Staircase in mammalian muscle without light chain phosphorylation

In disuse atrophied skeletal muscle, the staircase response is virtually absent and light chain phosphorylation does not occur. The purpose of the present study was to determine if staircase could be restored in atrophied muscle with continued absence of myosin light chain phosphorylation, by reducing what appears to be an otherwise enhanced calcium release. Control (untreated) and sham-operated female Sprague-Dawley rats were compared with animals after 2 weeks of complete inactivity induced by tetrodotoxin (TTX) application to the left sciatic nerve. In situ isometric contractile responses of rat gastrocnemius muscle were analyzed before and after administration of dantrolene sodium (DS), a drug which is known to inhibit Ca2+ release in skeletal muscle. Twitch active force (AF) was attenuated by DS from 2.2 ± 0.2 N, 2.7 ± 0.1 N and 2.4 ± 0.2 N to 0.77 ± 0.2 N, 1.05 ± 0.1 N and 1.01 ± 0.2 N in TTX (N = 5), sham (N = 11) and control (N = 7) muscles, respectively. Following dantrolene treatment, 10 s of 10-Hz stimulation increased AF to 1.32 ± 0.2 N, 1.52 ± 0.1 N and 1.45 ± 0.2 N for the TTX, sham and control groups, respectively, demonstrating a positive staircase response. Regulatory light chain (R-LC) phosphorylation was lower for TTX-treated (5.5 ± 5.5%) than for control (26.1 ± 5.3%) and sham (20.0 ± 5%) groups. There was no significant change from resting levels for any of the groups after DS treatment (P = 0.88). This study shows that treatment with dantrolene permits staircase in atrophied muscle as well as control muscle, by a mechanism which appears to be independent of R-LC phosphorylation.

Role of nitric oxide in hypoxia-induced hyperventilation and hypothermia: participation of the locus coeruleus

Hypoxia elicits hyperventilation and hypothermia, but the mechanisms involved are not well understood. The nitric oxide (NO) pathway is involved in hypoxia-induced hypothermia and hyperventilation, and works as a neuromodulator in the central nervous system, including the locus coeruleus (LC), which is a noradrenergic nucleus in the pons. The LC plays a role in a number of stress-induced responses, but its participation in the control of breathing and thermoregulation is unclear. Thus, in the present study, we tested the hypothesis that LC plays a role in the hypoxia-induced hypothermia and hyperventilation, and that NO is involved in these responses. Electrolytic lesions were performed bilaterally within the LC in awake unrestrained adult male Wistar rats weighing 250-350 g. Body temperature and pulmonary ventilation (VE) were measured. The rats were divided into 3 groups: control (N = 16), sham operated (N = 7) and LC lesioned (N = 19), and each group received a saline or an NG-nitro-L-arginine methyl ester (L-NAME, 250 µg/µl) intracerebroventricular (icv) injection. No significant difference was observed between control and sham-operated rats. Hypoxia (7% inspired O2) caused hyperventilation and hypothermia in both control (from 541.62 ± 35.02 to 1816.18 ± 170.7 and 36.3 ± 0.12 to 34.4 ± 0.09, respectively) and LC-lesioned rats (LCLR) (from 694.65 ± 63.17 to 2670.29 ± 471.33 and 36 ± 0.12 to 35.3 ± 0.12, respectively), but the increase in VE was higher (P<0.05) and hypothermia was reduced (P<0.05) in LCLR. L-NAME caused no significant change in VE or in body temperature under normoxia, but abolished both the hypoxia-induced hyperventilation and hypothermia. Hypoxia-induced hyperventilation was reduced in LCLR treated with L-NAME. L-NAME also abolished the hypoxia-induced hypothermia in LCLR. The present data indicate that hypoxia-induced hyperventilation and hypothermia may be related to the LC, and that NO is involved in these responses.

Role of the hypothalamic pituitary adrenal axis in the control of the response to stress and infection

The release of adrenocorticotropin (ACTH) from the corticotrophs is controlled principally by vasopressin and corticotropin-releasing hormone (CRH). Oxytocin may augment the release of ACTH under certain conditions, whereas atrial natriuretic peptide acts as a corticotropin release-inhibiting factor to inhibit ACTH release by direct action on the pituitary. Glucocorticoids act on their receptors within the hypothalamus and anterior pituitary gland to suppress the release of vasopressin and CRH and the release of ACTH in response to these neuropeptides. CRH neurons in the paraventricular nucleus also project to the cerebral cortex and subcortical regions and to the locus ceruleus (LC) in the brain stem. Cortical influences via the limbic system and possibly the LC augment CRH release during emotional stress, whereas peripheral input by pain and other sensory impulses to the LC causes stimulation of the noradrenergic neurons located there that project their axons to the CRH neurons stimulating them by alpha-adrenergic receptors. A muscarinic cholinergic receptor is interposed between the alpha-receptors and nitric oxidergic interneurons which release nitric oxide that activates CRH release by activation of cyclic guanosine monophosphate, cyclooxygenase, lipoxygenase and epoxygenase. Vasopressin release during stress may be similarly mediated. Vasopressin augments the release of CRH from the hypothalamus and also augments the action of CRH on the pituitary. CRH exerts a positive ultrashort loop feedback to stimulate its own release during stress, possibly by stimulating the LC noradrenergic neurons whose axons project to the paraventricular nucleus to augment the release of CRH.

Dermal dendritic cell number correlates with serum autoantibody titers in Brazilian pemphigus foliaceus patients

Pemphigus foliaceus (PF) is an autoimmune bullous disease endemic in Brazil. Since serum IL-12 is increased in patients with PF and Langerhans cells (LC) produce IL-12, we titrated serum autoantibodies by indirect immunofluorescence, and quantified epidermal dendritic cells, known as LC, and dermal dendritic cells (DC). Biopsies of blistering lesions were obtained from 22 patients, 13 of whom were submitted to biopsy of both injured and of apparently healthy skin. The control groups consisted of skin from 8 cadavers and from 12 women submitted to breast plastic surgery. LC and DC were identified with anti-CD1a antibody and quantified by morphometric analysis. LC number in the lesion and in apparently healthy skin from PF patients was similar to that of both control groups. DC number in the injured skin (median = 0.94 DC/mm basement membrane) was higher than that of the cadaver group (median = 0.13 DC/mm basement membrane). In the 13 patients with biopsies of both injured and apparently healthy skin, LC and DC were present in larger numbers in the lesion. There was a direct correlation between DC number in the lesion of the PF group and serum autoantibody titers. This correlation was not observed for LC number. The increased number of DC in the lesion, as well as its direct correlation with serum autoantibody titers suggest the participation of DC in the pathogenesis of PF. The relationship between increased DC number and IL-12 in PF needs to be clarified.

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7%) and a specificity of 94.7% (95% CI, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

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 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM 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 µM 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 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

Nitric oxide (·NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

The MTR A2756G polymorphism is associated with an increase of plasma homocysteine concentration in Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) present decreased homocysteine (Hcy) concentration, reflecting a functional folate deficiency secondary to overexpression of the cystathionine ß-synthase gene. Since plasma Hcy may be influenced by genetic polymorphisms, we evaluated the influence of C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR), of A2756G polymorphism in the methionine synthase gene (MTR), and of A80G polymorphism in the reduced folate carrier 1 gene on Hcy concentrations in Brazilian DS patients. Fifty-six individuals with free trisomy 21 were included in the study. Plasma Hcy concentrations were measured by liquid chromatography_tandem mass spectrometry with linear regression coefficient r² = 0.9996, average recovery between 92.3 to 108.3% and quantification limits of 1.0 µmol/L. Hcy concentrations >15 µmol/L were considered to characterize hyperhomocystinemia. Genotyping for the polymorphisms was carried out by polymerase chain reaction followed by enzyme digestion and allele-specific polymerase chain reaction. The mean Hcy concentration was 5.2 ± 3.3 µmol/L. There was no correlation between Hcy concentrations and age, gender or MTHFR C677T, A1298C and reduced folate carrier 1 A80G genotype. However, Hcy concentrations were significantly increased in the MTR 2756AG heterozygous genotype compared to the MTR 2756AA wild-type genotype. The present results suggest that the heterozygous genotype MTR 2756AG is associated with the increase in plasma Hcy concentrations in this group of Brazilian patients with DS.

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Occult hepatitis B virus (HBV) infection has been reported among patients with hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). Our aim was to evaluate the presence of occult HBV infection in patients with HCV-related liver cirrhosis (LC) with or without HCC in São Paulo, Brazil. Serum and liver tissue samples from 50 hepatitis B surface antigen-negative patients with HCV-related LC who underwent liver transplantation at the University of São Paulo School of Medicine Hospital from 1993 to 2004 were divided into groups with LC only (N = 33) and with LC plus HCC (N = 17). HBV DNA was assayed for serum and paraffin-embedded liver tissue (tumoral and non-tumoral) using real time PCR and only 1 case with HCC had HBV DNA-positive serum. All liver samples were negative. HCV genotype 3 was detected in 17/39 (43.7%) cases. In conclusion, using a sensitive real time PCR directed to detect HBV variants circulating in Brazil, occult hepatitis B infection was not found among HCV-positive cirrhotic patients and was rarely found among HCV-positive HCC patients. These results are probably related to the low prevalence of HBV infection in our population. Furthermore, we have also shown that HCV genotype 3 is frequently found in Brazilian cirrhotic patients, particularly when they also have HCC. More studies involving a large number of cases should be carried out to confirm these data and to further characterize Brazilian HCV genotype isolates to elucidate genetic features that might be related to its carcinogenic potential.

Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma, respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats

The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

AVALIAÇÃO SENSORIAL DO SUCO DE MAÇÃ PROCESSADO COM CASCA DE ARROZ COMO COADJUVANTE DE PRENSAGEM

Suco de maçã da variedade Fuji processado com casca de arroz como auxiliar na prensagem foi avaliado sensorialmente. Na prensagem foi utilizada pressão de trabalho de 150 e 200kgf/cm² sobre a polpa triturada e adicionado casca de arroz nas concentrações de 0; 1; 3 e 5%. Após o processamento foram efetuadas avaliações de rendimento e sensoriais por equipe de julgadores treinados. A maior concentração de casca de arroz proporcionou maior incremento no rendimento de extração do suco de maçã, superior às médias das testemunhas. Na pressão de 150kgf/cm² a testemunha obteve um rendimento de 59,91%, enquanto a concentração com 5% de casca de arroz teve um rendimento de 76,09%. Para a pressão de 200kgf/cm², a testemunha apresentou média de 66,52% e a concentração com 5% de casca de arroz obteve o maior rendimento de extração, média de 73,19%. Na avaliação sensorial, observa-se diferença significativa entre as testemunhas e os tratamentos, especialmente quanto aos atributos cor, odor e sabor. Na qualidade geral, os sucos obtidos com o tratamento testemunha foram considerados de melhor qualidade que aqueles obtidos com os tratamentos onde foi adicionado auxiliar de prensagem casca de arroz, indicando que este tratamento influencia negativamente na qualidade final do produto.

EFEITO DO TRATAMENTO TÉRMICO SOBRE A ATIVIDADE DE PEROXIDASE (POD) E POLIFENOLOXIDASE (PPO) EM MAÇÃ (Mallus comunis)

O extrato enzimático foi preparado a partir da polpa e casca da maçã de cultivares Fuji e Gala utilizando tampão fosfato de sódio 100mM, pH 5,0 como solução extratora. Dentre as análises determinou-se a concentração de proteína nos extratos enzimáticos concentrados de polpa e casca, sendo que o cultivar Fuji apresentou teores mais elevados em comparação ao cultivar Gala. Os tratamentos térmicos foram realizados nas temperaturas de 60, 65, 70 e 75°C por períodos que variaram de 1 a 10 minutos, sendo observado diminuição da atividade de POD e PPO com o aumento da temperatura e tempo; no entanto a POD não chegou a ser inativada em nenhum dos tratamentos realizados. A PPO foi inativada totalmente após 10 minutos de tratamento a 75°C. A eletroforese mostrou uma composição diferente de isoenzimas aniônicas e catiônicas da peroxidase.

Potencial da variedade Joaquina para o processamento de suco clarificado e vinho seco de maçã

Na classificação das maçãs para o comércio doméstico, uma parcela de até 30% pode ser descartada. As frutas de descarte, também chamadas de industriais, apresentam potencial tecnológico e podem ser aproveitadas na fabricação de diversos produtos, como sucos e fermentados alcoólicos. Cerca de 90% da produção brasileira de maçãs correspondem as cultivares Fuji e Gala, que apresentam baixa aptidão industrial, entretanto outras cultivares podem apresentar características que tornem o produto final com qualidade superior. Assim, foi avaliado o potencial tecnológico da cultivar Joaquina para a fabricação de suco clarificado e de vinho de maçã em comparação com as cultivares Fuji e Gala. Maçãs das cultivares Fuji e Gala foram obtidas no comercio local e da cultivar Joaquina, colocadas à disposição pela Estação Experimental de São Joaquim (Epagri). O suco clarificado e o vinho de maçã foram obtidos em bancada de laboratório com protocolos definidos. O suco varietal da Joaquina, quando comparado físico-quimicamente com os da Fuji e da Gala não diferiu significativamente, porém os julgadores em avaliação sensorial atribuíram-lhe as menores notas (3,78), com alto grau de rejeição (64%). O vinho varietal da maçã Joaquina, físico-quimicamente idêntico aos da Fuji e da Gala, foi considerado de aceitação semelhante ao da Gala (4,52) sendo o da Fuji (3,80) absolutamente rejeitado, com 67%. Assim, a maçã da variedade Joaquina pode ser usada com parcimônia no processamento de suco e com segurança na produção de vinho.

Avaliação sensorial e determinação de vida-de-prateleira de maçãs desidratadas

O objetivo deste trabalho foi determinar a preferência sensorial e vida-de-prateleira de fatias de maçãs das cultivares Fuji e Gala desidratadas. A secagem foi feita à temperatura de 60 °C, com velocidade do ar no interior do secador de bandejas de 0,121 m/s. Para a realização do teste de preferência sensorial e aceitabilidade, mediante uso de uma escala de avaliação, foram utilizadas as maçãs com e sem pré-tratamento, obtendo-se um total de 4 amostras para análise. A maçã com maior índice de aceitação foi a maçã da cultivar Fuji sem tratamento, e a maçã com índice elevado de rejeição foi a maçã Gala sem tratamento, com 25%. De acordo com o resultado da análise sensorial, a vida-de-prateleira experimental das maçãs desidratadas foi determinada para as maçãs Fuji sem pré-tratamento e para as maçãs Gala com tratamento em diferentes tipos e tamanhos de embalagens. Em 360 dias de armazenamento, à temperatura ambiente, observou-se principalmente o significativo aumento do ganho de umidade nas maçãs de ambas as cultivares em embalagens de Polietileno Tereftalato e Copolímero de Cloreto de Vinil e Vinilideno/ Polietileno (Pet Saran/Pe) e não se observou diferenças significativas entre as embalagens de Polietileno Tereftalato/Alumínio/Polietileno (Pet/Al/Pe) e Polipropileno/Alumínio/Polietileno (PP/Al/Pe).