Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Peripheral markers of oxidative stress in chronic mercuric chloride intoxication

The present study was designed to evaluate the time course changes in peripheral markers of oxidative stress in a chronic HgCl2 intoxication model. Twenty male adult Wistar rats were treated subcutaneously daily for 30 days and divided into two groups of 10 animals each: Hg, which received HgCl2 (0.16 mg kg-1 day-1), and control, receiving the same volume of saline solution. Blood was collected at the first, second and fourth weeks of Hg administration to evaluate lipid peroxidation (LPO), total radical trapping antioxidant potential (TRAP), and superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT). HgCl2 administration induced a rise (by 26%) in LPO compared to control (143 ± 10 cps/mg hemoglobin) in the second week and no difference was found at the end of the treatment. At that time, GST and GPx were higher (14 and 24%, respectively) in the Hg group, and Cu,Zn-SOD was lower (54%) compared to control. At the end of the treatment, Cu,Zn-SOD and CAT were higher (43 and 10%, respectively) in the Hg group compared to control (4.6 ± 0.3 U/mg protein; 37 ± 0.9 pmol/mg protein, respectively). TRAP was lower (69%) in the first week compared to control (43.8 ± 1.9 mM Trolox). These data provide evidence that HgCl2 administration is accompanied by systemic oxidative damage in the initial phase of the process, which leads to adaptive changes in the antioxidant reserve, thus decreasing the oxidative injury at the end of 30 days of HgCl2 administration. These results suggest that a preventive treatment with antioxidants would help to avoid oxidative damage in subjects with chronic intoxication.

Three-dimensional kinematic analysis of upper and lower limb motion during gait of post-stroke patients

The aim of this study was to analyze the alterations of arm and leg movements of patients during stroke gait. Joint angles of upper and lower limbs and spatiotemporal variables were evaluated in two groups: hemiparetic group (HG, 14 hemiparetic men, 53 ± 10 years) and control group (CG, 7 able-bodied men, 50 ± 4 years). The statistical analysis was based on the following comparisons (P ≤ 0.05): 1) right versus left sides of CG; 2) affected (AF) versus unaffected (UF) sides of HG; 3) CG versus both the affected and unaffected sides of HG, and 4) an intracycle comparison of the kinematic continuous angular variables between HG and CG. This study showed that the affected upper limb motion in stroke gait was characterized by a decreased range of motion of the glenohumeral (HG: 6.3 ± 4.5, CG: 20.1 ± 8.2) and elbow joints (AF: 8.4 ± 4.4, UF: 15.6 ± 7.6) on the sagittal plane and elbow joint flexion throughout the cycle (AF: 68.2 ± 0.4, CG: 46.8 ± 2.7). The glenohumeral joint presented a higher abduction angle (AF: 14.2 ± 1.6, CG: 11.5 ± 4.0) and a lower external rotation throughout the cycle (AF: 4.6 ± 1.2, CG: 22.0 ± 3.0). The lower limbs showed typical alterations of the stroke gait patterns. Thus, the changes in upper and lower limb motion of stroke gait were identified. The description of upper limb motion in stroke gait is new and complements gait analysis.

Anmärckningar samlade under en resa til China

Invokaatio: J.J.N.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Use of the Wasserman equation in optimization of the duration of the power ramp in a cardiopulmonary exercise test: a study of Brazilian men

This study aimed to analyze the agreement between measurements of unloaded oxygen uptake and peak oxygen uptake based on equations proposed by Wasserman and on real measurements directly obtained with the ergospirometry system. We performed an incremental cardiopulmonary exercise test (CPET), which was applied to two groups of sedentary male subjects: one apparently healthy group (HG, n=12) and the other had stable coronary artery disease (n=16). The mean age in the HG was 47±4 years and that in the coronary artery disease group (CG) was 57±8 years. Both groups performed CPET on a cycle ergometer with a ramp-type protocol at an intensity that was calculated according to the Wasserman equation. In the HG, there was no significant difference between measurements predicted by the formula and real measurements obtained in CPET in the unloaded condition. However, at peak effort, a significant difference was observed between oxygen uptake (V˙O2)peak(predicted)and V˙O2peak(real)(nonparametric Wilcoxon test). In the CG, there was a significant difference of 116.26 mL/min between the predicted values by the formula and the real values obtained in the unloaded condition. A significant difference in peak effort was found, where V˙O2peak(real)was 40% lower than V˙O2peak(predicted)(nonparametric Wilcoxon test). There was no agreement between the real and predicted measurements as analyzed by Lin’s coefficient or the Bland and Altman model. The Wasserman formula does not appear to be appropriate for prediction of functional capacity of volunteers. Therefore, this formula cannot precisely predict the increase in power in incremental CPET on a cycle ergometer.

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (ΔBP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 μM). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control.

Mercúrio total em cação comercializado em São Paulo - SP, Brasil

Foram determinados os teores de mercúrio total em espécies de cação - anjo, cação-azul, cambeva, caçoa, machote e anequim - comercializadas em São Paulo - SP, Brasil. Os níveis encontrados variaram de 0,04 a 4,71mg Hg/kg, sendo que 54% estavam acima de 1mg Hg/kg, que é o limite aceito pela legislação brasileira especificamente para peixes predadores, para o propósito de consumo. Os resultados mostram que espécies de cação têm sido comercializadas em desacordo com a legislação, expondo os consumidores aos riscos tóxicos provocados pelo mercúrio. Foram feitas considerações em relação aos elevados níveis de mercúrio observados e o consumo correspondente, baseado na Ingestão Semanal Tolerável Provisional de 0,3mg de mercúrio total por pessoa, que no máximo não deverá ser superior a 0,2mg na forma de metilmercúrio, recomendada pela FAO/OMS. Considerações também foram feitas em relação à necessidade de inspeção e monitoramento das espécies brasileiras de cação usadas como alimento.

Migração de minerais de panelas brasileiras de aço inoxidável, ferro fundido e pedra-sabão (esteatito) para simulantes de alimentos

Os utensílios culinários podem liberar alguns componentes inorgânicos durante a cocção de alimentos. A migração destes pode ser interessante desde que ocorra em quantidades adequadas às necessidades nutricionais do usuário ou não existam implicações toxicológicas. Foi avaliada a migração de metais (Fe, Mg, Mn, Cr, Ni, Ca, Zn, Pb, Cd e Hg) por espectrometria de emissão óptica, utilizando ácidos láctico e acético como simulantes do alimento em panelas de aço inoxidável (304), ferro fundido (GG-10) e pedra-sabão (esteatito), durante 15 cocções seqüenciais. O comportamento da migração durante cinco ciclos de estocagem a 22ºC também foi estudado. Análise de regressão dos dados revelou que, em função do uso, as panelas de inox apresentam redução da migração de metais de forma rápida, enquanto as de pedra-sabão mostram um declínio suave na migração, contrastando com as de ferro, que apresentam aumento na migração de Fe e Mn com o uso contínuo. Em nenhum utensílio foi observada a liberação de metais pesados, como Pb, Cd ou Hg.

Produção de poligalacturonase, pelo termofílico Bacillus sp. e algumas de suas propriedades

A produção de poligalacturonase pelo termofílico Bacillus sp. SMIA-2, cultivado em meio líquido contendo pectina cítrica como única fonte de carbono, alcançou a sua máxima atividade enzimática em 30 horas, com níveis de 42 IU.mL-1. Entre as várias fontes orgânicas e inorgânicas de nitrogênio testadas, o sulfato de amônio foi a que proporcionou maior atividade da poligalacturonase. O aumento da concentração da pectina cítrica, no meio de cultura, acima de 0,5% não proporcionou um aumento da atividade da enzima. O microrganismo foi capaz de utilizar uma variedade de fontes de carbono, mas a atividade da poligalacturonase variou com cada fonte. Pectina de maçã foi a melhor fonte de carbono para a secreção da poligalacturonase (56 IU.mL-1), enquanto frutose e maltose não foram muito efetivas. Galactose, rafinose e glicose inibiram a síntese da enzima. Estudos sobre a caracterização da poligalacturonase revelaram que a temperatura ótima dessa enzima foi 70 ºC e que ela manteve 62 e 58% de sua atividade máxima quando incubada por 2 horas a 40 e 90 ºC, respectivamente. O pH ótimo para atividade da enzima foi 7,0. A enzima manteve 90 e 75% de sua atividade máxima quando incubada a pH 8,0 e 8,5, respectivamente, por 24 horas, à temperatura ambiente. A atividade enzimática foi estimulada pelos íons Mg2+ e Zn2+. Por outro lado, foi inibida pelos íons Cs+2, Hg+2, Li+2 e Sr+2.

Concentração e retenção do selênio em peixes marinhos

O pescado contém substancialmente maiores concentrações de selênio (Se) do que outras carnes e é, por isso, considerado a maior fonte dietária desse nutriente. Ao mesmo tempo, o pescado também é a maior fonte de ingestão de mercúrio (Hg) na dieta humana, cuja toxidez pode ser reduzida pelo Se, por interação entre ambos. Em consequência, a biodisponibilidade do Se é reduzida. No presente estudo, a ocorrência do Se foi avaliada em espécies marinhas de peixes brasileiros, comercialmente disponíveis, e determinada sua retenção em cação-mangona. Os níveis médios de Se encontrados nos peixes ósseos, corvina (Micropogonias furnieri), peixe-porco (Balistes capricus), pescada-branca e sardinha (Sardinella brasiliensis), 0,48 ± 0,18 μg.g-1, e nos peixes cartilaginosos, cação-anjo, cação-cambeva, cação-machote e cação-mangona, 0,36 ± 0,07 μg.g-1, não diferiram entre si (p > 0,05) e situaram-se na mesma faixa de ocorrência indicada na literatura para peixes marinhos em geral. Os níveis de Se detectados nos cações (espécies predadoras) não permitem o estabelecimento de relação estequiométrica suficiente para a interação com o Hg, visando à proteção tóxica esperada. A retenção efetiva do Se medida em cação-mangona, após extração com água, foi considerada elevada (mínimo de 68,81%) e importante sob os pontos de vista nutricional e toxicológico.

Inverkan av indirekt rekombination på fotoinducerad absorption

I organiska halvledare påverkas mängden laddningsbärare kraftigt av indirekt rekombination, det vill säga processen då fria laddningsbärare försvinner genom att kombineras med orörliga laddningsbärare av motsatt laddning. De orörliga laddningsbärarna uppstår när laddningsbärare fastnar i fällor, som är energitillstånd med låg energi och densitet. Utöver indirekt rekombination sker även direkt rekombination mellan fria laddningsbärare. Då man tillverkar solceller av organiska halvledare påverkas effektiviteten av energidistributionen och rekombinationsprocesserna i materialen. Utveckling av olika metoder för undersökning av dessa egenskaper är således till nytta i jakten på bättre solcellsmaterial. Målet med detta arbete var att vidareutveckla dataanalysen för cwPA-mätningar(från engelska continuous-wave Photoinduced Absorption) för att ur resultaten få information om indirekt rekombination och fälldistributioner. I cwPA-mätningar studerar man fotoinducerad absorption, det vill säga förändringen i absorption hos ett prov då densiteten av fotogenererade laddningsbärare varierar. Laddningsbärarna genereras av ett pumpljus vars intensitet ges av en fyrkantsvåg som växlar mellan 0 och I med vinkelfrekvensen omega. Resultaten fås i form av i-fas-signal (PAI), som har samma frekvens och fas som pumpljuset, och kvadratur (PAQ), som har samma frekvens som pumpljuset men är fasförskjuten 90 grader. Fördelen med denna mätning är förutom känsligheten att den är kontaktlös, vilket gör att den visar egenskaperna hos det undersökta materialet utan att påverkas av elektriska kontakter. För att undersöka inverkan av indirekt rekombination på cwPA-mätningar simulerades mätresultat genom att använda numeriska beräkningar. Grunden för simuleringarna var att lösa differentialekvationer för densiteter av laddningsbärare i olika tillstånd. Beräkningarna använde en modell med transporttillstånd och fällor placerade så att energidistributionen var symmetrisk för elektroner och hål. Modellen antog att laddningsbärare inte kunde röra sig direkt mellan fällor utan endast via transporttillstånd. Från simuleringarna erhölls användbara samband mellan fotoinducerad absorption och olika fälldistributioner. Särskilt påverkade distributionerna i-fas-signalen för hög intensitet på pumpljuset och kvadraturen för låg frekvens på fyrkantsvågen. För en exponentiell fälldistribution hittades samband mellan mätresultat och distributionens karakteristiska energi (Ech) i förhållande till temperaturen (T). Dessa är för hög intensitet PAI~I^(1+Ech/kT) och för låg frekvens PAQ~omega^(kT/Ech). Resultaten visade att man kan skilja på en exponentiell fälldistribution, en gaussisk fälldistribution och ett system som domineras av direkt rekombination genom att göra cwPA-mätningar vid olika temperaturer.

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