Antiophidic potential of chlorogenic acid and rosmarinic acid against Bothrops leucurus snake venom.

Bothrops leucurus is responsible for most cases of snakebite in Northeast Brazil; however, this species is not included in the pool of venoms used in antivenom production in Brazil. The serotherapy has logistical and effectiveness limitations, which stimulates the search for therapeutic alternatives. Chlorogenic acid and rosmarinic acid present several biological activities, but their antiophidic potential has been poorly explored. Thus, the aim of this approach was to evaluate the potential inhibitory effects of these compounds on B. leucurus venom. Initially, the enzymatic inhibition of toxins was evaluated in vitro. Then, anti-hemorrhagic, anti-myotoxic, and anti-edematogenic assays were performed in vivo, as well analysis of several biochemical markers and hemostatic parameters. In addition, the interaction of inhibitors with SVMP and PLA2 was investigated by docking analysis. Results revealed that compounds inhibited in vitro the enzymatic activities and venom-induced edema, with a decrease in both myeloperoxidase and interleukin quantification. The inhibitors also attenuated the hemorrhagic and myotoxic actions and mitigated changes in serum biochemical and hemostatic markers, as well as decreased lipid peroxidation in liver and kidney tissues. Docking analysis revealed attractive interactions of both inhibitors with the zinc-binding site of SVMP and, in the case of PLA2, chlorogenic acid showed a similar inhibition mechanism to that described for rosmarinic acid. The results evidenced the antiophidic potential of both compounds, which showed higher efficiency than antivenom serum. Thus, both inhibitors are promising candidates for future adjuvants to be used to complement antivenom serotherapy.

Evaluation of oxidative stress, inflammation, apoptosis, oxidative DNA damage and metalloproteinases in the lungs of rats treated with cadmium and carvacrol.

BACKGROUND: The potential protective properties of carvacrol (CRV), which possesses various biological and pharmacological properties, against lung toxicity caused by cadmium (Cd), a major environmental pollutant, were investigated in the present study. METHODS AND RESULTS: In the study, rats were given 25 or 50 mg/kg CRV orally 30 min after administrating 25 mg/kg cadmium chloride for seven days. Subsequently, the levels of 8-OHdG, MMP-2, and MMP-9, as well as markers of oxidative stress, inflammation, and apoptosis, were analyzed in the lung tissue of the animals. The results revealed that CRV exhibited antioxidant characteristics and raised SOD, CAT, GPx, and CAT levels and decreased the MDA levels induced by Cd. It also suppressed proinflammatory cytokines by lowering the levels of CRV NF-κB and p38 MAPK, thus exerting an anti-inflammatory effect against Cd. It was found that the levels of Bax, Caspase-3, and cytochrome c increased by Cd were decreased by the application of CRV. CRV also showed an anti-apoptotic effect by increasing Bcl-2 levels. The levels of 8-OHdG, MMP2, and MMP9, which increased with Cd administration, were observed to reduce after treatment with CRV. CONCLUSIONS: The results indicate that CRV has protective properties against Cd-induced lung toxicity.

Improvement in organic solvent resistance and activity of metalloprotease by directed evolution.

Improving enzyme stability in the presence of organic solvent is crucial for non-aqueous catalysis. In this study, directed evolution was applied to improve the tolerance of metalloprotease PT121 towards organic solvent. In presence of acetonitrile and acetone, three mutants (T46Y, H224 F, and H224Y) of PT121 showed excellent solvent stability, which increased their half-lives by 1.2-3.5-fold as compared to the wild-type enzyme. Kinetic constants (KM and kcat values) of the caseinolysis reaction presented H224 F and H224Y mutants have higher affinity than the wild-type, but T46Y mutant were similar to those of the wild-type enzyme. Interestingly, combined mutants T46Y/H224 F and T46Y/H224Y mutants presented awesome stability and excellent caseinolytic activity. Molecular dynamic simulation suggest that improved enzyme stability may be attributed to extensive non-covalent bond network resulting in a more compact structure. Disruption of the disulphide bond formation between Cys-30 and Cys-58 residues in the F56 V mutant is possibly the reason behind its low stability among all the selected mutants. Additionally, T46Y/H224 F and T46Y/H224Y showed a higher peptide synthetic activity in the presence of organic solvents than the wild-type, which renders these mutant enzymes as promising biocatalysts for biotechnological applications.

[Effect of the management of class I obesity with metformin on metalloproteinase activity in patients with chronic periodontitis]. / Efecto del manejo de la obesidad clase I con metformina sobre actividad de metaloproteinasas en pacientes con periodontitis crónica.

INTRODUCTION: Introduction: in Mexico the main problem in public health is obesity and other diseases that are associated whit this condition, including oral health. Objective: to evaluate the effect of metformin treatment in patients with class I obese on the activity of metalloproteinases present in periodontium with chronic periodontitis. Methods: a clinical study was conducted in 68 patients with class I obesity and periodontal disease. They were divided into 4 groups. 2 of them, in addition to the periodontal treatment, were administered metformin 850 mg per day for six weeks; 2 samples were taken per patient of periodontal tissue before and after each treatment, body mass index, plaque index and inflammation were measured. Acrylamide gel zymography was used to measure the activity of metalloproteinases in the sample of tissue collected. The data obtained were analyzed by descriptive statistics, student t for related samples and one-way ANOVA was performed considering p < 0.01 as statistically significant. Results: in the group of patients who were administered metformin at the end of the treatment, there was a decrease in the body mass index, the degree of inflammation and lower metalloproteinase activity, compared with the control group (65% vs 25%; < 0.01). Conclusions: treatment with metformin in patients with obesity class I and periodontal disease decreases BMI, improves the symptoms of chronic periodontitis and decreases the activity of metalloproteinases 1, 3, 8, V present in periodontium of these patients.

INTRODUCCIÓN: Introducción: el principal problema de salud pública en México es la obesidad y sus enfermedades asociadas, incluyendo las bucales. Objetivo: evaluar el efecto del tratamiento con metformina en pacientes obesos de clase I sobre la actividad de las metaloproteinasas presentes en el periodonto con periodontitis crónica. Métodos: se realizó un estudio clínico con 68 pacientes mujeres con obesidad de clase I y enfermedad periodontal. Se dividieron en 4 grupos; a 2 de ellos, además del tratamiento periodontal, se les administro metformina de 850 mg al día durante seis semanas. Se tomaron 2 muestras por paciente de tejido periodontal antes y después de cada tratamiento y se midió el índice de masa corporal (IMC), el índice de placa dentobacteriana y de inflamación. Mediante zimografía en gel de acrilamida se midió la actividad de las metaloproteinasas en la muestra de tejido recolectada. Los datos obtenidos fueron analizados mediante estadística descriptiva t de student para muestras relacionadas y se realizó ANOVA de una vía considerando p < 0,01 como estadísticamente significativa. Resultados: en el grupo de pacientes a las que se les administro metformina al final del tratamiento se observó una disminución del índice de masa corporal, del grado de inflamación y menor actividad de metaloproteinasas respecto al grupo control (65% frente a 25%; p < 0,01). Conclusiones: el tratamiento con metformina en pacientes con obesidad de clase I y enfermedad periodontal disminuye el IMC, mejora los síntomas de la periodontitis crónica y disminuye la actividad de las metaloproteinasas 1, 3, 8 y V presentes en el periodonto de estos pacientes.

Carbon monoxide inhibits the anticoagulant activity of Mojave rattlesnake venoms type A and B.

The Mojave rattlesnake is a unique species of pit viper that expresses either a highly potent phospholipase A2 (PLA2)-dependent neurotoxin containing venom nearly devoid of fibrinogenolytic metalloproteinases (venom type A) or a hemotoxic venom with a high percentage of metalloproteinases and PLA2 without any neurotoxin present (venom type B) depending on its geographical location in the Southwestern United States and Mexico. Given that PLA2 have been demonstrated to affect coagulation, it was hypothesized that the anticoagulant effects of both type A and B venoms could be assessed by thrombelastography, and determination made if these venoms were heme modulated. Both venom types were exposed to carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM) in isolation and then placed in human plasma with consequent coagulation kinetics assessed by thrombelastography. It was determined that type A venom was twice as potent as an anticoagulant compared to type B venom, and that both venoms were inhibited by carbon monoxide releasing molecule-2 but not its inactivated molecule. Given the lack of proteolytic activity of type A venom and the dependence of neurotoxicity on PLA2 activity, it may be possible that carbon monoxide could inhibit neurotoxicity based on inhibition of PLA2 anticoagulant activity. These data may serve as the rationale for extension of these observations into animal models to determine if CO may inhibit not just hemotoxic venom, but also PLA2-dependent neurotoxic venom.

Warhead biosynthesis and the origin of structural diversity in hydroxamate metalloproteinase inhibitors.

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are  potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining.

YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-oligopeptidase.

BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. RESULTS: In this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. CONCLUSIONS: The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.

Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers.

The treatment and outcomes of patients with rheumatoid arthritis (RA) have been transformed over the past two decades. Low disease activity and remission are now frequently achieved, and this success is largely the result of the evolution of treatment paradigms and the introduction of new therapeutic agents. Despite the rapid pace of change, the most commonly used drug in RA remains methotrexate, which is considered the anchor drug for this condition. In this Review, we describe the known pharmacokinetic properties and putative mechanisms of action of methotrexate. Consideration of the pharmacodynamic perspective could inform the development of biomarkers of responsiveness to methotrexate, enabling therapy to be targeted to specific groups of patients. Such biomarkers could revolutionize the management of RA.

Effect of Metalloproteinase Inhibitors on the Microtensile Bond Strength of Composite Resin to Er:YAG Laser-Irradiated Dentin.

This study evaluated the effect of matrix metalloproteinase (MMP) inhibitors - 2% (CHX) and sodium fluoride (NaF) (5000 ppm) - on microtensile bond strength (µTBS) of composite resin to Er:YAG laser-irradiated dentin after chemical degradation of the bond interface. The occlusal surface of forty sound human molars was removed exposing the dentin surface (n=10), which was polished, irradiated with Er:YAG laser, acid etched and dried. Twenty specimens were rewetted with 2% CHX (control group) and 20 were rewetted with NaF (5000 ppm). The adhesive system was applied and a 4-mm-high plateau of light-cured composite resin was built up. Resin-dentin sticks were obtained with a rectangular cross-sectional area (0.8-1 mm2) and were either stored in water at 37 ?#61616;C for 24 h or submitted to chemical degradation. For chemical degradation, they were immersed in 10% NaOCl aqueous solution for 5 h and rinsed in water for 1 h. The sticks were submitted to microtensile test in a mechanical testing machine at 0.5 mm/min until failure. Fracture pattern was analyzed using SEM. µTBS values were calculated in MPa and submitted to analysis of variance ANOVA (α=0.05). The variance analysis showed that the 'MMP inhibitor' and 'degradation' factors (p=0.214 and p=0.093, respectively) and interaction between the factors were not statistically significant (p=0.143). Mixed failure predominated in all groups. In conclusion, the 2% CHX and NaF 5000 ppm presented similar µTBS of composite resin to laser-irradiated dentin before and after chemical degradation.

Effect of Metalloproteinase Inhibitors on the Microtensile Bond Strength of Composite Resin to Er: YAG Laser-Irradiated Dentin

Abstract This study evaluated the effect of matrix metalloproteinase (MMP) inhibitors - 2% (CHX) and sodium fluoride (NaF) (5000 ppm) - on microtensile bond strength (μTBS) of composite resin to Er:YAG laser-irradiated dentin after chemical degradation of the bond interface. The occlusal surface of forty sound human molars was removed exposing the dentin surface (n=10), which was polished, irradiated with Er:YAG laser, acid etched and dried. Twenty specimens were rewetted with 2% CHX (control group) and 20 were rewetted with NaF (5000 ppm). The adhesive system was applied and a 4-mm-high plateau of light-cured composite resin was built up. Resin-dentin sticks were obtained with a rectangular cross-sectional area (0.8-1 mm2) and were either stored in water at 37 ?#61616;C for 24 h or submitted to chemical degradation. For chemical degradation, they were immersed in 10% NaOCl aqueous solution for 5 h and rinsed in water for 1 h. The sticks were submitted to microtensile test in a mechanical testing machine at 0.5 mm/min until failure. Fracture pattern was analyzed using SEM. μTBS values were calculated in MPa and submitted to analysis of variance ANOVA (α=0.05). The variance analysis showed that the 'MMP inhibitor' and 'degradation' factors (p=0.214 and p=0.093, respectively) and interaction between the factors were not statistically significant (p=0.143). Mixed failure predominated in all groups. In conclusion, the 2% CHX and NaF 5000 ppm presented similar μTBS of composite resin to laser-irradiated dentin before and after chemical degradation.

Resumo Este estudo avaliou o efeito dos inibidores de metaloproteinase, clorexidina 2% e fluoreto de sódio (5000 ppm), na resistência de união entre a dentina irradiada por laser Er:YAG e a resina composta após a degradação química da interface de união. A superfície oclusal de quarenta molares humanos hígidos (n=10) foi removida expondo uma superfície de dentina, que foi polida, irradiada com laser Er:YAG, condicionada com ácido e seca. Vinte espécimes foram re-umedecidos com clorexidina 2% (Grupo controle) e 20 com fluoreto de sódio (5000 ppm). O sistema adesivo foi aplicado e um platô de resina composta fotopolimerizável de 4 mm de altura foi construído. Palitos de resina-dentina foram obtidos com secção transversal retangular (0,8-1 mm2). Eles foram armazenados em água (24 h a 37 ?#61616;C) ou submetidos a degradação química. Para a degradação química, foram imersos em solução aquosa de hipoclorito de sódio a 10% durante 5 horas e lavados em água durante 1 h. Os palitos foram submetidos ao teste de microtração em uma máquina de ensaios mecânicos a 0,5 mm/min até a fratura. O padrão de fratura foi analisado em MEV. Os valores de resistência de união foram calculados em MPa e submetidos à análise de variância ANOVA (α=0,05). A análise de variância mostrou que os fatores inibidor de metaloproteinases e degradação (p=0,214 e p=0,093, respectivamente), e a interação entre os fatores não foram estatisticamente significantes (p=0,143). A predominância de falha mista foi detectada para todos os grupos. Em conclusão, a clorexidina a 2% e fluoreto de sódio (ppm 5000) apresentaram resistência de união entre dentina irradiada e resina composta semelhante antes e após a degradação química.

Limited efficacy of COX-2 inhibitors on nerve growth factor and metalloproteinases expressions in human synovial fibroblasts.

BACKGROUND: Nerve growth factor (NGF) is associated with arthritic pain and metalloproteinases are implicated in collagen and aggrecan degradation. Although selective COX-2 inhibitors are recommended for the treatment of arthritic diseases, their effects on NGF and metalloproteinases remain unclear. This study investigated the regulations of NGF and metalloproteinases by selective COX-2 inhibitors in isolated human synovial cells. METHODS: The isolated human synovial cells were stimulated with IL-1ß in the presence of selective COX-2 inhibitors (NS-398 or celecoxib) with or without exogenous PGE2 or its receptor (EP1-4) agonists. The expressions of NGF, MMP-1, -3, -13, ADAMTS-4, and -5 were quantified by real-time PCR and their proteins were determined by Western blotting. The amount of PGE2 released was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The IL-1ß inductions of NGF and MMP-1 and MMP-13 were augmented by the COX-2 inhibitors, whereas the inductions of ADAMTS-4 and ADAMTS-5 were inhibited. These actions were reversed by supplementing PGE2 or the EP4 agonist exogenously. CONCLUSION: Our comprehensive analysis revealed that COX-2 inhibitors may be beneficial for suppressing aggrecan degradation and for reducing inflammatory pain by inhibiting PGE2 release, although they may have limited efficacy in suppressing collagen degradation and nerve growth. This study suggests the feedback roles of PGE2 in the negative regulation of NGF and MMP-1 and MMP-13 and the positive regulation of ADAMTS-4 and ADAMTS-5.

Effects of metoclopramide on the expression of metalloproteinases and interleukins in left colonic anastomoses. An experimental study.

PURPOSE: To evaluate the effects of metoclopramide on metalloproteinases (MMP) and interleukins (IL) gene expression in colonic anastomoses in rats. METHODS: Eighty rats were divided into two groups for euthanasia on the 3rd or 7th postoperative day (POD), then into two subgroups for sepsis induction or not, and then into subgroups to receive either metoclopramide or saline solution. Left colonic anastomosis were performed and then analyzed. RESULTS: On the 3rd POD, metoclopramide was associated with increased expression of MMP-1a, MMP-13, and TNF-α. On the 7th POD, the transcripts of all MMPs, TNF-α, IL-1ß, IFN-γ, and IL-10 of the treated animals became negatively modulated. In the presence of sepsis, metoclopramide did not change MMPs and decreased IL-6, IL-1ß, IFN-γ and IL-10 gene expression on the 3rd POD. On the 7th POD, increased expression of all MMPs, IFN-γ and IL-10 and negative modulated TNF-α and IL-6 gene expression. CONCLUSION: Administration of metoclopramide increased metalloproteinases and interleukins gene expression on the 3rd postoperative day and negatively modulated them on the 7th POD. In the presence of abdominal sepsis, metoclopramide did not change MMPs and decreased ILs gene expression on the 3rd POD. On the 7th POD, the drug increased expression of all MMPs.

Effects of metoclopramide on the expression of metalloproteinases and interleukins in left colonic anastomoses. An experimental study

PURPOSE : To evaluate the effects of metoclopramide on metalloproteinases (MMP) and interleukins (IL) gene expression in colonic anastomoses in rats. METHODS : Eighty rats were divided into two groups for euthanasia on the 3rd or 7th postoperative day (POD), then into two subgroups for sepsis induction or not, and then into subgroups to receive either metoclopramide or saline solution. Left colonic anastomosis were performed and then analyzed. RESULTS : On the 3rd POD, metoclopramide was associated with increased expression of MMP-1a, MMP-13, and TNF-α. On the 7th POD, the transcripts of all MMPs, TNF-α, IL-1β, IFN-γ, and IL-10 of the treated animals became negatively modulated. In the presence of sepsis, metoclopramide did not change MMPs and decreased IL-6, IL-1β, IFN-γ and IL-10 gene expression on the 3rd POD. On the 7th POD, increased expression of all MMPs, IFN-γ and IL-10 and negative modulated TNF-α and IL-6 gene expression. CONCLUSION : Administration of metoclopramide increased metalloproteinases and interleukins gene expression on the 3rd postoperative day and negatively modulated them on the 7th POD. In the presence of abdominal sepsis, metoclopramide did not change MMPs and decreased ILs gene expression on the 3rd POD. On the 7th POD, the drug increased expression of all MMPs.

Second generation steroidal 4-aminoquinolines are potent, dual-target inhibitors of the botulinum neurotoxin serotype A metalloprotease and P. falciparum malaria.

Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 µM). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the Ki of compound 67 is 0.10 µM). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds' in vitro potencies. In addition to specific residue contacts, coordination of the enzyme's catalytic zinc and expulsion of the enzyme's catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2.

Class I histone deacetylase inhibition modulates metalloproteinase expression and blocks cytokine-induced cartilage degradation.

OBJECTIVE: To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro. METHODS: A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro. RESULTS: Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression. CONCLUSION: Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

Chronic ethanol consumption induces histopathological changes and increases nitric oxide generation in the rat liver.

In the present work we evaluated the effect of ethanol consumption in histopathological liver changes and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochondrial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a significant increase on hepatic nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostaining of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase (MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels) and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury, the increased levels of NO described in our study might contribute to a progressive hepatic damage. Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage.

Studies on the parameters controlling the stability of the TET peptidase superstructure from Pyrococcus horikoshii revealed a crucial role of pH and catalytic metals in the oligomerization process.

The TET proteases from Pyrococcus horikoshii are metallopeptidases that form large dodecameric particles with high thermal stability. The influence of various physico-chemical parameters on PhTET3 quaternary structure was investigated. Analytical ultracentrifugation and biochemical analyses showed that the PhTET3 quaternary structure and enzymatic activity are maintained in high salt and that the complex is stable under extreme acidic conditions. Under basic pH conditions the complex disassembled into a low molecular weight species that was identified as folded dimer. Metal analyses showed that the purified enzyme only contains two equivalent of zinc per monomer, corresponding to the metal ions responsible for catalytic activity. When these metals were removed by EDTA treatment, the complex dissociated into the same dimeric species as those observed at high pH. Dodecameric TET particles were obtained from the metal free dimers when 2mM of divalent ions were added to the protein samples. Most of the dimers remained assembled at high temperature. Thus, we have shown that dimers are the building units in the TET oligomerization pathway and that the active site metals are essential in this process.

Evaluating antimicrobial efficacy and cost of 3 dressings containing silver versus a novel antimicrobial hydrogel impregnated gauze dressing containing Oakin, an oak extract.

PURPOSE: Antimicrobial wound dressings are becoming more popular and are routinely used in the treatment of chronic and problematic wounds. Despite the ever-growing number and types of these antimicrobial products, many practitioners often do not report significant clinical differences between various common antimicrobial wound dressings despite wide variations in cost. Although these dressings use different active ingredients or different presentations of a particular active ingredient, all attempt to protect the wound from bacterial colonization and promote wound repair. With so many topical antimicrobial dressings to choose from in the clinical setting (many having already fallen into disfavor due to their cytotoxic characteristics) it was of prime interest to determine if there was a substantial difference between some of the more commonly used antimicrobial dressings, with silver versus an antimicrobial wound dressing using Oakin (oak extract [Amerx Health Care Corporation, Clearwater, Florida]) as the active ingredient. METHODS: This article compares the antimicrobial efficacy of 4 commonly used wound dressings in vitro, utilizing a corrected zone of inhibition test followed by a cost analysis. RESULTS: In vitro testing demonstrated that there were no substantial differences in the corrected zone of inhibition measurements between the silver wound dressings and the less expensive Oakin-impregnated gauze dressing. CONCLUSION: Despite obvious limitations of this study, these results suggest that the biggest differences between many antimicrobial dressings on the market may be more in cost than in antimicrobial efficacy. The differences in cost are due to variances in cost per application and frequency of applications per week.

Advanced or metastatic pancreatic cancer: molecular targeted therapies.

Patients with pancreatic cancer normally present with advanced disease that is lethal and notoriously difficult to treat. However, clinical developments over the past decade have been modest and advances are incremental at most. The core drug and the backbone of treatment in all settings of pancreatic adenocarcinoma-adjuvant, locally advanced, and metastatic-remains gemcitabine. The past decade of research focused initially on combining cytotoxic therapies with gemcitabine, and during that time a large number of studies have been published that aimed to target the molecular abnormalities implicated in pancreatic tumor growth, invasion, metastasis, angiogenesis, and resistance to apoptosis. This research is of particular importance, as data suggest that a large number of genetic alterations affect only a few major signaling pathways and processes involved in pancreatic tumorigenesis. Although laboratory results of targeted therapies have been impressive, until now only erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, has demonstrated marginal survival benefit in combination with gemcitabine in phase III clinical trials. Even though the failures of targeted therapies in the clinical setting are discouraging, it is reasonable to surmise that major progress will evolve as the molecular biology of pancreatic adenocarcinoma continues to be unraveled. This review describes some of the important developments and targeted agents for pancreatic cancer that have been tested in clinical trials.