Clinical and genetic analysis of a case of dihydrolipoamide dehydrogenase deficiency caused by novel variant of DLD gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical and genetic characteristics of a patient with dihydrolipoamide dehydrogenase deficiency.@*METHODS@#Potential variants of the DLD gene were detected by whole exome sequencing and verified by Sanger sequencing.@*RESULTS@#Compound heterozygous variants, c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr), were detected in the DLD gene. The c.1058T>C (p.Ile353Thr) variant was derived from his mother and known to be pathogenic. The c.704_705delTT (p.Leu235Argfs*8) variant was derived from his father and was unreported previously.@*CONCLUSION@#The compound heterozygous variants of c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr) of the DLD gene probably underlay the disease in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for the family.

Hypertrophy of NADH-diaphorase positive myenteric neurons in rat jejunum after acute infection caused by Toxoplasma gondii

Toxoplasmosis, a globally distributed feline-associated zoonosis caused by the protozoan Toxoplasma gondii, affects birds and mammals, including humans. This study assesses the consequences of acute T. gondii infection for NADH-diaphorase positive myenteric neurons in rat jejunum. Ten male Wistar rats (Rattus norvegicus) were divided into two groups: G1 (n = 5) and G2 (n = 5). Animals from G2 were orally inoculated with 500 genotype III (M7741) T. gondii oocysts. Twenty-four hours after inoculation, the animals were euthanized and had their jejuna removed, through laparotomy, and measured (length and width) to calculate their areas. Intestinal segments were submitted to NADH-diaphorase histochemistry to evidence the most metabolically active subpopulation of myenteric neurons. No changes were found in body weight; intestinal length, width or area; or neuron population density. Increase of body cell area and cytoplasm and decrease of nuclear area of the myenteric neurons of infected animals were observed by morphometric analysis.

Effect of different carbon sources on pyruvic acid production by using lpdA gene knockout Escherichia coli / 生物工程学报

We studied the ability of lpdA gene knockout Escherichia coli to ferment different sugars in mineral salts medium for the production of pyruvate. The sugars studied were glucose, fructose, xylose and mannose at a concentration of 10 g/L. At the same time, effect of inoculum size on lpdA fermentation with glucose was studied. The strain was able to use all sugars for biomass generation and pyruvate production. The lpdA knockout mutant converted glucose, fructose, xylose and mannose to pyruvate with yields of 0.884 g/g, 0.802 g/g, 0.817 g/g and 0.808 g/L, respectively. The pyruvate accumulation curve coupled with cell growth except for mannose as carbon source. When the inoculation size increased, the rate of glucose consumption, pyruvate accumulation and cell growth increased but lower pyruvate concentration. This study demonstrates that E. coli lpdA mutant has the potential to produce pyruvic acid from xylose and mannose.

Caracterização dos genes que codificam a enzima dihidrolipomida desidrogenase (TcLip DH) e a proteína transportadora de hexoses (TcrHT1) em cepas do Trypanosoma Cruzi sensíveis e resistentes ao benzonidazol / Characterization of genes encoding the enzyme dihidrolipomida dehydrogenase (TcLip DH) and hexose transporter protein (TcrHT1) of Trypanosoma cruzi in strains sensitive and resistant to benznidazole

A glicose e outras hexoses são importantes para o funcionamento das células eucariotas. Os transportadores de hexoses são proteínas transmembrana que levam o substrato para dentro da célula. Na glicólise, o açúcar captado é convertido em piruvato e este, por sua vez, é convertido a acetil-CoA para o ciclo do ácido cítrico. A conversão do piruvato a acetil-CoA conta com a participação da enzima lipoamida desidrogenase(LipDH), uma flavoenzima homodimérica da família das FAD-dissulfeto oxiredutase que é também importante para o mecanismo de oxiredução da célula. Murta et al. (2008, in press), utilizando a metodologia Differential Display (DD) selecionaram os genes que codificam o transportador de hexoses (TcrHT1) e a enzima lipoamida desidrogenase (TcLipDH) como genes mais expressos em uma cepa do T. cruzi com resistência induzida in vitro ao benzonidazol BZ e, que, portanto, seriam alvos em potencial para quimioterapia. Neste estudo, a fim de caracterizar os genes TcrHT1 e TcLipDH, foram analisados os níveis de mRNA; a organização genômica, a amplificação e a presença de polimorfismos; o nível de expressão ou atividade da proteína e a localização cromossômica em cepas do T. cruzi sensíveis e resistentes ao BZ.

Ensaios de Northern blot e PCR em Tempo Real confirmaram os resultados de DD e mostraram que ambos estão cerca de 2X mais expressos na cepa com resistência induzida in vitro ao BZ, 17LER, quando comparada a seu par sensível, 17WTS. Por Southern blot confirmamos que o gene TcLipDH possui duas cópias e o TcrHT1, um arranjo em “tandem” e não estão amplificados no genoma de nenhuma cepa resistente. Além disso, foi encontrado um polimorfismo para o gene TcrHT1 que não está relacionado ao fenótipo de resistência a drogas e sim com o zimodema ao qual as cepas analisadas pertencem. Os resultados de Western blot mostraram que a enzima TcLipDH está igualmente expressa em todas as cepas analisadas à exceção da cepa 17LER na qual a proteína está 2X menos expressa em comparação à 17WTS. Devido à dificuldade de obter anticorpos anti-TcrHT1, os ensaios de Western blotting foram substituídos pelo ensaio de atividade enzimática no qual constatamos que a eficiência de captação de glicose é 40% menor na cepa 17LER quando comparada à 17WTS. A localização cromossômica desses genes não está relacionada à resistência e é zimodema-dependente para o gene TcrHT1. Análises de bioinformática permitiram contextualizar evolutivamente ambos os genes e geraram informações interessantes sobre filogenia e anotação no genoma

Caracterização dos genes que codificam a enzima dihidrolipomida desidrogenase (TcLip DH) e a proteína transportadora de hexoses (TcrHT1) em cepas do Trypanosoma Cruzi sensíveis e resistentes ao benzonidazol

A glicose e outras hexoses são importantes para o funcionamento das células eucariotas. Os transportadores de hexoses são proteínas transmembrana que levam o substrato para dentro da célula. Na glicólise, o açúcar captado é convertido em piruvato e este, por sua vez, é convertido a acetil-CoA para o ciclo do ácido cítrico. A conversão do piruvato a acetil-CoA conta com a participação da enzima lipoamida desidrogenase(LipDH), uma flavoenzima homodimérica da família das FAD-dissulfeto oxiredutase que é também importante para o mecanismo de oxiredução da célula. Murta et al. (2008, in press), utilizando a metodologia Differential Display (DD) selecionaram os genes que codificam o transportador de hexoses (TcrHT1) e a enzima lipoamida desidrogenase (TcLipDH) como genes mais expressos em uma cepa do T. cruzi com resistência induzida in vitro ao benzonidazol BZ e, que, portanto, seriam alvos em potencial para quimioterapia. Neste estudo, a fim de caracterizar os genes TcrHT1 e TcLipDH, foram analisados os níveis de mRNA; a organização genômica, a amplificação e a presença de polimorfismos; o nível de expressão ou atividade da proteína e a localização cromossômica em cepas do T. cruzi sensíveis e resistentes ao BZ.

Ensaios de Northern blot e PCR em Tempo Real confirmaram os resultados de DD e mostraram que ambos estão cerca de 2X mais expressos na cepa com resistência induzida in vitro ao BZ, 17LER, quando comparada a seu par sensível, 17WTS. Por Southern blot confirmamos que o gene TcLipDH possui duas cópias e o TcrHT1, um arranjo em “tandem” e não estão amplificados no genoma de nenhuma cepa resistente. Além disso, foi encontrado um polimorfismo para o gene TcrHT1 que não está relacionado ao fenótipo de resistência a drogas e sim com o zimodema ao qual as cepas analisadas pertencem. Os resultados de Western blot mostraram que a enzima TcLipDH está igualmente expressa em todas as cepas analisadas à exceção da cepa 17LER na qual a proteína está 2X menos expressa em comparação à 17WTS. Devido à dificuldade de obter anticorpos anti-TcrHT1, os ensaios de Western blotting foram substituídos pelo ensaio de atividade enzimática no qual constatamos que a eficiência de captação de glicose é 40% menor na cepa 17LER quando comparada à 17WTS. A localização cromossômica desses genes não está relacionada à resistência e é zimodema-dependente para o gene TcrHT1. Análises de bioinformática permitiram contextualizar evolutivamente ambos os genes e geraram informações interessantes sobre filogenia e anotação no genoma

Expressions of c-Fos and NADPH-d in the related brainstem during vestibular compensation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the mechanism of vestibular compensation and to observe the changes of c-Fos and NADPH-d expressions in the brainstem of the vestibular deafferentation rats in static status or following angular acceleration stimulation.</p><p><b>METHODS</b>Totally 60 SD rats were randomly divided into control group (labyrinthine intact), complete unilateral vestibular deafferentation (UVD) group, simultaneous complete bilateral vestibular deafferentation (BVD) group (n = 20 in each group). Subgroups (n = 10 in each subgroup) were set for static status or following angular acceleration stimulation in each group. Double labeling with histochemistry-immunohistochemistry was performed to observe c-Fos/NADPH-d neurons.</p><p><b>RESULTS</b>No positive c-Fos/NADPH-d expression was observed in the both sides of medial vestibular nucleus (MVN) and prepositus hypoglossi (PrH) of normal rats in static status and BVD rats whether following canal rotation or not. c-Fos/ NADPH-d expression was observed in the ipsilesional MVN and the contralesional PrH of UVD rats. However, c-Fos/NADPH-d were detected in both sides of MVN and PrH in UVD rats and normal rats following angular acceleration stimulation.</p><p><b>CONCLUSION</b>In the ipsilesional MVN and the contralesional PrH, c-Fos plays an important role in vestibular compensation, in which nitric oxide acts as a key neurotransmitter.</p>

Histological, enzymohistochemical and biomechanical observation of skeletal muscle injury in rabbits / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To explore the pathophysiological and biomechanical features of skeletal muscular injury for providing a rational basis for its treatment, prevention and rehabilitation.</p><p><b>METHODS</b>In 70 adult rabbits, the left tibialis anterior (TA) muscle was stretched to injury, while the right TA muscle served as control. Histological, enzymohistochemical and biomechanical changes were observed on days 0, 1, 2, 3, and 7 after injury. Cytochrome oxidase (CCO), acid phosphatase (ACP), ATPase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH), NADH-diaphorase (NADHD), glutamatedehydrogenase (GDH), alpha-glycerophosphate dehydrogenase (alpha-GPD) and lactate dehydrogenase (LDH) were measured. The examined biomechanical parameters included maximal contractile force, ultimate load, length, energy absorption, tangent stiffness, and rupture site.</p><p><b>RESULTS</b>Partial or complete rupture of TA muscle occurred near the muscle-tendon junction. There was an intense inflammatory reaction on day 1 and 2 after injury. Endomysium fibrosis and myotube formation were observed on day 3, and developed further on day 7. The activity of cell oxidases (CCO, ATPase, MDH, alpha-GPD, SDH, NADHD and GDH) showed a significant drop from day 0 to 2, and resumed with different levels on day 3. The increment of enzymatic activities continued on day 7 and the levels of NADHD and alpha-GPD reached to the levels of control muscle. Maximal contractile force was 70.17%+/-3.82% of controls immediately after injury, 54.82%+/-3.09% at 1 day, 66.41%+/-4.36% at 2 days, 78.39%+/-4.90% at 3 days and 93.64%+/-5.02% at 7 days. Ultimate load was 85.78%+/-7.54% of controls at the moment of injury, 61.44%+/-5.91% at 1 day, 49.17%+/-4.26% at 2 days, 64.43%+/-5.02% at 3 days, and 76.71%+/-6.46% at 7 days.</p><p><b>CONCLUSIONS</b>Endomysium fibrosis and scar formation at the injured site are responsible for frequent recurrence of skeletal muscle injury. Recovery of tensile load slower than that of maximal contractile force may be another cause. Whether the injured muscle returns to normal exercise is mainly determined by the tensility on which the muscle-tendon can bear rather than the maximal contractile force.</p>

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate theneuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6 ) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.

Discrete analysis of bile acid in serum and bile with 3 alpha-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads.

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD+ during oxidation of bile acid by immobilized 3alpha-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in 0.065 M sodium phosphate buffer (pH 7.0). The minimum detection limit of the method was 4.8 pmol/L in the serum and 19.5 micromol/L in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were <1.0% and <0.2% in the serum and <0.2% and <0.6% in bile, respectively. A good correlation for bile acid in the serum (r1= 0.95) and in bile (r2 = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3alpha-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A).

Microwave coagulation at different temperatures for hepatocellular carcinoma management: efficacy evaluation by enzyme histochemical staining / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the application of HE and enzyme histochemical staining in assessing the viability of hepatocellular carcinoma (HCC) cells coagulated by microwave ablation at different temperatures.</p><p><b>METHODS</b>Two groups of mice (n=6) with transplanted homogenic HCC were treated by microwave ablation at 60 degrees C and 50 degrees C for 3 min, respectively. Before and after microwave ablation, paraffin sections and frozen sections of the tumors were prepared for routine HE staining and enzyme histochemical staining with nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase), respectively, and observed under microscope.</p><p><b>RESULTS</b>Shortly after microwave ablation, the morphology and arrangements of the nucleus of the ablated tumor cells in the two groups showed no obvious alteration in HE stained sections, but in sections with enzyme histochemical staining, the activity of NADH-diaphorase in ablated tumor tissue at 60 degrees C disappeared, suggesting the death of HCC cells; sporadic activity of the enzyme was detected in the coagulated tumor at 50 degrees C, indicating tumor cells surviving the ablation. The ablation effect was markedly different between the two groups (P<0.01).</p><p><b>CONCLUSION</b>HE staining is not suitable for evaluation of HCC destruction immediately after microwave ablation, and detection of NADH-diaphorase activity with the enzyme histochemical method better suits this purpose.</p>

Inactivación de la dihidrolipoamida deshidrogenasa de Trypanosoma cruzi por fenotiazinas en presencia de citocromo c y peróxido de hidrógeno: efectos de los antioxidantes / Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by phenothiazines in the presence of cytochrome c and hydrogen peroxide: effects of antioxidants

El citocromo c catalizó la oxidación de las fenotiazinas (FTZ) en presencia de peróxido de hidrógeno. La formación del radical catiónico de promazina (PZ+.) se demostró por espectrofo-tometría y por su conversión a promazina sulfóxido La dihidrolipoamida deshidrogenasa (LADH) del Trypanosoma cruzi es inhibida irreversiblemente por el sistema citocromo c/H2O2 complementado con fenotiazinas. La inactivación de la LADH del parásito varía según la estructura de las FTZ, el tiempo de incubación del sistema pro-oxidante con la LADH, y la presencia de un antioxidante supresor de radicales FTZ+. Entre las 12 FTZ ensayadas, la promazina (PZ), tioridazina (TRDZ) y trimeprazina (TMPZ) fueron las más efectivas produciendo inactivaciones de 82 por ciento,76 por ciento y 72 por ciento, respectivamente, a los 90 min de incubación. El efecto de PZ (con grupo alquilamino en la posición N 10) disminuyó por modificación de su estructura en la posición 2 (efecto inactivante de PZ > cloropromazina (CPZ) > propionilpromazina (PPZ) > trifluopromazina (TFPZ) o en la posición 10 ( efecto inactivante de PZ > TMPZ > prometazina (PMTZ).El efecto de las FTZ con sustituyente piperidinil en N 10 dependió del grupo de la posición 2 ( SCH3, en TRDZ de mayor efecto; CN, en propericiazina (PCYZ), la de menor efecto entre las FTZ estudiadas). Parece que la presencia del sustituyente piperazinil en posición N 10 no tiene función importante en el efecto inactivante de las FTZ, el cual dependió de la estructura del grupo en la posición 2. El efecto de los compuestos con Cl en posición 2 (CPZ, procloroperazina (PCP), perfenazina (PFZ)) fue mayor que el obtenido con los compuestos CF3 (TFPZ, trifluoroperazina (TFP), flufenazina (FFZ), e independiente de la estructura del sustituyente N 10.El efecto de las FTZ sobre la LADH de T. cruzi depende, por lo menos en parte, de la estabilidad de los radicales FTZ+. generados por la actividad peroxidasa. La LADH T c, en comparación con la LADH de mamífero...

Cytochrome c catalyzed the oxidation of phenothiazines (PTZ) in the presence of hydrogen peroxide. The transient formation of the promazine radical cation (PZ+.) has been demonstrated by light absorption measurements as well as by its conversión to promazine sulfoxide. Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH T c) was irreversibly inhibited by treatment with cytochrome c (cyt c)/H2O2 system supplemented with PTZ. LADH T c inactivation depended on a) The PTZ structure b) Time of incubación with the complete oxidant system c) The presence of an antioxidant that intercept free radicals. PZ, thioridazine (TRDZ) and trimeprazine (TMPZ), were the most effective systems out of twelve PTZ studied, with inactivation values of 82, 76 and 72%, respectively, after 90 min of incubation. LADH T c inactivation by PZ (with alkylamine substituent at N 10 position) decreased by its structural modification at 2 position (inactivation PZ > chlorpromazine (CPZ) > propionylpromazine (PPZ)>trifluopromazine (TFPZ)) or at N 10 position (inactivation PZ > TMPZ > promethazine (PMTZ)) PTZ activity with piperidinyl substituent at N10 position depended on the group at 2 position (TRDZ, with thiomethyl group, has high inactivating effect on LADH T c; propericyazine (PCYZ), with cyano group, is much less active). Apparently, piperazinyl substituent at the N10 position on the phenothiazine have not an important function in the compound's inactivating effect on LADH T c. The effect of PTZ with Cl at 2 position (CPZ, prochlorperazine (PCP), perphenazine (PFZ)) was higher than the effect of compounds with CF3 in the same position (TFPZ,trifluoperazine (TFP),fluphenazine (FFZ) ) independent on the structure of substituents at N10 position. Production of PTZ+. radicals was essential for LADH T c inactivation and this effect depended on the stability of these free radicals. Comparision of inactivation values for LADH T c and mammalian LADH demonstrated...

Resposta dos neurônios mioentéricos do duodeno de ratos ao diabetes de curto prazo / Response of the myenteric neurons of the rat duodenal to short-term diabetes

Foram avaliados os neurônios mioentéricos do duodeno de ratos com diabetes de curto prazo induzido por estreptozootocina com e sem tratamento com insulina. A densidade de neurônios NADH diaforase positivos no duodeno dos ratos diabéticos aumentou em relação aos controles, indicando uma resposta neuronal precoce ao estado diabético. Por outro lado, a sub-população neuronal NADPH diaforase positiva não mostrou alterações significativas de densidade quando os grupos controle diabético não tratado e diabético tratado com insulina foram comparados, indicando que esses neurônios são resistentes ao diabetes de curto prazo. O tratamento com insulina melhorou a condição fisiológica dos ratos diabéticos tratados em comparação com o grupo diabético não tratado, mas não evitou o aumento na densidade neuronal, avaliada pela coloração com a NADH diaforase, no duodeno dos ratos diabéticos tratados comparados com os não tratados

Effect of protein and vitamin B deficiency on the morpho-quantitative aspects of the myenteric plexus of the descending colon of adult rats

We carried out this work with the purpose of studying the effects of protein and vitamin B deficiency on the morphologic and quantitative aspects of the myenteric plexus of the descending colon of adult Rattus norvegicus. Twenty-eight rats were divided in two groups, one of them receiving chow with 22 percent protein level (control) and the other fed with chow having 8 percent protein level without vitamin B supplementation, during 120 days. Whole-mounts of the descending colon were prepared and stained with Giemsa, NADH-diaphorase and NADPH-diaphorase. The undernourished rats had a body weight 11.84 percent less than the control group. Relative to the controls, the experimental group had a colonic area 48 percent smaller, 51.9 percent less Giemsa-stained neurons, 28.3 percent less NADH-diaphorase positive neurons and 24.2 percent less NADPH-diaphorase positive neurons

Morphometry and quantification of the myenteric neurons of the duodenum of adult rats fed with hypoproteic c how

Morphometric and quantitative analyses were carried out on the myenteric neurons of the duodenum, to study possible alterations resulting from a supply of low-protein level chow to adult Wistar rats. The animals were divided in two groups: nourished (N) fed with ration containing 22 per cent protein for 210 days and the undernourished (D) fed with ration containing 8 per cent protein from the 90th day of age on and during the next 120 days. Whole-mounts were stainedby the method of Giemsa and the histochemical reaction of NADH-diaphorase enzyme, to estimate the neuronal population, which revealed a greater neuronal density in undernourished rats, regardless of the technique employed. The morphometric studies of the cell body profiles of 500 neurons of each group indicated a reduction on the cell body size and an increase on the proportion of small neurons on the rats subjected to the hypoproteic chow.

Myeloperoxidase-generated phenothiazine cation radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase

Peroxidase/H2O2/phenothiazine systems irreversibly inhibit Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). Inactivation of the parasite enzyme depended on (a) phenothiazine structure; (b) peroxidase nature; (c) incubation time and (d) the presence of a cation radical scavenger. With the myeloperoxidase/H2O2/system, promazine, trimeprazine, thioridazine, promethiazine, prochlorperazine, chlorpromazine and perphenazine were the most effective derivatives out of twelve phenothiazines studied. An electronegative substituent at position 2 of the phenothiazine ring such as Cl, or trifluoromethyl, propionyl and nitrile groups decreased or nullified phenothiazine activity. Myeloperoxidase/H2O2/, horseradish peroxidase/H2O2/, and myoglobin/H2O2/systems activated phenothiazines producing the corresponding cation radicals, myeloperoxidase being the most selective one with respect to phenothiazine structure. The myoglobin/H2O2/system activated phenothiazines that were scarcely active or inactivate with the MPO/H2O2/system, such as the trifluoromethyl derivatives. Production of phenothiazine cation radicals was demonstrated by optical spectroscopy. Phenothiazine cation radical stability depended on their structure as illustrated by promazine and thioridazine. Thiol compounds (GSH, N-acetyl-cysteine and penicillamine), aromatic aminoacids (L-tyrosine, L-tryptophan, and the corresponding peptides) and ascorbate scavenged phenothiazine cation radicals, thus preventing LADH inactivation. Comparison of the summarized phenothiazine effects with those of phenothiazines on T. cruzi suggest the role of cation radicals in phenothiazines chemotherapeutic actions.

NADH diaphorase positive myenteric neurons of the aglandular region of the stomach of rats subjected to desnutrition

El estómago realiza movimientos caracterizados por potentes ondas peristálticas lentas, útiles en la mezcla del bolo alimenticio, la digestión mecánica y su lenta liberación por el píloro. El control de este movimiento se realiza por acción hormonal y por actividad nerviosa, destacando la inervación intrísica que representan las neuronas del plexo miotérico. Considerando que un estado de desnutrición puede provocar alteraciones morfológicas, incluso neuronas, se ha propuesto este trabajo, con el objetivo de verificar los efectos provocados por la desnutrición proteínica durante el período de 120 días, en el aspecto cuantiativo de las neuronas NADH-diaforasa positivas. Para este estudio se utilizaron 10 ratones adultos, los cuales, a los 90 días de vida fueron divididos en dos grupos; control y desnutridos. A los ratones del grupo desnutridos (n=5) se les alimentó con una dieta proteínica de, aproximadamente, 8 por ciento y a los ratones del grupo control (n=5) con dieta de valor proteínico normal, 22 por ciento. Transcurridos 120 días, al estómago aglandular se le hizo un ensayo de histoenzimología del NADH-diaforasa, para verificar características neuronales. La región glandular fue dividida en dos áreas: una proxima al lóbulo gástrico mayor y la otra, anterior al repliegue límite. En el recuento por muestreo, las neuronas observadas en 40 campos microscópicos (6,64mm²) de cada área, de las muestas de los ratones de los grupos control y desnutridos, fueron contadas con un microscopio de 40x. Constatamos entonces que las neuronas pueden estar aisladas o agrupadas en sus ganglios. Cerca del repliegue límite encontramos un promedio de 570,8 neuronas en el grupo control y 718,4 en el grupo desnutridos. Por otra parte, cerca del lóbulo gástrico mayor, encontramos 25,8 neuronas en el grupo control y 52,6 en el grupo desnutridos. La dieta hipoproteínica implicó un menor crecimiento físico, con una descompensación de alrededor de 30,33 por ciento, al compararlo con ratones del grupo control, como también hubo una reducción de la superficie del perfil estomacal, de aproximadamente 20.13 por ciento. Concluimos que, las neuronas no están distribuidas uniformemente en la pared estomacal y que en los ratones desnutridos presentaron una menor dispersión, presentando por lo tanto una mayor densidad neuronal por mm²

Effects of protein and vitamin B deficiency on blood parameters and myenteric neurons of the colon of rats

The aims of this work were to evaluate the effects of the deficient ingestion of protein and vitamin B on the biochemical and hematologic parameters and on the NADH- and NADPH-diaphorase positive myenteric neurons. The control animals (n=10) received commercial chow and the experimental rats (n=10) received chow with protein level reduced to 8 percent during 120 days. At the time of killing blood was collected for assessment of the blood and hematologic parameters and the ascending colon for quantitative analysis of the neurons of the myenteric plexus. It was observed that the reduction of the protein level to 8 percent coupled to the reduction of the levels of vitamin B in adult rats neither led to qualitative or quantitative changes on red or white blood cells, nor decreased globulin levels, induced the formation of edema or gave rise to clinical signs typical of protein or vitamin B deficiency. On the other hand, the experimental protocol led to less weight gain, change on the body composition with fat deposition; decrease of the values of serum total protein and albumin; reduction of the area of colon and density of nitrergic and NADH-diaphorase myenteric neurons inferior to the expected

Quantitative study of the myenteric plexus of the stomach of rats with streptozotocin-induced diabetes

The purpose of the present study was to investigate the morphological and quantitative alterations of the myenteric plexus neurons of the stomach of rats with streptozotocin-induced chronic diabetes and compare them to those of non-diabetic animals. Samples from the body of the stomach were used for whole-mount preparations stained with NADH-diaphorase and for histological sections stained with hematoxylin-eosin. It was observed that diabetes cause a significant decrease on the number of neurons

Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8 percent; 12 animals) or a low-salt diet (LS; 0.15 percent; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 +/- 11 vs 119 +/- 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.