Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 �� 0.48 vs food-restricted group = 4.84 �� 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 �� 0.44 vs food-restricted group = 7.96 �� 0.45, and control = 1.52 �� 0.06 vs food-restricted group = 1.53 �� 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of &#946;2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by &#946;2- and &#946;3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-&#945;-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor &#945; (TNF-&#945;). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-&#945; (100 &#956;g/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 ��M), KN-93 (0.2 ��M) and cyclosporin A (CsA, 0.2 ��M). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKII&#948;B and CaN were evaluated by the Lowry method, [��H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-&#945; induced a significant increase in protein content in a dose-dependent manner from 10 ��g/L (53.56 ��g protein/well) to 100 &#956;g/L (72.18 ��g protein/well), and in a time-dependent manner from 12 h (37.42 ��g protein/well) to 72 h (42.81 ��g protein/well). TNF-&#945; (100 &#956;g/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [��H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 ��M BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [��H]-leucine incorporation were abolished by 0.2 ��M KN-93 or 0.2 ��M CsA. TNF-&#945; increased the expression of CaMKII&#948;B by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 ��M BAPTA, which itself had no effect. These results suggest that TNF-&#945; induces increases in [Ca2+]i, CaMKII&#948;B and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-&#945;.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (&#8764;115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 ��M). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

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 ��m/s) and decreased its amplitude (HC=0.260��0.08 vs NC=0.324��0.10 &#916;F/F0), full width at half-maximum amplitude (HC=1.05��0.08 vs NC=1.26��0.01 ��m), 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 ��m/s, amplitude=0.282��0.10 &#916;F/F0, full width at half-maximum amplitude=1.14��0.01 ��m, 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.

Phytomass production and nutrient accumulation by green manure species

Green manuring is recognized as a viable alternative to improve nutrient cycling in soils. The aim of this study was to evaluate the phytomass production and nutrient accumulation in shoots of the summer green manures jack bean &#91;Canavalia ensiformis (L.) DC.&#93;, dwarf pigeon pea (Cajanus cajanvar var. Flavus DC.), dwarf mucuna &#91;Mucuna deeringiana (Bort) Merr&#93; and sunn hemp (Crotalaria juncea L.), under nitrogen fertilization and/or inoculation with N-fixing bacteria. A split plot design was arranged with the four Fabaceae species as main plots and nitrogen fertilization (with and without) and inoculation with diazotrophic bacteria (with and without) as the subplots, in a 2�� factorial. The experiment was arranged as a randomized complete block design with four replications. In the conditions of this trial, the sunn hemp had the highest production of shoot phytomass (12.4 Mg ha-1) and nutrient accumulation, while the dwarf mucuna had the lowest production of shoot phytomass (3.9 Mg ha-1) and nutrient accumulation. The results showed no effect of nitrogen fertilization or inoculation with N-fixing bacteria on the production of shoot phytomass and nutrient accumulation, except for inoculation without nitrogen fertilization, resulting in greater P accumulation (p <0.05) in the sunn hemp and greater Zn and Mn accumulation in the dwarf mucuna. These findings indicate that N fertilization or inoculation with N2-fixing bacteria for Fabaceae are low efficiency practices in the edaphoclimatic conditions of this study.

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum &#946;-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

Dynamic behavior of sylvatic yellow fever in Brazil (1954-2008)

INTRODUCTION: Sylvatic yellow fever (SYF) is enzootic in Brazil, causing periodic outbreaks in humans living near forest borders or in rural areas. In this study, the cycling patterns of this arbovirosis were analyzed. METHODS: Spectral Fourier analysis was used to capture the periodicity patterns of SYF in time series. RESULTS: SYF outbreaks have not increased in frequency, only in the number of cases. There are two dominant cycles in SYF outbreaks, a seven year cycle for the central-western region and a 14 year cycle for the northern region. Most of the variance was concentrated in the central-western region and dominated the entire endemic region. CONCLUSIONS: The seven year cycle is predominant in the endemic region of the disease due the greater contribution of variance in the central-western region; however, it was possible identify a 14 cycle that governs SYF outbreaks in the northern region. No periodicities were identified for the remaining geographical regions.

Varia����o temporal de nutrientes na ��gua escorrida pelo caule em floresta prim��ria explorada no nordeste do Par��

Foi enfocada a contribui&#231;&#227;o de nutrientes da &#225;gua escorrida pelo caule (CE), na ciclagem hidroqu&#237;mica em floresta prim&#225;ria explorada, em Benevides, PA. A concentra&#231;&#227;o de nutrientes na CE foi avaliada de dezembro de 1993 a abril de 1995, mediante coletores tipo colarinho, constru&#237;dos com espuma de silicone, acoplados por tubos a recipientes pl&#225;sticos. O monitoramento de CE se restringiu a doze &#225;rvores, nas quais foram coletadas mensalmente amostras para an&#225;lise qu&#237;mica de K+, Na+, Ca2+ e Mg2+ por espectrofotometria de absor&#231;&#227;o at&#244;mica, o N-NH4+, N-NO3 - e P-PO4 3- por colorimetria, em espectofot&#244;metro de fluxo cont&#237;nuo, o N-total por micro Kjeldahl e pH por potenciometria. A quantidade de nutrientes trazidos pela CE foi maior no in&#237;cio da &#233;poca chuvosa, exibindo marcante variabilidade temporal para K+, Na+, Ca2+, Mg2+, N-total, SO4 2-, enquanto que o oposto aconteceu com PO4 3-. A magnitude na concentra&#231;&#227;o dos nutrientes decresceu na seguinte ordem: K+ > Na+ > Ca2+ > N-t > SO4 2- > Mg2+ > PO4 3-. A distribui&#231;&#227;o e a intensidade de chuva n&#227;o influenciam marcantemente o pH na CE.

Contribui����o �� hidroqu��mica de drenagens no Munic��pio de Manaus - AM

O rio Puraquequara, e seu afluente direito, o igarap�� ��gua Branca, localizam-se fora da zona urbana de Manaus e est��o com a bacia ainda protegida por floresta prim��ria. T��m caracter��sticas geol��gicas, pedol��gicas e climatol��gicas de igarap��s naturais de terra-firme da Amaz��nia Central. Foram coletadas amostras de ��gua nos meses de novembro de 1998 (per��odo seco) e abril de 1999 (per��odo chuvoso) e determinados os par��metros temperatura, pH, turbidez, condutividade, alcalinidade, dureza, DQO, Ca2+, Mg2+, Na+, K+, SiO2, NO3-, NO2-, PO4(3-), SO4(2-), Cl-, NH4+ e Fe total. As ��guas apresentaram pH entre 3,8 e 4,1 passando a menos ��cidas na estiagem. A alcalinidade e a turbidez s��o, em geral, mais elevadas na estiagem, enquanto a condutividade, dureza e DQO s��o maiores no per��odo mais chuvoso. SiO2 e o Cl- foram os ��nions mais abundantes, com contribui����o maior do primeiro no Puraquequara e menor no ��gua Branca, enquanto o Cl- tem comportamento oposto. Na+, Fe total e NH4+ s��o os c��tions mais abundantes e predominam, no geral, no per��odo mais seco. O NH4+ �� o ��nico c��tion que tende a aumentar sua contribui����o para jusante do igarap�� no per��odo ��mido. Os teores de K+ s��o mais elevados do que os de Mg2+ e este que os de Ca2+, e s��o todos superados pelo Na+. Os teores da NH4+ e NO3-, acima de 0,2 mg/L e 0,5 mg/L, respectivamente, s��o ind��cios de contamina����o. Essas caracter��sticas definem as ��guas da bacia como muito dilu��das, com predomin��ncia dos ��nions sobre os c��tions e correlacion��veis as ��guas de cor preta.