Proteolytic enzyme engineering : a tool for wool

One of the goals of protein engineering is to tailor the structure of enzymes to optimize industrial bioprocesses. In the present work, we present the construction of a novel high molecular weight subtilisin, based on the fusion of the DNA sequences coding for Bacillus subtilis prosubtilisin E and for an elastin-like polymer (ELP). The resulting fusion protein was biologically produced in Escherichia coli, purified and used for wool finishing assays. When compared to the commercial protease Esperase, the recombinant subtilisinE-VPAVG220 activity was restricted to the cuticle of wool, allowing a significant reduction of pilling, weight loss and tensile strength loss of wool fibers. Here we report, for the first time, the microbial production of a functionalized high molecular weight protease for controlled enzymatic hydrolysis of wool surface. This original process overcomes the unrestrained diffusion and extended fiber damage which are the major obstacles for the use of proteases for wool finishing applications

Study on COgnition and Prognosis in the Elderly (SCOPE): baseline characteristics

Blood Press. 2000;9(2-3):146-51. Study on COgnition and Prognosis in the Elderly (SCOPE): baseline characteristics. Hansson L, Lithell H, Skoog I, Baro F, Bánki CM, Breteler M, Castaigne A, Correia M, Degaute JP, Elmfeldt D, Engedal K, Farsang C, Ferro J, Hachinski V, Hofman A, James OF, Krisin E, Leeman M, de Leeuw PW, Leys D, Lobo A, Nordby G, Olofsson B, Opolski G, Prince M, Reischies FM. University of Uppsala, Department of Public Health, Clinical Hypertension Research, Sweden. Abstract The Study on COgnition and Prognosis in the Elderly (SCOPE) is a multi-centre, prospective, randomized, double-blind, parallel-group study. The primary objective of SCOPE is to assess the effect of the angiotensin II type 1 (AT1) receptor blocker, candesartan cilexetil 8-16 mg once daily, on major cardiovascular events in elderly patients (70-89 years of age) with mild hypertension (DBP 90-99 and/or SBP 160-179 mmHg). The secondary objectives of the study are to test the hypothesis that antihypertensive therapy can prevent cognitive decline (as measured by the Mini Mental State Examination, MMSE) and dementia, and to assess the effect of therapy on total mortality, myocardial infarction (MI), stroke, renal function, and hospitalization. A total of 4964 patients from 15 participating countries were recruited during the randomization phase of SCOPE, exceeding the target population of 4000. The mean age of the patients at enrolment was 76 years, the ratio of male to female patients was approximately 1:2, and 52% of patients were already being treated with an antihypertensive agent at enrolment. The majority of patients (88%) were educated to at least primary school level. At randomization, mean sitting blood pressure values were SBP 166 mmHg and DBP 90 mmHg, and the mean MMSE score was 28. Previous cardiovascular disease in the study population included myocardial infarction (4%), stroke (4%) and atrial fibrillation (4%). Men, more often than women, had a history of previous MI, stroke and atrial fibrillation. A greater percentage of men were smokers (13% vs 6% in women) and had attended university (11% vs 3% of women). Of the randomized patients, 21% were 80 years of age. In this age group smoking was less common (4% vs 10% for 70-79-year-olds) and fewer had attended university (4% vs 7% for 70-79-year-olds). The incidence of MI was similar in both age groups. However, stroke and atrial fibrillation had occurred approximately twice as frequently in the older patients. The patients' mean age at baseline was similar in the participating countries, and most countries showed the approximate 1:2 ratio for male to female patients. There was also little inter-country variation in terms of mean SBP, DBP or MMSE score. However, there was considerable regional variation in the percentage of patients on therapy prior to enrolment. PMID: 10855739 [PubMed - indexed for MEDLINE

Study on COgnition and Prognosis in the Elderly (SCOPE)

Blood Press. 1999;8(3):177-83. Study on COgnition and Prognosis in the Elderly (SCOPE). Hansson L, Lithell H, Skoog I, Baro F, Bánki CM, Breteler M, Carbonin PU, Castaigne A, Correia M, Degaute JP, Elmfeldt D, Engedal K, Farsang C, Ferro J, Hachinski V, Hofman A, James OF, Krisin E, Leeman M, de Leeuw PW, Leys D, Lobo A, Nordby G, Olofsson B, Zanchetti A, et al. University of Uppsala, Department of Public Health, Sweden. Abstract The Study on COgnition and Prognosis in the Elderly (SCOPE) is a multicentre, prospective, randomized, double-blind, parallel-group study designed to compare the effects of candesartan cilexetil and placebo in elderly patients with mild hypertension. The primary objective of the study is to assess the effect of candesartan cilexetil on major cardiovascular events. The secondary objectives of the study are to assess the effect of candesartan cilexetil on cognitive function and on total mortality, cardiovascular mortality, myocardial infarction, stroke, renal function, hospitalization, quality of life and health economics. Male and female patients aged between 70 and 89 years, with a sitting systolic blood pressure (SBP) of 160-179 mmHg and/or diastolic blood pressure (DBP) of 90-99 mmHg, and a Mini-Mental State Examination (MMSE) score of 24 or above, are eligible for the study. The overall target study population is 4000 patients, at least 1000 of whom are also to be assessed for quality of life and health economics data. After an open run-in period lasting 1-3 months, during which patients are assessed for eligibility and those who are already on antihypertensive therapy at enrolment are switched to hydrochlorothiazide 12.5 mg o.d., patients are randomized to receive either candesartan cilexetil 8 mg once daily (o.d.) or matching placebo o.d. At subsequent study visits, if SBP remains >160 mmHg, or has decreased by 85 mmHg, study treatment is doubled to candesartan cilexetil 16 mg o.d. or two placebo tablets o.d. Recruitment was completed in January 1999. At that time 4964 patients had been randomized. All randomized patients will be followed for an additional 2 years. If the event rate is lower than anticipated, the follow-up will be prolonged. PMID: 10595696 [PubMed - indexed for MEDLINE

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Protective Intestinal Effects of Pituitary Adenylate Cyclase Activating Polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide widely distributed throughout the body, including the gastrointestinal tract. Several effects have been described in human and animal intestines. Among others, PACAP infl uences secretion of intestinal glands, blood fl ow, and smooth muscle contraction. PACAP is a well-known cytoprotective peptide with strong anti-apoptotic, anti-infl ammatory, and antioxidant effects. The present review gives an overview of the intestinal protective actions of this neuropeptide. Exogenous PACAP treatment was protective in a rat model of small bowel autotransplantation. Radioimmunoassay (RIA) analysis of the intestinal tissue showed that endogenous PACAP levels gradually decreased with longer-lasting ischemic periods, prevented by PACAP addition. PACAP counteracted deleterious effects of ischemia on oxidative stress markers and cytokines. Another series of experiments investigated the role of endogenous PACAP in intestines in PACAP knockout (KO) mice. Warm ischemia–reperfusion injury and cold preservation models showed that the lack of PACAP caused a higher vulnerability against ischemic periods. Changes were more severe in PACAP KO mice at all examined time points. This fi nding was supported by increased levels of oxidative stress markers and decreased expression of antioxidant molecules. PACAP was proven to be protective not only in ischemic but also in infl ammatory bowel diseases. A recent study showed that PACAP treatment prolonged survival of Toxoplasma gondii infected mice suffering from acute ileitis and was able to reduce the ileal expression of proinfl ammatory cytokines. We completed the present review with recent clinical results obtained in patients suffering from infl ammatory bowel diseases. It was found that PACAP levels were altered depending on the activity, type of the disease, and antibiotic therapy, suggesting its probable role in infl ammatory events of the intestine

Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis

BACKGROUND: Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC(50) for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential. CONCLUSIONS/SIGNIFICANCE: The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function

Obesity and Gastroesophageal Reflux: Quantifying the Association Between Body Mass Index, Esophageal Acid Exposure, and Lower Esophageal Sphincter Status in a Large Series of Patients with Reflux Symptoms

Obesity and gastroesophageal reflux disease (GERD) are increasingly important health problems. Previous studies of the relationship between obesity and GERD focus on indirect manifestations of GERD. Little is known about the association between obesity and objectively measured esophageal acid exposure. The aim of this study is to quantify the relationship between body mass index (BMI) and 24-h esophageal pH measurements and the status of the lower esophageal sphincter (LES) in patients with reflux symptoms. Data of 1,659 patients (50% male, mean age 51 ± 14) referred for assessment of GERD symptoms between 1998 and 2008 were analyzed. These subjects underwent 24-h pH monitoring off medication and esophageal manometry. The relationship of BMI to 24-h esophageal pH measurements and LES status was studied using linear regression and multiple regression analysis. The difference of each acid exposure component was also assessed among four BMI subgroups (underweight, normal weight, overweight, and obese) using analysis of variance and covariance. Increasing BMI was positively correlated with increasing esophageal acid exposure (adjusted R 2 = 0.13 for the composite pH score). The prevalence of a defective LES was higher in patients with higher BMI (p < 0.0001). Compared to patients with normal weight, obese patients are more than twice as likely to have a mechanically defective LES [OR = 2.12(1.63–2.75)]. An increase in body mass index is associated with an increase in esophageal acid exposure, whether BMI was examined as a continuous or as a categorical variable; 13% of the variation in esophageal acid exposure may be attributable to variation in BMI

HIV gp41 Engages gC1qR on CD4+ T Cells to Induce the Expression of an NK Ligand through the PIP3/H2O2 Pathway

CD4+ T cell loss is central to HIV pathogenesis. In the initial weeks post-infection, the great majority of dying cells are uninfected CD4+ T cells. We previously showed that the 3S motif of HIV-1 gp41 induces surface expression of NKp44L, a cellular ligand for an activating NK receptor, on uninfected bystander CD4+ T cells, rendering them susceptible to autologous NK killing. However, the mechanism of the 3S mediated NKp44L surface expression on CD4+ T cells remains unknown. Here, using immunoprecipitation, ELISA and blocking antibodies, we demonstrate that the 3S motif of HIV-1 gp41 binds to gC1qR on CD4+ T cells. We also show that the 3S peptide and two endogenous gC1qR ligands, C1q and HK, each trigger the translocation of pre-existing NKp44L molecules through a signaling cascade that involves sequential activation of PI3K, NADPH oxidase and p190 RhoGAP, and TC10 inactivation. The involvement of PI3K and NADPH oxidase derives from 2D PAGE experiments and the use of PIP3 and H2O2 as well as small molecule inhibitors to respectively induce and inhibit NKp44L surface expression. Using plasmid encoding wild type or mutated form of p190 RhoGAP, we show that 3S mediated NKp44L surface expression on CD4+ T cells is dependent on p190 RhoGAP. Finally, the role of TC10 in NKp44L surface induction was demonstrated by measuring Rho protein activity following 3S stimulation and using RNA interference. Thus, our results identify gC1qR as a new receptor of HIV-gp41 and demonstrate the signaling cascade it triggers. These findings identify potential mechanisms that new therapeutic strategies could use to prevent the CD4+ T cell depletion during HIV infection and provide further evidence of a detrimental role played by NK cells in CD4+ T cell depletion during HIV-1 infection

Aldosterone Antagonists in Monotherapy Are Protective against Streptozotocin-Induced Diabetic Nephropathy in Rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers