol plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (Cmax) of miglitol (100 mg oral dose) is 1740 ng/mL, time to observed maximum plasma concentration (tmax) is 3.5 h and elimination half-life (t(1/2)) is 2.5 h.     

Comparative toxicity studies in birds using nimesulide and diclofenac sodium

Nimesulide, a sulfonanilide derivative, was compared with diclofenac sodium for toxicity in poultry. In this study, Vanaraja and PB1 birds of 6 weeks old (either sex) were mixed and equally divided into 5 groups of 10 birds each. The birds were inoculated with nimesulide, @ 5 and 2mg; vehicle @ 0.5ml; and diclofenac sodium @ 5mg on kg bwt basis. One group served as untreated control. All the groups were observed for a period of 28days. Forty percent mortality was observed within 12 days in diclofenac-treated group. While birds inoculated with nimesulide remained normal. No significant differences in the weight gain, haematology, total protein contents in the nimesulide and diclofenac groups (survived birds) were observed when compared with the control group of birds. Serum creatinine, cholesterol, alkaline phosphatase, and aspartate aminotransferase levels were significantly (P<0.05) high in diclofenac-treated group compared to nimesulide (P>0.05) and control groups. Nimesulide-treated groups did not show any histopathological lesions, where as diclofenac-treated birds showed histopathological lesions in liver and kidney.     

Development and validation of a sensitive liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of olanzapine in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the quantification of olanzapine, atypical antipsychotic drug, in human plasma using loratadine as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/256 for olanzapine and m/z 383/337 for the IS. The assay exhibited a linear dynamic range of 0.1-30 ng/mL for olanzapine in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recovery of olanzapine from spiked plasma samples was 85.5+/-1.9%. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.