Post-mortem interval estimative through determination of catalase and Δ-aminolevulinate dehydratase activities in hepatic, renal, skeletal muscle and cerebral tissues of Swiss mice.

Purpose: Determining the post-mortem interval (PMI) is one of the challenging tasks in forensic science due to the lack of quick and inexpensive methods. Our objective is to develop innovative and alternative means for PMI evaluation. Methods: The relationship between PMI and enzymatic modifications in mice tissues was described. After being sacrificed, Swiss mice were randomly divided into groups according to the time elapsed since death. The activities of catalase (CAT) and δ-aminolevulinate dehydratase (δ-ALA-D) were determined in hepatic, renal, skeletal muscle and cerebral tissues. Results: CAT activity increased in kidney and brain 6 h after death and this increase remained for up to 24 h in the brain and 48 h in the kidney. δ-ALA-D had its activity decreased in the liver and kidneys in 6 h. In the skeletal muscle, δ-ALA-D activity was reduced only 48 h after death. Conversely, an increase on δ-ALA-D activity was observed in the brain at 6 h, followed by its decrease at 24 and 48 h. Conclusion: With the association of this set of results, it is possible to provide an estimate of PMI. Additionally, these results can be used as an auxiliary parameter associated with other methods to estimate PMI.

Oxidative stress mediated the inhibition of cerebral creatine kinase activity in silver catfish fed with aflatoxin B1-contaminated diet.

Aflatoxin B1 (AFB1) is an environmental toxicant and neurotoxic compound that induces the production of free radicals, causing oxidative stress. Creatine kinase (CK) is a central controller of energy metabolism in tissues with a large and fluctuating energy demand, and it is highly susceptible to inactivation by free radicals and oxidative damage. Thus, the aim of this study was to evaluate whether a diet for freshwater silver catfish (Rhamdia quelen) containing AFB1 inhibits cerebral CK activity, as well as the involvement of the oxidative stress on this inhibition. Brain CK activity was lower on days 14 and 21 post-feeding in animals that received AFB1-contaminated diet compared to the control group (basal diet), similarly to the brain sodium-potassium pump (Na+, K+-ATPase) activity. On the other hand, lipid peroxidation and protein carbonylation levels were higher on days 14 and 21 post-feeding in animals fed with AFB1-contaminated feed compared to the control group, while the antioxidant capacity against peroxyl radicals and thiol content was lower. Based on these evidences, the data demonstrated that diet containing AFB1 severely affects CK activity, an essential enzyme that plays an important role in brain energy homeostasis. Also, the impairment of energetic homeostasis linked with the use and generation of ATP via inhibition of CK activity elicited an inhibition of enzymes ATP-dependent, such as Na+, K+-ATPase. Moreover, the inhibition of brain CK activity appears to be mediated by the oxidation of lipids, proteins, and thiol group, as well as by a reduction in the antioxidant capacity.

Effects of Zinc and N-Acetylcysteine in Damage Caused by Lead Exposure in Young Rats.

This study investigated the toxicity of rats exposed to lead acetate (AcPb) during the second phase of brain development (8-12 days postnatal) in hematological and cerebral parameters. Moreover, the preventive effect of zinc chloride (ZnCl2) and N-acetylcysteine (NAC) was investigated. Pups were injected subcutaneously with saline (0.9% NaCl solution), ZnCl2 (27 mg/kg/day), NAC (5 mg/kg/day) or ZnCl2 plus NAC for 5 days (3rd-7th postnatal days), and with saline (0.9% NaCl solution) or AcPb (7 mg/kg/day) in the five subsequent days (8th-12th postnatal days). Animals were sacrificed 21 days after the last AcPb exposure. Pups exposed to AcPb presented inhibition of blood porphobilinogen-synthase (PBG-synthase) activity without changes in hemoglobin content. ZnCl2 pre-exposure partially prevented PBG-synthase inhibition. Regarding neurotoxicity biomarkers, animals exposed to AcPb presented a decrease in cerebrum acetylcholinesterase (AChE) activity and an increase in Pb accumulation in blood and cerebrum. These changes were prevented by pre-treatment with ZnCl2, NAC, and ZnCl2 plus NAC. AcPb exposure caused no alteration in behavioral tasks. In short, results show that AcPb inhibited the activity of two important enzymatic biomarkers up to 21 days after the end of the exposure. Moreover, ZnCl2 and NAC prevented the alterations induced by AcPb.

Galactose alters markers of oxidative stress and acetylcholinesterase activity in the cerebrum of rats: protective role of antioxidants.

We evaluated the in vitro effects of galactose at 0.1, 3.0, 5.0 and 10.0 mM on thiobarbituric acid-reactive substances (TBA-RS), total sulfhydryl content, protein carbonyl content, on the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and on acetylcholinesterase (AChE) activity in the cerebral cortex, cerebellum and hippocampus of rats. We also investigated the influence of the antioxidants (each at 1 mM), α-tocopherol, ascorbic acid and glutathione, on the effects elicited by galactose on the parameters tested. Results showed that galactose, at a concentration of 3.0 mM, enhanced TBA-RS levels in the hippocampus, cerebral cortex and cerebellum of rats. In the cerebral cortex, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS and protein carbonyl content, and at 10.0 mM increased CAT activity and decreased AChE activity. In the cerebellum, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS, SOD and GSH-Px activities. In the hippocampus, galactose at concentrations of 5.0 and 10.0 mM increased TBA-RS and CAT activity and at 10.0 mM decreased GSH-Px. Data showed that at the pathologically high concentration (greater than 5.0 mM), galactose induces lipid peroxidation, protein carbonylation, alters antioxidant defenses in the cerebrum, and also alters cholinesterase activity. Trolox, ascorbic acid and glutathione addition prevented the majority of alterations in oxidative stress parameters and the decrease in AChE activity that were caused by galactose. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by galactose.

Topological and histological description of preoptic area and hypothalamus in cardinal tetra Paracheirodon axelrodi (Characiformes: Characidae)

Topological and histological descriptions of the preoptic area and hypothalamus of the cardinal tetra Paracheirodon axelrodi were performed. Standard histological paraffin sections were used and stained with Nissl technique, and plastic sections for high-resolution optic microscopy (HROM). The preoptic area showed some differences related to the location of the magnocellular preoptic nucleus (PM) and the size of the neurons in this region, as they were the biggest in all the preoptic area. Additionally, within the preoptic area, the different structures that comprise the organum vasculosum of the lamina terminalis (OVLT) were identified and characterized. The hypothalamus could be subdivided in three regions - the ventral, the dorsal and the caudal hypothalamic regions - neuron morphology, size and staining pattern were similar in all of them.(AU)

Se realizó la descripción topológica e histológica del área preóptica e hipotálamo en el Neón cardenal Paracheirodon axelrodi. Se usaron cortes obtenidos con tecnicas histológicas estándar, coloreados con técnica de Nissl y secciones en resina con microscopía óptica de alta resolución (MOAR). El área preóptica muestra algunas diferencias relacionadas con la localización del núcleo preóptico magnocelular (PM) y el tamaño de algunas neuronas en esta región, puesto que estas eran las más grandes de toda el área preoptica. Adicionalmente, dentro del área preóptica, fue posible identificar y caracterizar las diferentes estructuras que componen el órgano vasculoso de la lámina media (OVLM). El hipotálamo puede sudividirse en tres zonas: la zona hipotalámica ventral, la zona hipotalámica dorsal y la zona hipotalámica caudal. La morfología de las neuronas de los núcleos que comprenden las diferentes zonas del hipotálamo tiene tamaño, forma y coloración similar.(AU)

Topological and histological description of preoptic area and hypothalamus in cardinal tetra Paracheirodon axelrodi (Characiformes: Characidae)

Topological and histological descriptions of the preoptic area and hypothalamus of the cardinal tetra Paracheirodon axelrodi were performed. Standard histological paraffin sections were used and stained with Nissl technique, and plastic sections for high-resolution optic microscopy (HROM). The preoptic area showed some differences related to the location of the magnocellular preoptic nucleus (PM) and the size of the neurons in this region, as they were the biggest in all the preoptic area. Additionally, within the preoptic area, the different structures that comprise the organum vasculosum of the lamina terminalis (OVLT) were identified and characterized. The hypothalamus could be subdivided in three regions - the ventral, the dorsal and the caudal hypothalamic regions - neuron morphology, size and staining pattern were similar in all of them.(AU)

ESUMEN Se realizó la descripción topológica e histológica del área preóptica e hipotálamo en el Neón cardenal Paracheirodon axelrodi. Se usaron cortes obtenidos con tecnicas histológicas estándar, coloreados con técnica de Nissl y secciones en resina con microscopía óptica de alta resolución (MOAR). El área preóptica muestra algunas diferencias relacionadas con la localización del núcleo preóptico magnocelular (PM) y el tamaño de algunas neuronas en esta región, puesto que estas eran las más grandes de toda el área preoptica. Adicionalmente, dentro del área preóptica, fue posible identificar y caracterizar las diferentes estructuras que componen el órgano vasculoso de la lámina media (OVLM). El hipotálamo puede sudividirse en tres zonas: la zona hipotalámica ventral, la zona hipotalámica dorsal y la zona hipotalámica caudal. La morfología de las neuronas de los núcleos que comprenden las diferentes zonas del hipotálamo tiene tamaño, forma y coloración similar.(AU)

Preventive effect of CuCl2 on behavioral alterations and mercury accumulation in central nervous system induced by HgCl2 in newborn rats.

This study investigated the benefits of Cu preexposition on Hg effects on behavioral tests, acetylcholinesterase (AChE) activity and Hg, and essential metal contents in the cerebrum and cerebellum of neonate rats. Wistar rats received (subcutaneous) saline or CuCl2 ·2H2O (6.9 mg/kg/day) when they were 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) when they were 8 to 12 days old. Mercury exposure reduced the performance of rats in the negative geotaxis (3-13 days) and beaker test (17-20 days), inhibited cerebellum AChE activity (13 days), increased cerebrum and cerebellum Hg (13 days), cerebrum Cu (13 days), and cerebrum and cerebellum Zn levels (33 days). The performance of rats in the tail immersion and rotarod tests as well as Fe and Mg levels were not altered by treatments. Copper prevented all alterations induced by mercury. These results are important to open a new perspective of prevention and/or therapy for mercury exposure.

Trypanosoma evansi: cholinesterase activity in acutely infected Wistar rats.

The aim of this study was to evaluate cholinesterase activity during the early acute phase of Trypanosoma evansi infection in rats. Fifteen male Wistar rats were randomly distributed into three groups (n=5 animals per group): two trypanosome-infected groups (T3 and T5) and uninfected controls (C). The animals were inoculated intraperitoneally with 10(6) trypanosomes. The blood was collected by cardiac puncture on the 3rd (T3) or 5th day post-infection (T5 and C). Cerebrum and cerebellum were removed for the evaluation of acetylcholinesterase (AChE) activity. AChE activity was also evaluated in whole blood and butyrylcholinesterase activity (BUChE) in plasma samples. Parasitemia were progressive increase and parasites were observed in the peripheral blood of all infected animals one day post-inoculation. AChE activity was not altered in cerebrum and cerebellum tissues. AChE activity in blood significantly decreased in the T3 and T5 groups (26.63 and 25.86mU/lmolHb) compared with the control (37.84mU/lmolHb). In addition BUChE activity in plasma was lower in the T3 (7.01micromol BTC hydrolyzed/h/mL) than the T5 and C groups (9.84 and 12.00micromol BTC hydrolyzed/h/mL). This study therefore, shows that reductions in the activity of cholinesterase occur in acute infection by T. evansi in rats and this demonstrates an important change occurring in animals infected by the protozoan and may indicate a potential role the enzymes play in the mechanism of disease.

Methylphenidate treatment increases Na(+), K (+)-ATPase activity in the cerebrum of young and adult rats.

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na(+), K(+)-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na(+), K(+)-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na(+), K(+)-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na(+), K(+)-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na(+), K(+)-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na(+), K(+)-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na(+), K(+)-ATPase activity may be associated with neuronal excitability caused by methylphenidate.

ZnCl2 exposure protects against behavioral and acetylcholinesterase changes induced by HgCl2.

This study examined the effects of inorganic mercury exposure on behavioral and biochemical parameters and investigated the possible preventive effects of zinc on the alterations induced by mercury. Pups were exposed from 3rd to 7th postnatal day to ZnCl2 (27 mg/kg/day, s.c.) and subsequently to HgCl2 (5 doses of 5 mg/kg/day, s.c.). Each litter contained two rats for each treatment. The rats were submitted to behavioral task and litters were killed at 13 or 33 days old for acetylcholinesterase activity assays and for the determination of metal levels. Based on the results obtained from 13-day-old rats, they were divided in two groups of litters that were defined at the end of the experimental period (33 days) as less sensitive rats to mercury and more sensitive rats to mercury in accordance with the recovery of body weight until day 33. The mercury exposure caused accumulation of this metal in cerebrum and cerebellum in all mercury treated rats, and inhibited the cerebellum acetylcholinesterase activity from 13-day-old rats. Besides, the mercury-animals of the most sensitive litters to mercury presented impairment in motor function and muscular strength verified in the beaker test, as well as a reduction of the locomotor and exploratory activities in the open field task. Zinc partially prevented all the alterations induced by mercury exposure and reduced the mercury level accumulated in cerebrum and cerebellum. This study confirms the preventive effect of zinc on behavioral alterations induced by mercury in young rats and demonstrates that the mercury behavioral effects are present even for a long time after the end of the exposure.

Distribution of NADPH-diaphorase in rat mesencephalon: a light and electron microscopical study

NADPH-diaphorase is a useful technique to reveal NO producing neurons at light microscopic level (LM). A modification of the technique using the tetrazolium salt BSPT as substrate, is useful to study the ultrastructure of NO neurons. The aim of this work was to perform a detailed analysis of NADPH-diaphorase reactive neurons in rat mesencephalon both at light and electron microscopic levels. NADPH-diaphorase reactive neurons were observed in superior colliculus, in central gray matter, in dorsal and medial raphe and in the pedunculopontine tegmental nucleus using two histochemical techniques at LM. Electron microscopy showed deposits on membranes of the endoplasmic reticulum, Golgi apparatus and nuclear envelope of dorsal raphe neurons. Presynaptic and postsynaptic terminals showed deposits on membranous elements but postsynaptic terminals also showed deposits on the inner surface of their membranes. Further physiological studies are needed to clarify the meaning of the ultrastructural findings such as the putative interaction of NOS with postsynaptic proteins, receptors or membranous channels.(AU)

Uso de una técnica de inmunopersoxidasa para la detección de virus de rabia en cortes gruesos de cerebro / The use of an immunoperoxydase technique for the detection of rabies virus in thick brain slices

Con el propósito de evaluar una técnica de avidina-peroxidasa biotinilada, se utilizó el conjugado antirrábico producido en el INS para la detección del virus de rabia en cortes gruesos de cerebros de ratón tratados con varios tipos de fijadores y con varias diluciones del conjugado. Los animales infectados experimentalmente fueron perfundidos vía intracardiaca con varios fijadores; los cerebros se recuperaron, se les hicieron cortes gruesos de 60 µm y se sometieron a la inmunodetección. Se encontró que en las diluciones normalmente usadas en fluorescencia se presentó un fuerte marcaje inespecífico sin mejorar la detección. En este trabajo, se presenta un protocolo fácil y rápido que pudiera ser útil para la observación de muestras sospechosas

Distúrbio do metabolismo de fosfolípides na doença de Alzheimer / Disordered metabolism of membrane phospholipds in Alzheimer's disease

A fosfolipase A2 (PLA2) é uma enzima fundamental no metabolismo dos fosfolípides da membrana celular. A PLA2 influencia o processamento e a secreção da proteína precursora do amilóide, que dá origem ao peptídeo ß-amilóide, o componente principal das placas senis na doença de Alzheimer (DA). Investigamos a atividade da PLA2 em duas amostras: a) num estudo post mortem, em tecido cerebral de 23 pacientes com uma DA e 20 controles não-dementes, e b) em plaquetas de 16 pacientes com uma DA provável, 13 controles sadios e 14 pacientes idosos com uma depressão. No cérebro, a atividade da PLA2 estava significantemente reduzida no córtex parietal e frontal de pacientes com DA. Esta redução estava correlacionada com um início precoce da doença, com um número mais elevado de neurofibrilas e placas senis, e com um óbito mais prematuro. Também nas plaquetas de pacientes com DA encontramos uma redução da atividade da PLA2 em comparação com os controles sadios e deprimidos. A redução da atividade da enzima em plaquetas estava correlacionada com um início precoce da doença, com o grau de atrofia cerebral e com um déficit cognitivo mais acentuado, indicando assim uma relação entre a redução da PLA2 e uma forma mais severa da doença. Nossos resultados indicam um distúrbio do metabolismo de fosfolípides na DA e sugerem que a redução da atividade da PLA2 poderia contribuir para a formação do peptídeo ß-amilóide na doença. Estudos futuros devem esclarecer se a atividade da PLA2 em plaquetas poderia ser útil como um marcador periférico para a DA, ou como preditor para o grau de severidade da doença

Antioxidant enzymes and their modification under oxidative stress conditions

There are many cellular defense strategies against processes mediated by active oxygen species, including scavenger molecules and the enzymes catalase, superoxide dismutase and glutathione peroxidase. The activity of these antioxidant enzymes allows to keep O2- and H2O2 steady state concentrations at low levels, compatible with cell function. In most pathologies considered, modifications in levels of enzymatic activity have been observed in relation to values of control patients. Both increased and decreased levels have been found and in almost all systems the response of the three enzymes has been parallel. Different models of chronic treatment have also been considered. Parallel variations could be noted again regarding the antioxidant activity of these three enzymes. Moreover, there exists a correlation between the increase in the enzymatic activities and the acquired protection by treatment with barbital as reflected by the measured values of spontaneous chemiluminescence. on the other hand, increased levels of chemiluminescence in liver homogenates and diminished enzymatic activities were found in other experimental models. The response to these treatments is not the same in different organs since the rates of production of active oxygen species as well as the antioxidant defense activities are different in each organ. Finally, data of acute models of oxidative stress were compiled. Some of them have shown a biphasic response. At first a decrease in levels of enzymatic activities could be seen in response to the injury. Afterwards, an increase in the activity of the antioxidant enzymes followed as a consequence of an enzymatic induction or activation.

Biochemical adjustments to hypoxia by Amazon cichlids

The isozyme distribution of cichlid lactate dehydrogenase (LDH) is related to species environmental preferences. Cichlasoma amazonarum. occurs in different environments and presents LDH tissue distribution patterns that correlate with oxygen tension at the capture location. Cichlasoma amazonarum was exposed to long term severe hypoxia (51 days at 36.4 +/- 5.9 mmHg), tissue LDH isozyme distribution was analyzed by electrophoresis and enzyme activities were measured by monitoring the oxidation of NADH as pyruvate was reduced to lactate. The exposure of Cichlasoma amazonarum to long-term severe hypoxia resulted in changes in the tissue distribution of LDH isozymes. The major changes in response to hypoxia occurred in heart, liver and brain: isozyme A4 was activated in heart and brain, whereas isozyme B4 was activated in liver. The most significant quantitative change occurred in brain LDH of hypoxia-exposed animals which adopted muscle type kinetics, reflecting a new LDH isozyme distribution. LDH activity was significantly reduced (P<0.05) in animals exposed to hypoxia (N = 8), suggesting an overall LDH suppression. Pyruvate inhibition decreased in all hypoxia-exposed tissues. Thus, the ability of Cichlasoma amazonarum to regulate LDH tissue expression according to oxygen availability allows the animal to survive chronic hypoxic environments. This phenotypic plasticity may occur in other hypoxia-tolerant fish species.

Cambios inducidos por rehabilitación nutricional temprana en la vía serotoninérgica cerebral activada por desnutrición gestacional / Changes induced by nutritional neonatal rehabilitation in the brain serotonergic path activated by gestational malnourished

Introducción. Se ha demostrado que la desnutrición gestacional produce desde la etapa fetal una aceleración de la síntesis de 5-hidroxitriptamina cerebral (5-HT), secundaria a un aumento de la afinidad de la triptófano-5-hidroxilasa (T5-H) por el L-triptófano (L-Trp) y una mayor capacidad de fosforilación. Estos hallazgos han sugerido un cambio conformacional de la enzima durante el desarrollo cerebral como mecanismo principal que explique la aceleración de la síntesis de 5-HT. El objetivo de este trabajo fue evaluar los diferentes cambios que se producen en el cerebro de las ratas desnutridas durante la gestación y que al nacer son sometidas a un esquema de rehabilitación nutricia, con el propósito de obtener información que nos permita apoyar la hipótesis de que el mecanismo de activación de la síntesis de 5-HT cerebral es debida a un cambio estructural de la T5-H. Material y Métodos. Se seleccionaron ratas cepa Wistar, adaptadas a condiciones ambientales estándar. Al término de este período se formaron dos grupos: uno con desnutrición proteínico-calórico (D) y el otro control (C). Las hembras fueron pareadas con machos normales. Al nacimiento las crías de ambos grupos fueron mezcladas y redistribuidas al azar a madres del mimsmo grupo; ademas se realizó un entrecruzamiento de las crías de estos grupos para formar dos subgrupos; el desnutrido recuperado (DR) y el desnutrido en la lactancia (DL). A las edades de 1, 10, 15 y 21 días, se obtuvo el encéfalo para los ensayos bioquímicos. Además se realizaron curvas de peso corporal, cerebral y de la longitud céfalo-sacra. Resultados. Los grupos D y DL mostraron un retraso significativo del crecimiento corporal, cerebral y de la longitud céfalo desde el primer día hasta los 21 días de edad en comparación a los controles. El mismo patrón se observó en las proteínas tisulares. El grupo DR alcanzó una recuperación física a los 15 días de edad. La actividad de la enzima en los desnutridos mostró un aumento significativo en todas las edades estudiadas; la misma elevación significativa persistió en el grupo DR hasta los 21 días en comparación al grupo control y una elevación significativa en la concentración de 5-HT. Conclusiones: Los resultados confirman que la desnutrición gestacional y posnatal producen una deficiencia en la composición corporal, cerebral y una aceleración en la síntesis de 5-hidroxitriptamina. Además apoyan el hecho de que la rebilitación nutricional neonatal produce una recuperación en la composición corporal. Sin embargo los hallazgos de que el L-Trp cerebral se normalice en el animal rehabilitado, de que la actividad de la T5-H permanezca elevada y persista un aumento en la síntesis de neurotransmisor, 5-HT, apoya indirectamente la hipótesis de que el mecanismo de activación de esta importante vía biosintética cerebral, es posiblemente debiido a un cambio relacionado a la estructura de la triptófano-5-hidroxilasa inducido por la desnutrición ontogénica. Desnutrición gestacional; 5-hidroxitriptamina; cerebro; rehabilitación nutricional; triptófano-5-hidroxilasa

Characterization of an endooligopeptidase A-like protein in PC12 cells Activity modulation by cAMP but not by basic fibroblast growth factor

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.

Inhibition of bovine brain acetylcholinesterase by aluminum

We report the in vitro inhibitory effect of very low concentrations of aluminum salts (IC50 = 4.1 x 10-12M) on bovine brain acetylcholinesterase (AChE). The enzymatic assays were performed using acetylcholine bromide in a buffered pH 7.4 solution at 37§C. The relevant enzyme interacting species is the Al3+ ion, whose concentrations were fixed at pM levels by a citrate metal ion buffer system. The IC50 demonstrates that Al3+ is a potent inhibitor of AChE

Effects of methylmercury exposure during the second stage of rapid postnatal brain growth on negative geotaxis and on delta-aminolevulinate dehydratase of suckling rats

In the present study, we examined the effects of exposure to methylmercury (0, 2.3, 4.6, 6.9 and 9.2 mg/kg, daily for 5 consecutive days, sc) during the second stage of rapid postnatal brain development (8 to 12 days of age) on the sulfhydryl-containing enzyme delta-aminolevulinate dehydratase (ALA-D, E.C. 4.2.1.24) from brain, liver and kidney and on motor performance (latency to complete a negative geotaxis response) of rats. ALA-D specific activity of 13-day old rats of both sexes (7-12 per group) was reduced significantly in rats treated with 6.9 mg/kg and 9.2 mg/kg in brain (about 40 per cent , P < 0.05) and in liver (about 25 per cent , P < 0.05). Renal ALA-D specific activity was not affected by methylmercury treatment. The in vitro IC50 for inhibition of brain, liver and renal ALA-D was 79.3, 81.8 and 39.1 microM, respectively. The latency to complete the negative geotaxis response of 12-day old rats was increased by 6.9 (7.9 +/- 0.7 s, mean +/- SEM) and 9.2 mg/kg methylmercury (7.8 +/- 0.5 s) when compared with control rats (5.8 +/- 0.3 s), suggesting an impairment in motor performance of exposed rats. These results demonstrate that exposure to relatively high doses of methylmercury during the second stage of brain development causes a significant reduction in brain and hepatic ALA-D. The absence of inhibition of ALA-D by lower doses may be related to the relatively low in vitro sensitivity of the enzyme to methylmercury. The possible involvement of ALA-D inhibition on the neurotoxicity of methylmercury deserves additional investigation

Equilibrium kinetics modal for the cGMP-stimulated phosphodiesterase of brain coated vesicles

An equilibrium kinetics model is proposed to described some of the enzymatic properties of the cyclic GMP-stimulated phosphodiesterase activity associated with brain clathrin coated vesicles. The model assumes the presence of pharmacologically distinct regultory and catalytic domains in the enzyme. The model contemplates that random fashion occupancy of the regulatory site by the substrate, cyclic GMP, induces a conformational change which leads to the generation of a actived catalytic state. Therefore, cyclic GMP is a positive allosteric modulator of the coated vesicle enzyme. Experimental data revealed that occupancy or activation of the regulatory site was not essential for catalysis to occur since hydrolysis occured after loss (200%) of the activation by cyclic GMP. This constitutes an example of non-essential substrate activation. Analysis of this PDE following activation by cGMP and after loss of the regulation, activation capacity of the enzyme allows the calculation of the various kinetic parameters inherent in the model