Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25��C in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Ambient particulate air pollution from vehicles promotes lipid peroxidation and inflammatory responses in rat lung

Oxidative stress plays a major role in the pathogenesis of particle-dependent lung injury. Ambient particle levels from vehicles have not been previously shown to cause oxidative stress to the lungs. The present study was conducted to a) determine whether short-term exposure to ambient levels of particulate air pollution from vehicles elicits inflammatory responses and lipid peroxidation in rat lungs, and b) determine if intermittent short-term exposures (every 4 days) induce some degree of tolerance. Three-month-old male Wistar rats were exposed to ambient particulate matter (PM) from vehicles (N = 30) for 6 or 20 continuous hours, or for intermittent (5 h) periods during 20 h for 4 consecutive days or to filtered air (PM &lt;10 ��m; N = 30). Rats continuously breathing polluted air for 20 h (P-20) showed a significant increase in the total number of leukocytes in bronchoalveolar lavage compared to control (C-20: 2.61 x 105 �� 0.51;P-20: 5.01 x 105 �� 0.81; P < 0.05) and in lipid peroxidation ([MDA] nmol/mg protein: C-20: 0.148 �� 0.01; P-20: 0.226 �� 0.02; P < 0.05). Shorter exposure (6 h) and intermittent 5-h exposures over a period of 4 days did not cause significant changes in leukocytes. Lipid damage resulting from 20-h exposure to particulate air pollution did not cause a significant increase in lung water content. These data suggest oxidative stress as one of the mechanisms responsible for the acute adverse respiratory effects of particles, and suggest that short-term inhalation of ambient particulate air pollution from street with high automobile traffic represents a biological hazard.

Comparison of serum hormone levels of captive and free-living maned wolves Chrysocyon brachyurus

Serum hormone levels were compared between captive and free-living maned wolves and seasonal variations of sex hormones were studied. Blood samples were collected from 16 male and 26 female adult animals from Brazilian zoos, and from 30 male and 24 female free-living adults to determine serum progesterone and testosterone by radioimmunoassay. Serum testosterone concentrations varied (P < 0.05) across seasons for 16 captive males, being higher in autumn (2184.7 �� 355.1 pg/mL) than in summer (1080.7 �� 205.4 pg/mL), winter (1270.1 �� 276.6 pg/mL) and spring (963.9 �� 248.1 pg/mL), although they did not differ between summer, winter and spring. Testosterone concentration of 30 free-living males differed (P < 0.05) between autumn (824.1 �� 512.2 pg/mL), winter (14.4 �� 8.0 pg/mL) and spring (151.9 �� 90.5 pg/mL). Comparison between captive and free-living animals showed no difference in autumn (P > 0.05). Sixteen captive males showed higher testosterone concentration during winter and spring compared with 30 free-living animals (P < 0.05). Progesterone concentration varied among seasons in 26 captive females (P < 0.05), being higher in autumn (15.3 �� 3.1 ng/mL) than in summer (6.6 �� 1.5 ng/mL), winter (5.3 �� 3.1 ng/mL) and spring (4.3 �� 0.7 ng/mL). Progesterone concentration of 24 free-living females varied between autumn (17.1 �� 6.0 ng/mL) and winter (1.7 �� 0.3 ng/mL) (P < 0.05), but we could not obtain data for spring or summer. No difference in progesterone levels was observed between captive and free-living females in autumn and winter.

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.

Vascular complications of black patients with type 2 diabetes mellitus in Southern Brazil

Ethnicity has been shown to be associated with micro- and macrovascular complications of diabetes in European and North American populations. We analyzed the contribution of ethnicity to the prevalence of micro- and macrovascular complications in Brazilian subjects with type 2 diabetes attending the national public health system. Data from 1810 subjects with type 2 diabetes (1512 whites and 298 blacks) were analyzed cross-sectionally. The rates of ischemic heart disease, peripheral vascular disease, stroke, distal sensory neuropathy, and diabetic retinopathy were assessed according to self-reported ethnicity using multiple logistic regression models. Compared to whites, black subjects [odds ratio = 1.72 (95%CI = 1.14-2.6)] were more likely to have ischemic heart disease when data were adjusted for age, sex, fasting plasma glucose, HDL cholesterol, hypertension, smoking habit, and serum creatinine. Blacks were also more likely to have end-stage renal disease [3.2 (1.7-6.0)] and proliferative diabetic retinopathy [1.9 (1.1-3.2)] compared to whites when data were adjusted for age, sex, fasting plasma glucose, HDL cholesterol, hypertension, and smoking habit. The rates of peripheral vascular disease, stroke and distal sensory neuropathy did not differ between groups. The higher rates of ischemic heart disease, end-stage renal disease and proliferative diabetic retinopathy in black rather than in white Brazilians were not explained by differences in conventional risk factors. Identifying which aspects of ethnicity confer a higher risk for these complications in black patients is crucial in order to understand why such differences exist and to develop more effective strategies to reduce the onset and progression of these complications.

The effect of a low dose of clenbuterol on rat soleus muscle submitted to joint immobilization

The aim of the present study was to evaluate the effect of joint immobilization on morphometric parameters and glycogen content of soleus muscle treated with clenbuterol. Male Wistar (3-4 months old) rats were divided into 4 groups (N = 6 for each group): control, clenbuterol, immobilized, and immobilized treated with clenbuterol. Immobilization was performed with acrylic resin orthoses and 10 ��g/kg body weight clenbuterol was administered subcutaneously for 7 days. The following parameters were measured the next day on soleus muscle: weight, glycogen content, cross-sectional area, and connective tissue content. The clenbuterol group showed an increase in glycogen (81.6%, 0.38 �� 0.09 vs 0.69 �� 0.06 mg/100 g; P < 0.05) without alteration in weight, cross-sectional area or connective tissue compared with the control group. The immobilized group showed a reduction in muscle weight (34.2%, 123.5 �� 5.3 vs 81.3 �� 4.6 mg; P < 0.05), glycogen content (31.6%, 0.38 �� 0.09 vs 0.26 �� 0.05 mg/100 mg; P < 0.05) and cross-sectional area (44.1%, 2574.9 �� 560.2 vs 1438.1 �� 352.2 ��m��; P < 0.05) and an increase in connective tissue (216.5%, 8.82 �� 3.55 vs 27.92 �� 5.36%; P < 0.05). However, the immobilized + clenbuterol group showed an increase in weight (15.9%; 81.3 �� 4.6 vs 94.2 �� 4.3 mg; P < 0.05), glycogen content (92.3%, 0.26 �� 0.05 vs 0.50 �� 0.17 mg/100 mg; P < 0.05), and cross-sectional area (19.9%, 1438.1 �� 352.2 vs 1724.8 �� 365.5 ��m��; P < 0.05) and a reduction in connective tissue (52.2%, 27.92 �� 5.36 vs 13.34 �� 6.86%; P < 0.05). Statistical analysis was performed using Kolmogorov-Smirnov and homoscedasticity tests. For the muscle weight and muscle glycogen content, two-way ANOVA and the Tukey test were used. For the cross-sectional area and connective tissue content, Kruskal-Wallis and Tukey tests were used. This study emphasizes the importance of anabolic pharmacological protection during immobilization to minimize skeletal muscle alterations resulting from disuse.

De anima rationali

Motto: Meisnerus Phil. sobr. part. 1. sect. 3. c. 6. p. m. 913. Si enim in universo naturae latifundio quicquam ...

How auditory temporal processing deficits relate to dyslexia

Studies have shown that dyslexic children present a deficiency in the temporal processing of auditory stimuli applied in rapid succession. However, discussion continues concerning the way this deficiency can be influenced by temporal variables of auditory processing tests. Therefore, the purpose of the present study was to analyze by auditory temporal processing tests the effect of temporal variables such as interstimulus intervals, stimulus duration and type of task on dyslexic children compared to a control group. Of the 60 children evaluated, 33 were dyslexic (mean age = 10.5 years) and 27 were normal controls (mean age = 10.8 years). Auditory processing tests assess the abilities of discrimination and ordering of stimuli in relation to their duration and frequency. Results showed a significant difference in the average accuracy of control and dyslexic groups considering each variable (interstimulus intervals: 47.9 �� 5.5 vs 37.18 �� 6.0; stimulus duration: 61.4 �� 7.6 vs 50.9 �� 9.0; type of task: 59.9 �� 7.9 vs 46.5 �� 9.0) and the dyslexic group demonstrated significantly lower performance in all situations. Moreover, there was an interactive effect between the group and the duration of stimulus variables for the frequency-pattern tests, with the dyslexic group demonstrating significantly lower results for short durations (53.4 �� 8.2 vs 48.4 �� 11.1), as opposed to no difference in performance for the control group (62.2 �� 7.1 vs 60.6 �� 7.9). These results support the hypothesis that associates dyslexia with auditory temporal processing, identifying the stimulus-duration variable as the only one that unequally influenced the performance of the two groups.

Flow rate, pH and calcium concentration of saliva of children and adolescents with type 1 diabetes mellitus

Alterations in salivary parameters may increase the caries risk in diabetic children, but, contradictory data on this issue have been reported. The aims of this study were to compare salivary parameters (flow rate, pH and calcium concentration) between healthy and type 1 diabetes mellitus (T1DM) individuals. The sample consisted of 7- to 18-year-old individuals divided into two groups: 30 subjects with T1DM (group A) and 30 healthy control subjects (group B). Fasting glucose levels were determined. Unstimulated and stimulated saliva was collected. The pH of unstimulated saliva was measured with paper strips and an electrode. Calcium concentrations in stimulated saliva were determined with a selective electrode. Group A individuals had inadequate blood glucose control (HbA1C >9%), with means �� SD unstimulated salivary flow rate of 0.15 �� 0.1 mL/min compared to 0.36 �� 0.2 mL/min for group B (P < 0.01). Stimulated salivary flow rate was similar by both groups and above 2.0 mL/min. Saliva pH was 6.0 �� 0.8 for group A and significantly different from 7.0 �� 0.6 for group B (P < 0.01). Salivary calcium was 14.7 �� 8.1 mg/L for group A and significantly higher than 9.9 �� 6.4 mg/L for group B (P < 0.01). Except for elevated calcium concentrations in saliva, salivary parameters favoring caries such as low saliva pH and unstimulated salivary flow rate were observed in T1DM individuals.

Effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats

This study evaluated the effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats. Overfeeding was induced by reducing the litter size to 3 male pups per mother during the suckling period. The litter size of control rats was adjusted to 10 male pups per mother. Seven weeks after birth overfed and normally fed rats were selected and assigned to a sedentary protocol or to a low-intensity treadmill training protocol (60 min, 5 times/week, for 9 weeks). Four groups (overfed sedentary, N = 23; normally fed sedentary, N = 32; overfed exercised, N = 18, and normally fed exercised, N = 18) were evaluated at 18 weeks. Data are reported as means �� SEM. Initial body weight was similar in control and overfed rats [8.0 �� 0.2 g (N = 42) vs 8.0 �� 0.1 g (N = 50); P > 0.05] and body weight gain during the suckling period was higher in the overfed rats (30.6 �� 0.9 vs 23.1 �� 0.3 g; P < 0.05). Exercise attenuated the body weight gain of overfed compared to sedentary rats (505 �� 14 vs 537 �� 12 g; P < 0.05). The sedentary overfed rats showed higher visceral fat weight compared to normally fed animals (31.22 �� 2.08 vs 21.94 �� 1.76 g; P < 0.05). Exercise reduced visceral fat by 36.5% in normally fed rats and by 35.7% in overfed rats. Exercise attenuated obesity in overfed rats and induced an important reduction of visceral fat.

Zete��mata quaedam ex spatioso ac fructuoso philosophiae agro excerpta

Invokaatio: Cum Deo.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 ��M Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 ��M L-NAME, 10 ��M losartan, or 10 ��M enalaprilat. Gd significantly increased the maximum response (control: 72.3 �� 3.5; Gd: 101.3 �� 6.4%) and sensitivity (control: 6.6 �� 0.1; Gd: 10.5 �� 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 �� 4.1; Gd: -47.4 �� 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 �� 8.6; Gd: 122.4 �� 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 �� 28.8; L-NAME + Gd: 67.9 �� 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Stability of relative oxygen pulse curve during repeated maximal cardiopulmonary testing in professional soccer players

During cardiopulmonary exercise testing (CPET), stroke volume can be indirectly assessed by O2 pulse profile. However, for a valid interpretation, the stability of this variable over time should be known. The objective was to analyze the stability of the O2 pulse curve relative to body mass in elite athletes. VO2, heart rate (HR), and relative O2 pulse were compared at every 10% of the running time in two maximal CPETs, from 2005 to 2010, of 49 soccer players. Maximal values of VO2 (63.4 �� 0.9 vs 63.5 �� 0.9 mL O2&#8226;kg-1&#8226;min-1), HR (190 �� 1 vs188 �� 1 bpm) and relative O2 pulse (32.9 �� 0.6 vs 32.6 �� 0.6 mL O2&#8226;beat-1&#8226;kg-1) were similar for the two CPETs (P &gt; 0.05), while the final treadmill velocity increased from 18.5 �� 0.9 to 18.9 �� 1.0 km/h (P < 0.01). Relative O2 pulse increased linearly and similarly in both evaluations (r�� = 0.64 and 0.63) up to 90% of the running time. Between 90 and 100% of the running time, the values were less stable, with up to 50% of the players showing a tendency to a plateau in the relative O2 pulse. In young healthy men in good to excellent aerobic condition, the morphology of the relative O2 pulse curve is consistent up to close to the peak effort for a CPET repeated within a 1-year period. No increase in relative O2pulse at peak effort could represent a physiologic stroke volume limitation in these athletes.

Involvement of &#946;3-adrenergic receptors in the control of food intake in rats

This study examined the food intake changes evoked by intracerebroventricular (icv) injection of a selective agonist (BRL37344, 2 and 20 nmol) or antagonist (SR59230A, 10 and 50 nmol) of &#946;3-adrenergic receptors in 24-h fasted rats (adult male Wistar rats, 200-350 g, N = 6/treatment). The animals were also pretreated with saline icv (SAL) or SR59230A (50 nmol) followed by BRL37344 (20 nmol) or SAL in order to determine the selectivity of the effects evoked by BRL37344 on food intake or the selectivity of the effects evoked by SR59230A on risk assessment (RA) behavior. The highest dose of BRL37344 (N = 7) decreased food intake 1 h after the treatment (6.4 �� 0.5 g in SAL-treated vs 4.2 �� 0.8 g in drug-treated rats). While both doses of SR59230A failed to affect food intake (5.1 �� 1.1 g for 10 nmol and 6.0 �� 1.8 g for 50 nmol), this treatment reduced the RA frequency (number/30 min) (4 �� 2 for SAL-treated vs 1 �� 1 for 10 nmol and 0.5 �� 1 for 50 nmol SR59230A-treated rats), an ethological parameter related to anxiety. While pretreatment with SR59230A (7.0 �� 0.5 g) abolished the hypophagia induced by BRL37344 (3.6 �� 0.9 g), BRL37344 suppressed the reduction in RA frequency caused by SR59230A. These results show that the hypophagia caused by BRL37344 is selectively mediated by &#946;3-adrenergic receptors within the central nervous system. Moreover, they suggest the involvement of these receptors in the control of anxiety.

Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 ��g/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 �� 1.4 vs control: 23.1 �� 2.5) and hippocampus (1 LPS: 165.0 �� 3.0 vs control: 137.5 �� 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 �� 4.3; hippocampus = 182.2 �� 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 �� 0.8 vs 46.7 �� 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 �� 1.7 vs 28.1 �� 1.9) or repeated (47.6 �� 1.1 vs 28.1 �� 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 �� 4.1 vs 65.2 �� 2.2) and hippocampus (99.4 �� 4.4 vs 73.8 �� 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 �� 4.6 vs 36.6 �� 2.2; ARC: 62.4 �� 6.0 vs 37.0 �� 2.2; PVN: 100.7 �� 4.4 vs 65.2 �� 2.2; hippocampus: 123.0 �� 3.8 vs 73.8 �� 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.