Aerobic exercise prevents cardiomyocyte damage caused by oxidative stress in early cardiovascular disease by increasing vascularity while L-arginine supplementation prevents it by increasing activation of the enzyme nitric oxide synthase

L-Arginine and chronic exercise reduce oxidative stress. However, it is unclear how they affect cardiomyocytes during cardiovascular disease (CVD) development. The aim of this research was to investigate the possible effects of L-arginine supplementation and aerobic training on systemic oxidative stress and their consequences on cardiomyocytes during cardiometabolic disease onset caused by excess fructose. Wistar rats were allocated into four groups: control (C), fructose (F, 10% fructose in water), fructose training (FT; moderate running, 50-70% of the maximal velocity), and fructose arginine (FA; 880 mg/kg/day). Fructose was given for two weeks and fructose plus treatments for the subsequent eight weeks. Body composition, blood glucose, insulin, lipid profile, lipid peroxidation, nitrite, metalloproteinase-2 (MMP-2) activity, left ventricle histological changes, microRNA-126, -195, and -146, eNOS, p-eNOS, and TNF-α expressions were analyzed. Higher abdominal fat mass, triacylglycerol level, and insulin level were observed in the F group, and both treatments reversed these alterations. Myocardial vascularization was impaired in fructose-fed groups, except in FT. Cardiomyocyte hypertrophy was observed in all fructose-fed groups. TNF-α levels were higher in fructose-fed groups than in the C group, and p-eNOS levels were higher in the FA than in the C and F groups. Lipid peroxidation was higher in the F group than in the FT and C groups. During CVD onset, moderate aerobic exercise reduced lipid peroxidation, and both training and L-arginine prevented metabolic changes caused by excessive fructose. Myocardial vascularization was impaired by fructose, and cardiomyocyte hypertrophy appeared to be influenced by pro-inflammatory and oxidative environments.

Enchytraeid abundance in Araucaria Mixed Forest determined by cold and hot wet extraction

Abstract Enchytraeids are small oligochaetes found worldwide in soils with sufficient moisture and organic matter, but scarcely studied in the Southern hemisphere. This is the third study on enchytraeid abundance in Brazil using wet extraction and the first carried out in Araucaria Mixed Forest (subtropical region). The sampling and extraction were based on the standard method ISO 23611-3/2007 using an adapted split soil corer and wet extraction with and without heat to assess the abundance of enchytraeids in a forest fragment at Embrapa Forestry in Colombo, Paraná State. The samplings were performed in 3 occasions between September 2011 and April 2012. The average numbers estimated by each method varied from appr. 2.000-12.000 (cold) and 5.000-12.000 ind./ m2 (hot), respectively, with a maximum of 44.000 ind./ m2 in one of the samples, the highest value reported so far in Brazil. The hot extraction was more advantageous, given the speed and preservation of the specimens in vivo, allowing taxonomic identification. Advantages and disadvantages of wet extractions compared to handsorting and formol methods are also discussed. Guaranidrilus, Hemienchytraeus, Enchytraeus, Fridericia and Achaeta were the genera identified in the samples.

Resumo Os enquitreídeos são pequenos oligoquetas encontrados no mundo todo em solos com suficiente umidade e matéria orgânica, porém muito pouco estudados no hemisfério Sul. Este é o terceiro estudo sobre a abundância de enquitreídeos no Brasil utilizando o método de extração úmida e o primeiro realizado em Floresta Ombrófila Mista (região subtropical). A amostragem e extração foram baseadas no método padrão ISO 23611-3/2007, utilizando-se um trado desmontável adaptado e extração úmida com e sem aquecimento para acessar a abundância de enquitreídeo em um fragmento de floresta na Embrapa Florestas em Colombo, Paraná. As amostragens foram realizadas em três ocasiões entre setembro, 2011 e abril 2012. Os números médios estimados através de cada método variaram de 2.000-12.000 (frio) e 5.000-12.000 ind./ m2 (quente), respectivamente, e o máximo de 44.000 ind./ m2 em uma das amostras, o mais alto já relatado no Brasil. A extração quente foi a mais vantajosa, considerando a rapidez e preservação dos exemplares in vivo. As vantagens e desvantagens das extrações úmidas comparadas aos métodos de triagem manual e extração com formol foram discutidas. Os gêneros Guaranidrilus, Hemienchytraeus, Enchytraeus, Fridericia e Achaeta foram identificados nas amostras.

Enchytraeid abundance in Araucaria Mixed Forest determined by cold and hot wet extraction / Abundância de enquitreídeos na Floresta Ombrófila Mista determinada por extração úmida quente e fria

Abstract Enchytraeids are small oligochaetes found worldwide in soils with sufficient moisture and organic matter, but scarcely studied in the Southern hemisphere. This is the third study on enchytraeid abundance in Brazil using wet extraction and the first carried out in Araucaria Mixed Forest (subtropical region). The sampling and extraction were based on the standard method ISO 23611-3/2007 using an adapted split soil corer and wet extraction with and without heat to assess the abundance of enchytraeids in a forest fragment at Embrapa Forestry in Colombo, Paraná State. The samplings were performed in 3 occasions between September 2011 and April 2012. The average numbers estimated by each method varied from appr. 2.000-12.000 (cold) and 5.000-12.000 ind./ m2 (hot), respectively, with a maximum of 44.000 ind./ m2 in one of the samples, the highest value reported so far in Brazil. The hot extraction was more advantageous, given the speed and preservation of the specimens in vivo, allowing taxonomic identification. Advantages and disadvantages of wet extractions compared to handsorting and formol methods are also discussed. Guaranidrilus, Hemienchytraeus, Enchytraeus, Fridericia and Achaeta were the genera identified in the samples.

Resumo Os enquitreídeos são pequenos oligoquetas encontrados no mundo todo em solos com suficiente umidade e matéria orgânica, porém muito pouco estudados no hemisfério Sul. Este é o terceiro estudo sobre a abundância de enquitreídeos no Brasil utilizando o método de extração úmida e o primeiro realizado em Floresta Ombrófila Mista (região subtropical). A amostragem e extração foram baseadas no método padrão ISO 23611-3/2007, utilizando-se um trado desmontável adaptado e extração úmida com e sem aquecimento para acessar a abundância de enquitreídeo em um fragmento de floresta na Embrapa Florestas em Colombo, Paraná. As amostragens foram realizadas em três ocasiões entre setembro, 2011 e abril 2012. Os números médios estimados através de cada método variaram de 2.000-12.000 (frio) e 5.000-12.000 ind./ m2 (quente), respectivamente, e o máximo de 44.000 ind./ m2 em uma das amostras, o mais alto já relatado no Brasil. A extração quente foi a mais vantajosa, considerando a rapidez e preservação dos exemplares in vivo. As vantagens e desvantagens das extrações úmidas comparadas aos métodos de triagem manual e extração com formol foram discutidas. Os gêneros Guaranidrilus, Hemienchytraeus, Enchytraeus, Fridericia e Achaeta foram identificados nas amostras.

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1 (p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.

Eccentric and concentric cardiac hypertrophy induced by exercise training: microRNAs and molecular determinants

Among the molecular, biochemical and cellular processes that orchestrate the development of the different phenotypes of cardiac hypertrophy in response to physiological stimuli or pathological insults, the specific contribution of exercise training has recently become appreciated. Physiological cardiac hypertrophy involves complex cardiac remodeling that occurs as an adaptive response to static or dynamic chronic exercise, but the stimuli and molecular mechanisms underlying transduction of the hemodynamic overload into myocardial growth are poorly understood. This review summarizes the physiological stimuli that induce concentric and eccentric physiological hypertrophy, and discusses the molecular mechanisms, sarcomeric organization, and signaling pathway involved, also showing that the cardiac markers of pathological hypertrophy (atrial natriuretic factor, β-myosin heavy chain and α-skeletal actin) are not increased. There is no fibrosis and no cardiac dysfunction in eccentric or concentric hypertrophy induced by exercise training. Therefore, the renin-angiotensin system has been implicated as one of the regulatory mechanisms for the control of cardiac function and structure. Here, we show that the angiotensin II type 1 (AT1) receptor is locally activated in pathological and physiological cardiac hypertrophy, although with exercise training it can be stimulated independently of the involvement of angiotensin II. Recently, microRNAs (miRs) have been investigated as a possible therapeutic approach since they regulate the translation of the target mRNAs involved in cardiac hypertrophy; however, miRs in relation to physiological hypertrophy have not been extensively investigated. We summarize here profiling studies that have examined miRs in pathological and physiological cardiac hypertrophy. An understanding of physiological cardiac remodeling may provide a strategy to improve ventricular function in cardiac dysfunction.

Tuberculosis determined by Mycobacterium bovis in captive waterbucks (Kobus ellipsiprymnus) in São Paulo, Brazil

Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus.

Aspectos produtivos e sanitários de vacas mestiças leiteiras tratadas com produtos homeopáticos / Productive and sanitary aspects of crossbred milking cows receiving homeopathic products

Nos dias de hoje, a população mundial exige o consumo de alimentos de qualidade, sem resíduos de produtos químicos. Desempenho (produção e qualidade do leite, peso e condição corporal) e sanidade (mastite e infestação por ecto e endoparasitas) foram acompanhados em oito vacas leiteiras mestiças de um rebanho de 40 (20%), que recebeu diariamente produtos homeopáticos comerciais no concentrado para o controle de endo e ectoparasitas e mastite. No período de nove meses, que correspondeu a toda uma lactação, não houve necessidade de medicar com produtos alopáticos nenhuma vaca do rebanho, o que demonstrou ser possível criar vacas leiteiras mestiças não utilizando produtos químicos para o combate a parasitas (carrapato, mosca-do-chifre e verminose), ou antibióticos para o controle de mastite, e sem interferir na produtividade dos animais.

Nowadays the world population demands food of good quality, without any residues from chemical products. Eight cows from a herd of 40, which were consuming commercial homeopathic medicines at the daily concentration for control of endoand ectoparasites, were monitored in terms of their performance (production and quality of milk, body score and body weight) and sanitary aspects (parasites infestation and mastitis) during the lactation period. It was not necessary to give any allopathic (conventional) medicines to control mastitis or parasites (ticks, horn-fly, verminosis) in any cow of the herd during the nine months of the lactation period. The results showed that it is possible to produce milk without using chemical products, and not interfering in the productivity of the crossbred animals.

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.

Tipificação genética, através da técnica de spoligotyping, de isolados de Mycobacterium bovis em animais abatidos na região metropolitana de Salvador, Bahia, Brasil / Use of spoligotyping for genetic typing of Mycobacterium bovis isolates from slaughtered animals in the metropolitan area of Salvador, Bahia, Brazil

A tuberculose é uma enfermidade infecciosa crônica, que afeta mamíferos e aves e constitui um sério problema de saúde pública e animal. Objetivando realizar um levantamento molecular da enfermidade em bovinos abatidos em matadouros frigoríficos no Estado da Bahia, Brasil, foram analisadas as lesões pulmonares e de linfonodos mediastínicos de 43 carcaças de animais abatidos em três matadouros-frigoríficos localizados na Região Metropolitana de Salvador, Bahia. Sete isolados de Mycobacterium bovis foram identificados, através da técnica do spolygotyping, e discriminados em três diferentes espoligotipos (SB1055, SB0120 e SB0268) descritos no Brasil e em diversas áreas do mundo. Os resultados indicam que o método de diagnóstico utilizado pode contribuir para a criação de uma base de dados para o estudo epidemiológico da tuberculose bovina no Estado da Bahia.

Tuberculosis is an infectious chronic disease that affects mammals and birds and constitutes a serious problem for public and animal health. Pulmonary and mediastinic lymph node lesions of 43 animals slaughtered in 3 slaughterhouses in the metropolitan region of the city of Salvador, Bahia, Brazil, were analyzed with the objective of obtaining a molecular survey of the disease in bovines slaughtered in slaughterhouses in the state. Seven isolates ofMycobacterium bovis were identified through the spoligotyping technique and classified into 3 different spoligotypes (SB1055, SB0120, SB0268), described in Brazil and in many areas worldwide. The results indicate that the diagnostic method utilized may contribute to the creation of a database for the epidemiologic study of bovine tuberculosis in the state of Bahia.

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 ¡À 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 ¡À 63 and 174 ¡À 57 g/m2 (P = 0.008), 19 ¡À 5 and 21 ¡À 5 mm (P = 0.02), and 10 ¡À 2 and 12 ¡À 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 ¡Ý190 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 myocardialhypertrophy in the patients with HCM.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37°C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 æM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 æM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 æM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Diabetes mellitus: new therapeutic approaches to treat an old disease

Diabetes mellitus is a widespread disease whose frequency increases constantly and is expected to reach alarming levels by the year 2025. Introduction of insulin therapy represented a major breakthrough; however, a very strict regimen is required to maintain blood glucose levels within the normal range and to prevent or postpone chronic complications associated with this disease. Frequent hyper- and hypoglycemia seriously affect the quality of life of these patients. Reversion of this situation can only be achieved through whole organ (pancreas) transplant or pancreatic islet transplant, the former being a high-risk surgical procedure, while the latter is a much simpler and may be accomplished in only 20-40 min. The advantages and perspectives of islet cell transplantation will be discussed, in the light of tissue engineering and gene therapy. Ongoing research carried out in our laboratory, aimed at developing clinical cell and molecular therapy protocols for diabetes will also be focused

Swimming training increases cardiac vagal activity and induces cardiac hypertrophy in rats

The effect of swimming training (ST) on vagal and sympathetic cardiac effects was investigated in sedentary (S, N = 12) and trained (T, N = 12) male Wistar rats (200-220 g). ST consisted of 60-min swimming sessions 5 days/week for 8 weeks, with a 5 percent body weight load attached to the tail. The effect of the autonomic nervous system in generating training-induced resting bradycardia (RB) was examined indirectly after cardiac muscarinic and adrenergic receptor blockade. Cardiac hypertrophy was evaluated by cardiac weight and myocyte morphometry. Plasma catecholamine concentrations and citrate synthase activity in soleus muscle were also determined in both groups. Resting heart rate was significantly reduced in T rats (355 ± 16 vs 330 ± 20 bpm). RB was associated with a significantly increased cardiac vagal effect in T rats (103 ± 25 vs 158 ± 40 bpm), since the sympathetic cardiac effect and intrinsic heart rate were similar for the two groups. Likewise, no significant difference was observed for plasma catecholamine concentrations between S and T rats. In T rats, left ventricle weight (13 percent) and myocyte dimension (21 percent) were significantly increased, suggesting cardiac hypertrophy. Skeletal muscle citrate synthase activity was significantly increased by 52 percent in T rats, indicating endurance conditioning. These data suggest that RB induced by ST is mainly mediated parasympathetically and differs from other training modes, like running, that seems to mainly decrease intrinsic heart rate in rats. The increased cardiac vagal activity associated with ST is of clinical relevance, since both are related to increased life expectancy and prevention of cardiac events.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Microencapsulation and tissue engineering as an alternative treatment of diabetes

In the 70's, pancreatic islet transplantation arose as an attractive alternative to restore normoglycemia; however, the scarcity of donors and difficulties with allotransplants, even under immunosuppressive treatment, greatly hampered the use of this alternative. Several materials and devices have been developed to circumvent the problem of islet rejection by the recipient, but, so far, none has proved to be totally effective. A major barrier to transpose is the highly organized islet architecture and its physical and chemical setting in the pancreatic parenchyma. In order to tackle this problem, we assembled a multidisciplinary team that has been working towards setting up the Human Pancreatic Islets Unit at the Chemistry Institute of the University of São Paulo, to collect and process pancreas from human donors, upon consent, in order to produce purified, viable and functional islets to be used in transplants. Collaboration with the private enterprise has allowed access to the latest developed biomaterials for islet encapsulation and immunoisolation. Reasoning that the natural islet microenvironment should be mimicked for optimum viability and function, we set out to isolate extracellular matrix components from human pancreas, not only for analytical purposes, but also to be used as supplementary components of encapsulating materials. A protocol was designed to routinely culture different pancreatic tissues (islets, parenchyma and ducts) in the presence of several pancreatic extracellular matrix components and peptide growth factors to enrich the beta cell population in vitro before transplantation into patients. In addition to representing a therapeutic promise, this initiative is an example of productive partnership between the medical and scientific sectors of the university and private enterprises.

Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients

Genetic damage caused by ionizing radiation and repair capacity of blood lymphocytes from 3 breast cancer patients and 3 healthy donors were investigated using the comet assay. The comets were analyzed by two parameters: comet tail length and visual classification. Blood samples from the donors were irradiated in vitro with a 60Co source at a dose rate of 0.722 Gy/min, with a dose range of 0.2 to 4.0 Gy and analyzed immediately after the procedure and 3 and 24 h later. The basal level of damage and the radioinduced damage were higher in lymphocytes from breast cancer patients than in lymphocytes from healthy donors. The radioinduced damage showed that the two groups had a similar response when analyzed immediately after the irradiations. Therefore, while the healthy donors presented a considerable reduction of damage after 3 h, the patients had a higher residual damage even 24 h after exposure. The repair capacity of blood lymphocytes from the patients was slower than that of lymphocytes from healthy donors. The possible influence of age, disease stage and mutations in the BRCA1 and BRCA2 genes are discussed. Both parameters adopted proved to be sensitive and reproducible: the dose-response curves for DNA migration can be used not only for the analysis of cellular response but also for monitoring therapeutic interventions. Lymphocytes from the breast cancer patients presented an initial radiosensitivity similar to that of healthy subjects but a deficient repair mechanism made them more vulnerable to the genotoxic action of ionizing radiation. However, since lymphocytes from only 3 patients and 3 normal subjects were analyzed in the present paper, additional donors will be necessary for a more accurate evaluation

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20 percent when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Perfil eletroforético das proteínas e concentraçäo das imunoglobulinas plasmáticas em pacientes assintomáticos e sintomáticos portadores de HIV / Proteins electrophoretic profile and immunoglobulins plasmatics concentration in asymptomatics and symptomatics patients infected by HIV

Estudos têm demonstrado que 81 por cento dos pacientes infectados pelo HIV apresentam um padräo proteico anormal devido ao estado hipermetabólico. Os achados laboratoriais revelam que as anormalidades da imunidade humoral precedem a imunidade mediada pelas células e portanto, o diagnóstico da infecçäo pelo HIV depende do estabelecimento da infecçäo definindo o estado imunológico e o quadro clínico do paciente que ocorre secundário à infecçä e à perda da funçäo imune. No presente trabalho, realizamos um estudo comparativo do perfil eletroforético das proteínas, bem como a determinaçäo da concentraçäo das imunoglobulinas plasmáticas em pacientes portadores de HIV (assintomáticos e sintomáticos) com o objetivo de investigarmos alteraçöes específicas das fraçöes protéicas (albumina e globulina) durante os diferentes estágios da doença. Foram analisadas 30 amostras de sangue coletado de pacientes HIV positivos divididos em dois grupos (assintomáticos e sintomáticos). As proteínas totais foram determinadas pelo método do Biureto, sendo observada uma hipoproteinemia em 60 por cento dos pacientes sintomáticos e valores normais em todos os assintomáticos. A eletroforese das proteínas foi realizada em gel de agarose mostrando hipoalbuminemia em 70 por cento dos pacientes assintomáticos e 90 por cento sintomáticos. A hipergamaglobulinemia predominou em 100 por cento dos sintomáticos e 90 por cento dos assintomáticos. As fraçöes alfa e beta globulinas apresentaram-se normais nos dois grupos. As imunoglobulinas foram avaliadas por turbidimetria e uma elevaçäo significativa foi verificada na concentraçäo de IgG em todos os pacientes sintomáticos. Por outro lado, os valores de IgA e IgM apresentaram-se dentro dos valores de referência. Os resultados estäo de acordo com vários trabalhos citados na literatura, comprovando que a AIDS é uma gamopatia monoclonal, caracterizada por hipoalbuminemia e hipergamaglobulinemia. O trabalho mostra portanto, que, a avaliaçäo de parâmetros laboratoriais mais simples säo úteis no monitoramento da síndrome em estudo, como também fortes indicadores do estágio da doença.

Effects of mercury on the arterial blood pressure of anesthetized rats

The available data suggests that hypotension caused by Hg2+ administration may be produced by a reduction of cardiac contractility or by cholinergic mechanisms. The hemodynamic effects of an intravenous injection of HgCl2 (5 mg/kg) were studied in anesthetized rats (N = 12) by monitoring left and right ventricular (LV and RV) systolic and diastolic pressures for 120 min. After HgCl2 administration the LV systolic pressure decreased only after 40 min (99 +or - 3.3 to 85 + or - 8.8 mmHg at 80 min). However, RV systolic pressure increased, initially slowly but faster after 30 min (25 + or - 1.8 to 42 + or - 1.6 mmHg at 80 min). Both right and left diastolic pressures increased after HgCl2 treatment, suggesting the development of diastolic ventricular dysfunction. Since HgCl2 could be increasing pulmonary vascular resistance, isolated lungs (N = 10) were perfused for 80 min with Krebs solution (continuous flow of 10 ml/min) containing or not 5 µM HgCl2. A continuous increase in pulmonary vascular resistance was observed, suggesting the direct effect of Hg2+ on the pulmonary vessels (12 + or - 0.4 to 29 + or - 3.2 mmHg at 30 min). To examine the interactions of Hg2+ and changes in cholinergic activity we analyzed the effects of acetylcholine (Ach) on mean arterial blood pressure (ABP) in anesthetized rats (N = 9) before and after Hg2+ treatment (5 mg/kg). Using the same amount and route used to study the hemodynamic effects we also examined the effects of Hg2+ administration on heart and plasma cholinesterase activity (N = 10). The in vivo hypotensive response to Ach (0.035 to 10.5 µg) was reduced after Hg2+ treatment. Cholinesterase activity (µM h-1 mg protein-1) increased in heart and plasma (32 and 65 percent, respectively) after Hg2+ treatment. In conclusion, the reduction in ABP produced by Hg2+ is not dependent on a putative increase in cholinergic activity. HgCl2 mainly affects cardiac function. The increased pulmonary vascular resistance and cardiac failure due to diastolic dysfunction of both ventricles are factors that might contribute to the reduction of cardiac output and the fall in arterial pressure