Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of £0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

Erythrocyte phosphoglucomutase activity of bipolar I patients currently using lithium or carbamazepine

Lithium has been used for the last five decades to treat bipolar disorder, but the molecular basis of its therapeutic effect is unknown. Phosphoglucomutase is a key enzyme in the metabolism of glycogen. In yeast, rabbit and human HEK293 cells, it is inhibited by lithium in the therapeutic concentration range. We measured the phosphoglucomutase activity in erythrocytes and the inhibitor constant for lithium in a population of healthy subjects and compared them to those of bipolar patients treated with lithium or carbamazepine. The specific activity of phosphoglucomutase measured in vitro in erythrocytes from control subjects presented a normal distribution, with the difference between the lowest and the highest activity being approximately 2-fold (0.53-1.10 nmol mg Hb-1 min-1). Comparison of phosphoglucomutase activity in untreated bipolar patients and control subjects showed no significant difference, whereas comparison between bipolar patients treated with carbamazepine or lithium revealed significantly lower mean values in patients treated with carbamazepine (747.3 ± 27.6 vs 879.5 ± 35.9 pmol mg Hb-1 min-1, respectively). When we studied the concentration of lithium needed to inhibit phosphoglucomutase activity by 50%, a bimodal distribution among the population tested was obtained. The concentration of LiCl needed to inhibit phosphoglucomutase activity by 50% was 0.35 to 1.8 mM in one group of subjects and in the other it was 3 to 4 mM. These results suggest that phosphoglucomutase activity may be significant in patients with bipolar disorder treated with lithium and carbamazepine.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 ± 5.06 vs 27.6 ± 3.25 nmol Hip-His Leu-1 min-1 mL-1 in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 ± 0.2 vs 3.43 ± 0.8 ng Ang I mL plasma-1 h-1 in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 ± 1.3 vs 22.7 ± 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.

Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Synergistic effect of mycophenolate mofetil and angiotensin-converting enzyme inhibitor in patients with chronic allograft nephropathy

Experimental data and few clinical non-randomized studies have shown that inhibition of the renin-angiotensin system by angiotensin-converting enzyme (ACE) associated or not with the use of mycophenolate mofetil (MMF) could delay or even halt the progression of chronic allograft nephropathy (CAN). In this retrospective historical study, we investigated whether ACE inhibition (ACEI) associated or not with the use of MMF has the same effect in humans as in experimental studies and what factors are associated with a clinical response. A total of 160 transplant patients with biopsy-proven CAN were enrolled. Eighty-one of them were on ACE therapy (G1) and 80 on ACEI_free therapy (G2). Patients were further stratified for the use of MMF. G1 patients showed a marked decrease in proteinuria and stabilized serum creatinine with time. Five-year graft survival after CAN diagnosis was more frequent in G1 (86.9 vs 67.7%; P < 0.05). In patients on ACEI-free therapy, the use of MMF was associated with better graft survival. The use of ACEI therapy protected 79% of the patients against graft loss (OR = 0.079, 95%CI = 0.015-0.426; P = 0.003). ACEI and MMF or the use of MMF alone after CAN diagnosis conferred protection against graft loss. This finding is well correlated with experimental studies in which ACEI and MMF interrupt the progression of chronic allograft dysfunction and injury. The use of ACEI alone or in combination with MMF significantly reduced proteinuria and stabilized serum creatinine, consequently improving renal allograft survival.

Association of angiotensin-converting enzyme activity and polymorphism with echocardiographic measures in familial and nonfamilial hypertrophic cardiomyopathy

Angiotensin-converting enzyme (ACE) activity and polymorphism contribute significantly to the prognosis of patients with cardiomyopathy. The aim of this study was to determine the activity and type of ACE polymorphism in patients with familial and nonfamilial hypertrophic cardiomyopathy (HCM) and to correlate these with echocardiographic measurements (echo-Doppler). We studied 136 patients (76 males) with HCM (69 familial and 67 nonfamilial cases). Mean age was 41 ± 17 years. DNA was extracted from blood samples for the polymerase chain reaction and the determination of plasma ACE levels. Left ventricular mass, interventricular septum, and wall thickness were measured. Mean left ventricular mass index, interventricular septum and wall thickness in familial and nonfamilial forms were 154 ± 63 and 174 ± 57 g/m² (P = 0.008), 19 ± 5 and 21 ± 5 mm (P = 0.02), and 10 ± 2 and 12 ± 3 mm (P = 0.0001), respectively. ACE genotype frequencies were DD = 35%, ID = 52%, and II = 13%. A positive association was observed between serum ACE activity and left ventricular mass index (P = 0.04). Logistic regression showed that ACE activity was twice as high in patients with familial HCM and left ventricular mass index ≥190 g/m² compared with the nonfamilial form (P = 0.02). No other correlation was observed between ACE polymorphisms and the degree of myocardial hypertrophy. In conclusion, ACE activity, but not ACE polymorphisms, was associated with the degree of myocardial hypertrophy in the patients with HCM.

Inhibition of cyclooxygenase activity by standardized hydroalcoholic extracts of four Asteraceae species from the Argentine Puna

We determined the anti-inflammatory activity of standardized extracts of four medicinal plant species (Baccharis incarum, B. boliviensis, Chuquiraga atacamensis, Parastrephia lucida) that grow in the Argentine Puna (3800 m above sea level) and that are used to reduce oxidative stress and alleviate gout and arthritic pain. The extracts of plant aerial parts were standardized in terms of total phenolic compounds and flavone/flavanone content and free radical scavenging activity. All extracts showed high phenolic compound concentration (0.5-1.6 mg/mL), mainly flavones and flavonols (0.1-0.8 mg/mL). The extracts showed hydrogen donating ability (DPPH and ABTS) and reactive oxygen species scavenging activity (O2●-, OH-, H2O2). The ability of the extracts to inhibit cyclooxygenase enzymes (COX-1 and COX-2) was determined by calculating percent inhibition of PGE2 production measured by enzyme immunoassay. All extracts inhibited both enzymes with IC50 values of 2.0 to 16.7 µg/mL. The anti-inflammatory activity of B. incarum and C. atacamensis extracts was higher than that of B. boliviensis and P. lucida. The IC50 values obtained for indomethacin were 0.11 and 0.78 µM for COX-1 and COX-2, respectively. The present results are consistent with the anecdotal use of these species in phytotherapic preparations.

Genetic polymorphism of alcohol-metabolizing enzyme and alcohol dependence in Polish men

Alcohol dependence poses a serious medical and sociological problem. It is influenced by multiple environmental and genetic factors, which may determine differences in alcohol metabolism. Genetic polymorphism of the enzymes involved in alcohol metabolism is highly ethnically and race dependent. The purpose of this study was to investigate the differences, if present, in the allele and genotype frequency of alcohol dehydrogenase 1B (ADH1B), ADH1C and the microsomal ethanol-oxidizing system (MEOS/CYP2E1) between alcohol-dependent individuals and controls and also to determine if these genotypes cause a difference in the age at which the patients become alcohol dependent. The allele and genotype frequencies of ADH1B, ADH1C, and CYP2E1 were determined in 204 alcohol dependent men and 172 healthy volunteers who do not drink alcohol (control group). Genotyping was performed by PCR-RFLP methods on white cell DNA. ADH1B*1 (99.3%) and ADH1C*1 (62.5%) alleles and ADH1B*1/*1 (N = 201) and ADH1C*1/*1 (N = 85) genotypes were statistically more frequent among alcohol-dependent subjects than among controls (99.3 and 62.5%, N = 201 and 85 vs 94.5 and 40.7%, N = 153 and 32, respectively). Differences in the CYP2E1 allele and genotype distribution between groups were not significant. The persons with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes became alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes (28.08, 25.67 years vs 36.0, 45.05, 34.45 years, respectively). In the Polish men examined, ADH1C*1 and ADH1B*1 alleles and ADH1C*1/*1 and ADH1B*1/*1 genotypes favor alcohol dependence. The ADH1B*2 allele may protect from alcohol dependence. However, subjects with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes become alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Effect of silybin on high-fat-induced fatty liver in rats

Silybin, a natural antioxidant, has been traditionally used against a variety of liver ailments. To investigate its effect and the underlying mechanisms of action on non-alcoholic fatty liver in rats, we used 60 4-6-week-old male Sprague-Dawley rats to establish fatty liver models by feeding a high-fat diet for 6 weeks. Hepatic enzyme, serum lipid levels, oxidative production, mitochondrial membrane fluidity, homeostasis model assessment-insulin resistance index (HOMA-IR), gene and protein expression of adiponectin, and resistin were evaluated by biochemical, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Compared with the model group, silybin treatment (26.25 mg·kg-1·day-1, started at the beginning of the protocol) significantly protected against high-fat-induced fatty liver by stabilizing mitochondrial membrane fluidity, reducing serum content of alanine aminotransferase (ALT) from 450 to 304 U/L, decreasing hepatic malondialdehyde (MDA) from 1.24 to 0.93 nmol/mg protein, but increasing superoxide dismutase (SOD) and glutathione (GSH) levels from 8.03 to 9.31 U/mg protein and from 3.65 to 4.52 nmol/mg protein, respectively. Moreover, silybin enhanced the gene and protein expression of adiponectin from 215.95 to 552.40, but inhibited that of resistin from 0.118 to 0.018. Compared to rosiglitazone (0.5 mg·kg-1·day-1, started at the beginning of the protocol), silybin was effective in stabilizing mitochondrial membrane fluidity, reducing SOD as well as ALT, and regulating gene and protein expression of adiponectin (P < 0.05). These results suggest that mitochondrial membrane stabilization, oxidative stress inhibition, as well as improved insulin resistance, may be the essential mechanisms for the hepatoprotective effect of silybin on non-alcoholic fatty liver disease in rats. Silybin was more effective than rosiglitazone in terms of maintaining mitochondrial membrane fluidity and reducing oxidative stress.