Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Role of 11β-hydroxysteroid dehydrogenase 2 renal activity in potassium homeostasis in rats with chronic renal failure

Aldosterone concentrations vary in advanced chronic renal failure (CRF). The isozyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which confers aldosterone specificity for mineralocorticoid receptors in distal tubules and collecting ducts, has been reported to be decreased or normal in patients with renal diseases. Our objective was to determine the role of aldosterone and 11β-HSD2 renal microsome activity, normalized for glomerular filtration rate (GFR), in maintaining K+ homeostasis in 5/6 nephrectomized rats. Male Wistar rats weighing 180-220 g at the beginning of the study were used. Rats with experimental CRF obtained by 5/6 nephrectomy (N = 9) and sham rats (N = 10) were maintained for 4 months. Systolic blood pressure and plasma creatinine (Pcr) concentration were measured at the end of the experiment. Sodium and potassium excretion and GFR were evaluated before and after spironolactone administration (10 mg·kg-1·day-1 for 7 days) and 11β-HSD2 activity on renal microsomes was determined. Systolic blood pressure (means ± SEM; Sham = 105 ± 8 and CRF = 149 ± 10 mmHg) and Pcr (Sham = 0.42 ± 0.03 and CRF = 2.53 ± 0.26 mg/dL) were higher (P < 0.05) while GFR (Sham = 1.46 ± 0.26 and CRF = 0.61 ± 0.06 mL/min) was lower (P < 0.05) in CRF, and plasma aldosterone (Pald) was the same in the two groups. Urinary sodium and potassium excretion was similar in the two groups under basal conditions but, after spironolactone treatment, only potassium excretion was decreased in CRF rats (sham = 0.95 ± 0.090 (before) vs 0.89 ± 0.09 µEq/min (after) and CRF = 1.05 ± 0.05 (before) vs 0.37 ± 0.07 µEq/min (after); P < 0.05). 11β-HSD2 activity on renal microsomes was lower in CRF rats (sham = 0.807 ± 0.09 and CRF = 0.217 ± 0.07 nmol·min-1·mg protein-1; P < 0.05), although when normalized for mL GFR it was similar in both groups. We conclude that K+ homeostasis is maintained during CRF development despite normal Pald levels. This adaptation may be mediated by renal 11β-HSD2 activity, which, when normalized for GFR, became similar to that of control rats, suggesting that mineralocorticoid receptors maintain their aldosterone selectivity.

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.

Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

Angiotensin-converting enzymes 1 (ACE1) and 2 (ACE2) are key enzymes of the renin-angiotensin system, which act antagonistically to regulate the levels of angiotensin II (Ang II) and Ang-(1-7). Considerable data show that ACE1 acts on normal skeletal muscle functions and architecture. However, little is known about ACE1 levels in muscles with different fiber compositions. Furthermore, ACE2 levels in skeletal muscle are not known. Therefore, the purpose of this study was to characterize protein expression and ACE1 and ACE2 activities in the soleus and plantaris muscles. Eight-week-old female Wistar rats (N = 8) were killed by decapitation and the muscle tissues harvested for biochemical and molecular analyses. ACE1 and ACE2 activities were investigated by a fluorometric method using Abz-FRK(Dnp)P-OH and Mca-YVADAPK(Dnp)-OH fluorogenic substrates, respectively. ACE1 and ACE2 protein expression was analyzed by Western blot. ACE2 was expressed in the skeletal muscle of rats. There was no difference between the soleus (type I) and plantaris (type II) muscles in terms of ACE2 activity (17.35 ± 1.7 vs 15.09 ± 0.8 uF·min-1·mg-1, respectively) and protein expression. ACE1 activity was higher in the plantaris muscle than in the soleus (71.5 ± 3.9 vs 57.9 ± 1.1 uF·min-1·mg-1, respectively). Moreover, a comparative dose-response curve of protein expression was established in the soleus and plantaris muscles, which indicated higher ACE1 levels in the plantaris muscle. The present findings showed similar ACE2 levels in the soleus and plantaris muscles that might result in a similar Ang II response; however, lower ACE1 levels could attenuate Ang II production and reduce bradykinin degradation in the soleus muscle compared to the plantaris. These effects should enhance the aerobic capacity necessary for oxidative muscle activity.

Effects of high-intensity intermittent training on carnitine palmitoyl transferase activity in the gastrocnemius muscle of rats

We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min-1·mg protein-1) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a "time-efficient" strategy inducing metabolic adaptation.

Effect of eccentric training on mitochondrial function and oxidative stress in the skeletal muscle of rats

The objective of the present study was to investigate the effects of eccentric training on the activity of mitochondrial respiratory chain enzymes, oxidative stress, muscle damage, and inflammation of skeletal muscle. Eighteen male mice (CF1) weighing 30-35 g were randomly divided into 3 groups (N = 6): untrained, trained eccentric running (16°; TER), and trained running (0°) (TR), and were submitted to an 8-week training program. TER increased muscle oxidative capacity (succinate dehydrogenase and complexes I and II) in a manner similar to TR, and TER did not decrease oxidative damage (xylenol and creatine phosphate) but increased antioxidant enzyme activity (superoxide dismutase and catalase) similar to TR. Muscle damage (creatine kinase) and inflammation (myeloperoxidase) were not reduced by TER. In conclusion, we suggest that TER improves mitochondrial function but does not reduce oxidative stress, muscle damage, or inflammation induced by eccentric contractions.

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Production of glucose and fructose syrups from cassava (Manihot esculenta Crantz) starch using enzymes produced by microorganisms isolated from Brazilian Cerrado soil

The high demands for sugars and the development of enzymatic technology have increased the production of sweeteners, especially for glucose and fructose syrups. This work describe a technology for glucose and fructose syrups from Brazilian cassava starch using enzymes produced by soil microrganisms isolated from the Brazilian Cerrado soil. Firstly, Aspergillus niger and Streptomyces sp. were isolated from the soil and used as glucoamylase (GA) and glucose isomerase (GI) producer sources. After characterization, GA and GI exhibited optimum pH 4.5 and 8.0, respectively. GA showed maximum activity at 60 ºC and GI at 85 ºC. GA and GI retained 65 and 80%, respectively, of initial activity after 180 minutes of incubation at 60 ºC. The kinetic parameters Km and Vmáx were 0.476 (mg.mL-1) and 8.58 (µmol/minute) for GA and 0.082 (M) and 48.20 (µmol/minute) for GI. The maximum glucose syrups production occurred after 24 hours of reaction with a 98% yield. The production of fructose syrups with 42% (w/v) was reached after 96 hours of reaction.

Microorganisms screening for limonene oxidation

Limonene is a monoterpene obtained in large amounts from essential oils and is used as a raw material for the synthesis of flavors and fine chemicals. Several pathways or routes for the microbial degradation of limonene making use of the cytochrome P450-dependent monooxygenases have been described. In this study, we present a fermentative screening of microorganisms in order to verify their ability to perform the desirable conversion. In parallel, the PCR technique was used to select the microorganisms that contain the limC gene, which is responsible for the conversion of carveol to carvone. The microorganisms selected by PCR were not able to bioconvert limonene. From this result, we can suppose that these strains do not have the gene that codifies the enzyme responsible for the transformation of limonene into carveol. The results obtained in the fermentative screening showed that 4 microorganisms were able to bioconvert limonene into carveol. In addition, the amplification results showed the presence of fragments of 800 pb, expected for the limC gene. Therefore, the results obtained in the bioconversion and evaluation of the limC gene did not allow a correlation showing that these strains do not contain all the enzymes responsible for the conversion of limonene to carvone.

Effect of pectinolitic enzymes on the physical properties of caja-manga (Spondias cytherea Sonn.) pulp

Cajá-manga, also known as golden apple and hog-plum, is an exotic fruit native from Îles de la Société (French Polynesia), which was first introduced in Brazil in 1985. The pulp of ripe fruit was treated with the commercial enzymatic pool and its effect was evaluated in terms of yield, as well as the physical properties viscosity, turbidity and color (L* values). Response surface methodology was used and three levels were adopted for the independent variables temperature (30, 40, and 50 ºC), incubation time (30, 60 and 90 minutes) and enzyme concentration (0.01, 0.05, 0.09 v/v%). A central composite statistical design was used to guide the experimental work. The enzyme treatment highly increased both juice yield (up to 56%) and color (up to 8.6%) and strongly decreased viscosity (up to 57.4%), clarity (up to 77%) and turbidity (up to 85.5%). Incubation time was the most interacting facto, whereas temperature was the least one. Optimization analysis was carried out to reduce enzyme concentration to a minimum by superposing the contour plots of the tested properties, and the recommended ranges of the variables enzyme concentration, process temperature and incubation time were, respectively, 0.042-0.068%, 47.0-49.0 ºC and 82-90 minutes.

Simultaneous α-amylase and protease production by the soil bacterium Bacillus sp. SMIA-2 under submerged culture using whey protein concentrate and corn steep liquor: compatibility of enzymes with commercial detergents

Protease and α-amylase production by a thermophilic Bacillus sp. SMIA-2 cultivated in liquid cultures containing 0.25% (w/v) starch as a carbon source reached a maximum at 18 hours (47 U.mg-1 Protein) and 36 hours (325 U.mg-1 Protein), respectively. Culture medium supplementation with whey protein concentrate (0.1%, w/v) and corn steep liquor (0.3%, w/v) not only improved the production of both enzymes but also enabled them to be produced simultaneously. Under these conditions, α-amylase and protease production reached a maximum in 18 hours with levels of 401 U.mg-1 protein and 78 U.mg-1 protein, respectively. The compatibility of the enzymes produced with commercial laundry detergent was investigated. In the presence of Campeiro® detergent, α-amylase activity increased while protease activity decreased by about 27%. These enzymes improved the cleaning power of Campeiro® detergent since they were able to remove egg yolk and tomato sauce stains when used in this detergent.

Changes in enzymes, phenolic compounds, tannins, and vitamin C in various stages of jambolan (Syzygium cumini Lamark) development

The physiological state of a fruit is closely related to ripening and climatic conditions during the growing period when the fruit undergo changes in color, texture, and flavor. The ripening of the fruit can involve a complex series of biochemical reactions with alteration in enzymes activities, phenols, tannins, and ascorbic acid. The activity of enzymes (carboximethylcellulase, polygalacturonase, and pectinlyase), the total concentration of phenolic compounds, condensed tannins, and vitamin C in five stages of maturation were studied. Significant changes were observed between the maturity stages. The phenolic compounds were higher at green stage (705.01 ± 7.41); tannins were higher at green/purple stage (699.45 ± 0.22). The results showed that the ascorbic acid levels of the pulp varied significantly from 50.81 ± 1.43 to 6.61 ± 1.04 mg.100 g-1 during maturation. The specific activity of pectin lyase was higher at green stage (1531.90 ± 5.83). The specific activity of polygalacturonase was higher at mature stage (1.83 ± 0.0018). The specific activity of carboximetilcelulose was higher at ripe mature stage (4.61 ± 0.0024). The low ascorbic acid content found in jambolan fruit indicates that this fruit is not a rich source of this nutrient; however, other characteristics can make jambolan products fit for human consumption.

Oxidative enzymes activity in sugarcane juice as a function of the planting system

In Brazil, the largest producer of sugarcane in the world, the industrial process transforms this crop into ethanol and/or granulated sugar. Some cultivars exhibit enzymatic browning in the extracted sugarcane juice at levels harmful to the manufacturing process of white granulated sugar. The objective of this study was to assess the effect of sugarcane straw used as soil coverage, the use of different planting systems, and treatments with hydrogel polymer on enzymatic activity. The cultivar RB 86 7515 was sampled for 8 months; the first sample was obtained by cutting the upper portion of the stalk at the internode, which was taken to the laboratory for determination of the enzymatic activity of polyphenoloxidase (PPO) and peroxidase (POD). The soil coverage with different forms of straw as well as the planting systems did not change the enzymatic activity of polyphenoloxidase (PPO) and peroxidase (POD). The polyphenoloxidase (PPO) activity increased with the use of a polymer due to increased polyphenoloxidase (PPO) activity in the groove system. The enzymes studied showed changes in activity during the experimental period. The production of sugar at the end of the season (August to November) avoids the periods of highest enzymatic activity.

Adhesion and production of degrading enzymes by bacteria isolated from biofilms in raw milk cooling tanks

Biofilms in milk cooling tanks compromise product quality even on farms. Due to the lack of studies of this topic, this study evaluated the microbiological conditions of raw milk cooling tanks on farms and characterized the microorganisms isolated from these tanks. Samples were wiped off with sterile swabs from seven milk cooling tanks in three different points in each tank. Mesophiles and psychrotrophic counts were performed in all samples. The isolation of Pseudomonas spp., Bacillus cereus and atypical colonies formed on selective media were also performed, totalizing 297 isolates. All isolates were tested for protease and lipase production and biofilm formation. Of the total isolates, 62.9% produced protease, 55.9% produced lipase, and 50.2% produced biofilm. The most widespread genus inside the milk cooling tank was Pseudomonas since it was not possible to associate this contamination with a single sampling point in the equipment. High counts of microorganisms were found in some cooling tanks, indicating poor cleaning of the equipment and providing strong evidences of microbial biofilm presence. Moreover, it is worth mentioning the milk potential contamination with both microbial cells and their degrading enzymes, which compromises milk quality.