Blockage of angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation.

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation.

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.

Use of multimetric indices to classify estuaries with different hydromorphological characteristics and different levels of human pressure.

The assessment of estuaries based on benthic communities is widely used to determine impacts caused by human pressure and is one of the required tools for the implementation of the European Water Framework Directive (WFD). Our study compared multimetric approaches (B-IBI and TICOR) to assess the benthic condition of three Portuguese estuaries (Mondego, Tejo, and Mira rivers) with different levels of natural and human induced stress. Benthic community condition was classified into quality status categories of the WFD and compared for consistency with a priori status categories based upon physical-chemical criteria. Both multimetric indices discriminated equally well between locations classified above or below the good status category but were unable to provide good separation between other quality classes (high/good, moderate, poor/bad). Metrics included in these indices are greatly affected by natural stress and we recommend the development of habitat-specific thresholds to increase the discriminatory ability of any benthic condition index.

Lactate-upregulation of lactate oxidation complex-related genes is blunted in left ventricle of myocardial infarcted rats.

Lactate modulates the expression of lactate oxidation complex (LOC)-related genes and cardiac blood flow under physiological conditions, but its modulatory role remains to be elucidated regarding pathological cardiac stress. The present study evaluated the effect of lactate on LOC-related genes expression and hemodynamics of hearts submitted to myocardial infarction (MI). Four weeks after MI or sham operation, isolated hearts of male Wistar rats were perfused for 60 min with Na+-lactate (20 mM). As expected, MI reduced cardiac contractility and relaxation with no changes in perfusion. The impaired cardiac hemodynamics were associated with increased reactive oxygen species (ROS) levels (Sham: 19.3±0.5 vs MI: 23.8±0.3 µM), NADPH oxidase (NOX) activity (Sham: 42.2±1.3 vs MI: 60.5±1.5 nmol·h-1·mg-1) and monocarboxylate transporter 1 (mct1) mRNA levels (Sham: 1.0±0.06 vs MI: 1.7±0.2 a.u.), but no changes in superoxide dismutase (SOD), catalase, NADH oxidase (NADox), and xanthine oxidase activities. Lactate perfusion in MI hearts had no additional effect on ROS levels, NADox, and NOX activity, however, it partially reduced mct1 mRNA expression (MI-Lactate 1.3±0.08 a.u.). Interestingly, lactate significantly decreased SOD (MI-Lactate: 54.5±4.2 µmol·mg-1·min-1) and catalase (MI: 1.1±0.1 nmol·mg-1·min-1) activities in MI. Collectively, our data suggest that under pathological stress, lactate lacks its ability to modulate the expression of cardiac LOC-related genes and the perfused pressure in hearts submitted to chronic MI. Together, these data contribute to elucidate the mechanisms involved in the pathogenesis of heart failure induced by MI.

Influence of seasonal variability in benthic invertebrate community structure on the use of biotic indices to assess the ecological status of a Portuguese estuary.

The present study focused on the use of benthic invertebrate communities to assess the ecological quality of a Portuguese estuary characterized by strong seasonal changes and with eutrophication problems. Seasonal benthic samples were collected during a flood year and the methodology proposed by the WFD Portuguese group was used to classify benthic assemblages into five different quality classes. Factor analysis was applied to classify stations based on their physical-chemical status. Different classifications were obtained with different indices and among seasons and there was low agreement between indices and index-season interactions. Diversity indices were better correlated to eutrophication related variables than AMBI and ABC method. Predictable responses of benthic indices to anthropogenic stress symptoms were stronger during the dry period.

Tissue-specific modulation of angiotensin-converting enzyme (ACE) in hyperthyroidism.

We have previously demonstrated the interaction between the RAS and thyroid hormones (TH). The present study was designed to determine the role of TH in the local regulation of ACE activity and expression in different tissues. Adult male Wistar rats were randomized into three groups: T4-25 and T4-100 (0.025 and 0.100mg/kg of body weight/day of l-thyroxine for 14 days, respectively) and control. Hemodynamic parameters as well as cardiac and renal hypertrophy were evaluated. ACE activity and mRNA levels were determined by Fluorimetric and Northern blot assays, respectively. Both doses increased SBP and HR, as well as inducing cardiac and renal hypertrophy. Pulmonary and serum ACE levels were comparable among the groups. Both doses promoted increased renal ACE activity and expression but surprisingly ACE was diminished in the heart in both hyperthyroid groups. This change was mediated by a tissue-specific transcription mechanism.

Naltrexone blocks the enhancing effect of novel experiences on performance in memory tests in humans.

Two experiments were carried out in healthy human volunteers in order to investigate the effect of novel experiences on retrieval, and the influence of naltrexone thereupon. Naltrexone (50 mg) and placebo (50 mg of starch) were given orally using a double blind design. In Experiment 1, the subjects were asked, on two consecutive days, to recall well-known facts or events, and to recall the year in which major events took place. On Day 2, some subjects were, and others were not, exposed to a nonsense text prior to testing, which was viewed as a novel experience by the subjects. Exposure to the text was followed by enhanced scores in both memory tests. The effect was blocked by naltrexone, but not by the placebo, given 1 hr prior to the novel experience; the treatments had no effect of their own in subjects unexposed to the nonsense text. In Experiment 2, the memory tests were the recognition of famous faces, and the dates test (see above); and the novel experience was being taken for 5 min to a room where they had never been before. Again, the novel experience was followed by increased scores in both memory tests in the untreated and placebo groups, but not in the naltrexone treated subjects. These results confirm previous findings on memory enhancement by pre-test exposure to novel experiences, and suggest that endogenous opioid, or at least naltrexone-sensitive, mechanisms are involved in the effect.

Thyroid hormone upregulates ecto-5'-nucleotidase/CD73 in C6 rat glioma cells.

Thyroid hormones have profound effects on the central nervous system, such as proliferation, secretion of growth factors and gene expression regulation. Ecto-NTPDases and ecto-5'-nucleotidase can control the extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigated the influence of T(3) on the enzyme cascade which catalyzes interconversion of purine nucleotides in rat C6 glioma cells. Exposure of C6 cells to T(3) caused a dose dependent increase of 30% in the AMP hydrolysis up to 0.25 nM, which was suppressed by actinomycin. No significant alteration was observed on ATP/ADP hydrolysis and T(4) at higher concentrations (10-1000 nM) promoted an increase in AMP hydrolysis that was not dose dependent. T(3) treatment also increased the expression of CD73 mRNA. Besides the importance of the ecto-5'-NT in the cell proliferation and differentiation, its overexpression can enhance extracellular adenosine levels, which could also be an important proliferation signal.

The effect of non-factual post-training negative comment on the recall of verbal information.

Healthy human volunteers were asked to study a 250-word account of the 1954 World Football Cup and submitted to a questionnaire on the verbally learned material 48 h later. The subjects were divided into 7 groups. One received no treatment between text and questionnaire. The others were shown either a brief laudatory or derogatory comment on the World Cup 0, 3, or 6 h after having read the text. The comment contained no facts relevant to the text. Subjects exposed to the derogatory comment 0 or 3 h after having studied the text performed much worse in the questionnaire than any of the other groups. Performance in another, unrelated, general knowledge memory test was not affected by reading the World Cup text or subsequent comments. Thus post-event non-factual information was able to affect recall of verbally acquired factual material even if presented 3 h later; the effect does not seem to be explicable by a general performance deficit, and may be due to integration of information acquired during and after the event into one single experience.

Memory dysfunction and autonomic neuropathy in non-insulin-dependent (type 2) diabetic patients.

Considering the nervous system as a unit, it might be expected that diabetic patients with autonomic neuropathy could have a central abnormality expressed as cognitive dysfunction. To determine whether autonomic neuropathy is independently associated with cognitive dysfunction, we studied a cross-section of 20 non-insulin-dependent diabetic patients with autonomic neuropathy (14 males and six females; age (mean) = 60 + or - 1 years); 29 non-insulin-dependent diabetic patients without autonomic neuropathy (14 males and 15 females; age = 59 + or - 1 years) and 34 non-diabetic patients (10 males and 24 females; age = 58 + or - 1 years), matched by age, education and duration of disease. Cognitive function was evaluated by tests of immediate, recent and remote memory: verbal (digit span; word span) and visual (recognition of towers and famous faces). Diabetic patients with autonomic neuropathy scored (median) lower in visual memory tests than diabetic patients without autonomic neuropathy and controls (towers immediate = 5 versus 7 and 6; towers recent = 4 versus 6 and 6; faces = 16 versus 18 and 18; respectively; Kruskal-Wallis; P < 0.05). There was no difference in verbal memory performance (Kruskal-Wallis; P > 0.05). Entering age, education, duration of disease and fasting plasma glucose in a stepwise multiple regression, the performance in these tests remained associated with autonomic neuropathy (towers immediate, P = 0.0054, partial r2 = 0.166; towers recent, P = 0.0076, partial r2 = 0.163). Scores in visual tests correlated negatively with the number of abnormal cardiovascular tests (faces, r = -0.25; towers recent, r = -0.24; Spearman; P < 0.05). Decreased visual cognitive function in non-insulin-dependent diabetic patients is associated with the presence and degree of autonomic neuropathy.

Cognitive Performance of Patients with Epilepsy and Calcified Neurocysticercotic Lesions: A Case-Control Study.

Neuropsychological tests were applied to 20 patients with focal epilepsy related to calcified neurocysticercosis (NCC) (mean: three lesions/patient; NCC group), 22 patients with focal epilepsy without NCC (EPI group), and 29 healthy controls matched for age, sex, and educational level. The EPI and NCC groups were matched for age at onset of epilepsy, epilepsy duration, frequency of attacks, seizure semiology, interictal EEG findings, and antiepileptic drugs used. There were no differences in the digit span, word span, calculus, and Mini-Mental State examination among the three groups studied. The NCC and EPI groups showed lower scores than controls in immediate and delayed verbal memory, famous faces test, spatial recognition span, abstractions and judgment, and visuoconstructional abilities. The EPI group, but not the NCC group, also had lower scores in a praxis tests. There were no differences between the NCC and EPI groups in any of the tests applied (P > 15), except for the spatial recognition span, which was lower in the former. Cognitive impairment is a prevalent neuropsychological feature of patients with epilepsy and NCC.

Mental health screening by self-report questionnaire among community adolescents in southern Brazil.

PURPOSE: To evaluate the frequency of symptoms of depression, suicidal ideation, suicide behavior, and hopelessness among adolescents in southern Brazil. METHODS: The Self-Report Questionnaire (SRQ) was administered to a random sample of 126 community youngsters to screen for mental problems, the Montgomery-Asberg Depression Rating scale for signs and symptoms of depression, an adapted version of DIS (Diagnostic Interview Schedule) for suicidal ideation and behavior, and the Back's Hopelessness scale (adapted version). Social class, cognitive performance, age, and sex were also analyzed. RESULTS: Levels of symptoms of depression, suicidal ideation and behavior, and hopelessness were higher among those who were SRQ positive (8%). Female youngsters presented higher SRQ scores and on the Montgomery-Asberg scale. Cognitive performance was similar among all groups. Differences were not found according to social classes. The 15-17 year-old individuals (middle stage) presented higher hopelessness than the other stages of adolescence. CONCLUSIONS: The present findings suggest that screening for mental health conditions by self-report questionnaires may be of value of identify groups at major risk for violent, self-destructive behavior among community adolescents.

Previous exposure to a novel experience enhances performance in two simple memory tests in humans.

Young healthy human volunteers (N = 52), non-amnesic patients (N = 53), and amnesic patients (N = 52) were submitted once a day on three consecutive days to two simple memory tests: recognition of the pictures of famous faces (actors, politicians, sports figures) cut from newspapers or news magazines, and the recall of the year in which widely publicized major events happened (the Pope's visit to Brazil, a famous fire that destroyed a block downtown, etc.). One, 3 or 6 h prior to testing either on the 2nd or the 3rd day, the subjects were exposed to one of the following experiences: staying in a room where they had never been before for 10 min, or a 5 min digit learning task to which they also had never been submitted. Exposure to either of the two novel experiences 1 or 3, but not 6, h before testing was followed by increased scores in both memory tests.

Mild depression levels alter self-perceptions of future but not the recall of verbal information in elderly inpatients.

In order to determine the correlation of levels of symptoms of depression and rate of forgetting and perception of the future, a total of 68 elderly inpatients without Major Depression admitted to a general hospital were evaluated by: 1) the Montgomery-Asberg Depression Rating Scale (MADRS), 2) the Mini-Mental State Examination (MMSE), 3) a questionnaire on future self-perceptions (FSPQ), and 4) a test on the recall of verbal information to estimate the rate of forgetting. They were grouped according to the clinical prognosis of their disease (good, N = 48, 25 women, 23 men, age mean +/- SD, 68 +/- 6.64; poor, N = 20, 10 women, 10 men, age mean +/- SD, 69 +/- 6.68) which correlates with morbidity-mortality rates (low/high). There was no relationship between mild levels of signs and symptoms of depression and increased forgetting. However, levels of depression were negatively correlated to the score of future perceptions (B = -0.18, beta = -0.29, P = 0.032). Patients with diseases with good prognosis did not present different levels of depression, rates of forgetting or future expectations from those of patients with poor prognosis (high mortality rates). However, individuals with negative FSPQ scores showed significantly higher MADRS scores, independent of the type of disease. These data suggest that the modifications in the processing of information related to the future are present in clinical patients without Major Depression but they occur within a small range of very mild signs and symptoms of depression.

Diazepam inhibits retroactive interference of memory in humans: pretreatment with naltrexone does not alter this effect.

1. In a double-blind study of the effects of diazepam and naltrexone on retroactive memory interference, 88 healthy human volunteers were asked to study a text on the 1954 World Soccer Cup and were submitted to a written questionnaire on the material 48 h later. Three hours after reading the text, 58 of the subjects were exposed to a non-factual, derogatory comment on the World Cup. 2. All subjects were given either placebo or naltrexone (50 mg) before reading the text, and either placebo or diazepam (5 mg), per os, 2 h after reading the text (1 h prior to the comment). Subjects were assigned to the different treatment groups by a double-blind design. 3. Exposure to the derogatory comment caused retroactive memory interference with the retention of material from the text. Diazepam blocked the retroactive interference and had no effect of its own on retention of the text. Pretreatment with naltrexone did not influence retention, retrograde interference, or the effect of diazepam on these variables. 4. The results obtained here extend to healthy adult humans observations made on rats and mice in which diazepam blocked retroactive memory interference.

The use of a new non-verbal test in the evaluation of recent memory.

A test for recent memory was developed using non-verbal material. The present report describes a series of experiments conducted to evaluate its reproducibility and the influences of changes in the interval between acquisition and retrieval, and age and level of instruction, and its sensitivity to the integrity of memory function. A total of 114 subjects participated in 5 experiments. The test of recent memory is reproducible over a period of at least 5 months and can be used with training-testing intervals of 24 to 48 h. The performance of the test is affected by age and level of instruction. Moreover, the memory test is sensitive to differences in the integrity of memory function. Thus, the memory test developed in this study may be used to evaluate the effects of behavioral and/or pharmacological manipulations on recent memory in homogeneous groups of subjects.

Construction and reconstruction of memories.

1. Recent evidence suggests that treatments given after training may influence memory in two ways: by becoming themselves incorporated to the experience, or by altering post-training mechanisms involved in the storage of the experience. The two processes may be called consolidation. 2. Some endogenous substances that are normally released during or after training (brain beta-endorphin; the peripheral stress hormones, ACTH, epinephrine and vasopressin) appear to be of particular importance. Their effect may become incorporated to the experiences as a conditioned stimulus (CS), generating state dependency. The effect of beta-endorphin appears to be physiological, since the substance is released by novel experiences. 3. Post-event information provided by other training experiences, in rats, or by comments or leading words, in humans, may also incorporate to the experiences, altering their content qualitatively or quantitatively. 4. A variety of substances including the stress hormones at low doses and analeptic drugs may facilitate retention when given after training. In this case, the effect is best explained by an enhancement of the post-training strengthening of memory traces. 5. The reiteration of part of the experiences at the time of testing facilitates retrieval. This may be viewed as a reconstruction of consolidation at the time of retrieval, and may be obtained using cognitive material ("priming"), or neurohumoral stimuli (a beta-endorphin injection, or a presumable release of brain beta-endorphin by an interpolated novel experience). The effect can be seen in animals rendered amnestic by electroconvulsive shock, and in humans with amnesia of organic and non-organic nature. 6. The human amnesic syndrome seems, thus, largely explainable by a deficit of retrieval. It is possible that the stimulation of retrieval by priming, or by drugs, through the "reconstruction" of consolidation, may be useful for the relief or treatment of the human amnesic syndrome.

Diagnosing dementia and normal aging: clinical relevance of brain ratios and cognitive performance in a Brazilian sample.

The main objective of the present study was to evaluate the diagnostic value (clinical application) of brain measures and cognitive function. Alzheimer and multi-infarct patients (N = 30) and normal subjects over the age of 50 (N = 40) were submitted to a medical, neurological and cognitive investigation. The cognitive tests applied were Mini-Mental, word span, digit span, logical memory, spatial recognition span, Boston naming test, praxis, and calculation tests. The brain ratios calculated were the ventricle-brain, bifrontal, bicaudate, third ventricle, and suprasellar cistern measures. These data were obtained from a brain computer tomography scan, and the cutoff values from receiver operating characteristic curves. We analyzed the diagnostic parameters provided by these ratios and compared them to those obtained by cognitive evaluation. The sensitivity and specificity of cognitive tests were higher than brain measures, although dementia patients presented higher ratios, showing poorer cognitive performances than normal individuals. Normal controls over the age of 70 presented higher measures than younger groups, but similar cognitive performance. We found diffuse losses of tissue from the central nervous system related to distribution of cerebrospinal fluid in dementia patients. The likelihood of case identification by functional impairment was higher than when changes of the structure of the central nervous system were used. Cognitive evaluation still seems to be the best method to screen individuals from the community, especially for developing countries, where the cost of brain imaging precludes its use for screening and initial assessment of dementia.

Stimulatory effect of dexamethasone on angiotensin-converting enzyme in neonatal rat cardiac myocytes.

Angiotensin-converting enzyme (ACE) plays a central role in cardiac remodeling associated with pathological conditions such as myocardial infarction. The existence of different cell types in the heart expressing components of the renin-angiotensin system makes it difficult to evaluate their relative role under physiological and pathological conditions. Since myocytes are the predominant cellular constituent of the heart by mass, in the present study we studied the effects of glucocorticoids on ACE activity using well-defined cultures of neonatal rat cardiac myocytes. Under steady-state conditions, ACE activity was present at very low levels, but after dexamethasone treatment ACE activity increased significantly (100 nmol/l after 24 h) in a time-dependent fashion. These results demonstrate the influence of dexamethasone on ACE activity in rat cardiac myocytes. This is consistent with the idea that ACE activation occurs under stress conditions, such as myocardial infarction, in which glucocorticoid levels may increase approximately 50-fold.

Evaluation of behavioral states among morning and evening active healthy individuals.

The Horne-Ostberg questionnaire partly covers some factors that may be important determinants of peak time and characterize patterns of behavior. We conducted a study for the evaluation of self-reported behavioral states (hunger sensation, availability for study, physical exercise, solving daily problems, and time preferences) as expressions of underlying cyclic activity. Three hundred and eighteen community subjects without history of medical, psychiatric, or sleep disorders were evaluated in a cross-sectional design. A self-report about daily highest level of activity was used to categorize individuals into morning, evening, and indifferently active. Time-related behavioral states were evaluated with 23 visual analog questions. The responses to most analogic questions were significantly different between morning and evening active subjects. Logistic regression analysis identified a group of behaviors more strongly associated with the self-reported activity pattern (common wake up time, highest subjective fatigue, as well as wake up, bedtime, exercise and study preferences). These findings suggested that the patterns of activity presented by normal adults were related to specific common behavioral characteristics that may contribute to peak time.