Unlocking the Anti - Prostate Cancer Potential of Some Tropical and Sub - Tropical Plants

Prostate cancer is the third leading cause of cancer death among men worldwide and is the leading cause of cancer mortality in males greater than 60 years in Trinidad and Tobago. Although there are some treatment options for the metastatic disease, the impact on overall survival rate has not improved in the last two decades. In particular, the mortality rate from prostate cancer is high among men of African descent in Trinidad and Tobago and the Americas. These groups manifest aggressive cancers that are often less responsive to available therapies. We hypothesize that phytochemical screening of tropical plants, coupled to cell culture studies would identify other potential treatments for prostate cancer. In separate experiments, the roots or leaves of five plants indigenous to the Caribbean and the skin of the muscadine grape, indigenous to North America were dried, crushed and suspended in 100% methanol. The respective methanol extracts were fractionated and the fractions dried. Next the ability of the extracts to halt the growth of or kill PC-3 prostate cancer cells was assessed by MTT assays. Colony formation, cell migration studies, cell cycle studies and western blots were performed to determine probable mechanism of action. The categories of chemical compounds present in the extracts were determined by Thin Layer chromatography (TLC). Results from MTT assays showed that compared to the control cells (ie those treated with DMSO only), treatment with at least four plant extracts significantly (p<0.05) inhibited the growth of the cancer cells; decreases as large as 95 % were observed. On the other hand these extracts had a limited or delayed effect on the "normal" PNT1A cells. Results from the cell cycle assay demonstrated that one extract, now identified as plant-A extract, caused the arrest of the G1/S phase of PC-3 cells i.e. leading to an increase in the number of cells that remained in the growth phase. On the other hand the total ethanol extract of muscadine grape skin decreased the expression of heat shock protein-40, possibly causing deactivation of the androgen receptor. This suggests that extracts prepared from Plant A and the muscadine grape have the potential for use as anticancer drugs.

Unlocking the anti-prostate cancer potential of some tropical and sub-tropical plants

Prostate cancer is the third leading cause of cancer death among men worldwide and is the leading cause of cancer mortality in males greater than 60 years in Trinidad and Tobago. Although there are some treatment options for the metastatic disease, the impact on overall survival rate has not improved in the last two decades. In particular, the mortality rate from prostate cancer is high among men of African descent in Trinidad and Tobago and the Americas. These groups manifest aggressive cancers that are often less responsive to available therapies. We hypothesize that phytochemical screening of tropical plants, coupled to cell culture studies would identify other potential treatments for prostate cancer. In separate experiments, the roots or leaves of five plants indigenous to the Caribbean and the skin of the muscadine grape, indigenous to North America were dried, crushed and suspended in 100% methanol. The respective methanol extracts were fractionated and the fractions dried. Next the ability of the extracts to halt the growth of or kill PC-3 prostate cancer cells was assessed by MTT assays. Colony formation, cell migration studies, cell cycle studies and western blots were performed to determine probable mechanism of action. The categories of chemical compounds present in the extracts were determined by Thin Layer chromatography (TLC). Results from MTT assays showed that compared to the control cells (ie those treated with DMSO only), treatment with at least four plant extracts significantly (p<0.05) inhibited the growth of the cancer cells; decreases as large as 95 % were observed. On the other hand these extracts had a limited or delayed effect on the "normal" PNT1A cells. Results from the cell cycle assay demonstrated that one extract, now identified as plant-A extract, caused the arrest of the G1/S phase of PC-3 cells i.e. leading to an increase in the number of cells that remained in the growth phase. On the other hand the total ethanol extract of muscadine grape skin decreased the expression of heat shock protein-40, possibly causing deactivation of the androgen receptor. This suggests that extracts prepared from Plant A and the muscadine grape have the potential for use as anticancer drugs.

Short term effects of cocoa consumption on blood pressure.

Hypertension, defined as diastolic pressure ≥ 90 mmHg and systolic pressure ≥ 140 mmHg, is a major cause of morbidity and mortality among black populations globally. Several studies have shown that prolonged consumption of cocoa or cocoa containing products leads to decreased blood pressure (BP) in hypertensives. In this study, we investigated the flavonoid content of the top selling cocoa/cocoa based products in Trinidad and Tobago and attempted to determine if consumption of cocoa had any immediate impact on blood pressure levels. The flavonoid content of three 100% cocoa powder products and four cocoa based formulas was measured using a modified Folin-Ciocalteu procedure. The brand with the highest flavonoid content, 372 gallic acid equivalents, was selected to evaluate the short-term impact of cocoa consumption on blood pressure. Thirty-six participants comprising nineteen hypertensives and seventeen persons with normal blood pressure had their blood pressure recorded on three separate days using ambulatory blood pressure monitors; the blood pressure was recorded every half hour for eight hours. On the first day, the participants received no intervention but on the second and third days, they received either the intervention (5 g cocoa in 125 ml water) or a placebo, in any order. Statistical analysis conducted using t-test statistic and a 95% confidence interval revealed that whether participants regularly took antihypertensive medication or not, a single intervention of cocoa induced decreases in both the diastolic and systolic BPs that were significant (p = 0.0001). Mean decreases of between 8 mmHg and 18 mmHg were observed.

Hydrogen/hydride ion relay--a mechanism for early electron transfer in cytochrome c oxidases.

Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.

Hydrogen/rydride ion relay - a mechanism for early electron transfer in cytochrome c oxidases / El reléuónico hidrógeno-hidruro: un mecanismo de transferencia temprana de electrones en las citocromo c oxidasas

Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.

La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.

An evaluation of treatment strategies for head and neck cancer in an African American population.

OBJECTIVE: This study evaluated treatment strategies for head and neck cancers in a predominantly African American population. METHODS: Data were collected utilizing medical records and the tumour registry at the Howard University Hospital. Kaplan-Meier method was used for survival analysis and Cox proportional hazards regression analysis predicted the hazard of death. RESULTS: Analysis revealed that the main treatment strategy was radiation combined with platinum for all stages except stage I. Cetuximab was employed in only 1% of cases. Kaplan-Meier analysis revealed stage II patients had poorer outcome than stage IV while Cox proportional hazard regression analysis (p = 0.4662) showed that stage I had a significantly lower hazard of death than stage IV (HR = 0.314; p = 0.0272). Contributory factors included tobacco and alcohol but body mass index (BMI) was inversely related to hazard of death. CONCLUSIONS: There was no difference in survival using any treatment modality for African Americans.

Elevated ferric, calcium and magnesium ions in the brain induce protein aggregation in brain mitochondria.

OBJECTIVE: Alzheimer's disease and Parkinson's disease are two of several neurodegenerative disorders that affect the elderly. Although their aetiology remains uncertain, studies suggest that elevated aluminium or other metal ions in the brain directly influence the development of the histological abnormalities normally associated with these diseases; other investigations suggest that metal-ion-induced-dysfunction of mitochondria might be a critical factor. METHODS: In this study, the impact of elevated aluminum (Al3+), ferric (Fe3+), calcium (Ca2+) and magnesium (Mg2+) ions on brain histology and on the protein composition of brain mitochondria were evaluated. Rabbits were injected intra-cerebrally with 1.4% solutions of either aluminium chloride (AlCl3), ferric chloride (FeCl3), calcium chloride (CaCl2) or magnesium chloride (MgCl2) and sacrificed 10 days later RESULTS: Histological analysis revealed that Al3+ but not the other ions induced neurofibrillary degeneration within the midbrain and medulla. Alternatively, SDS-PAGE revealed that Fe3+, Ca2+ and Mg2+ but not Al3+ induced alterations to the distribution of brain mitochondrial proteins. Both Fe3+ and Ca2+ triggered decreased concentration of three low molecular weight proteins (-7-14 kd) but Ca2+ precipitated their total absence. Both ions led to increased concentration of a high molecular weight protein (-110 kd). In contrast, Mg2 led to the total absence of the protein of lowest molecular weight (-7 kd) and increased concentration of a -36 kd protein. CONCLUSION: These results suggest that elevation of some metal ions in the brain induces protein aggregation with the nature of the aggregation being highly ion dependent. The results also point toward major differences between the histopathological effect of Al3+ and other ions.

Elevated ferric, calcium and magnesium ions in the brain induce protein aggregation in brain mitochondria / El aumento de iones férricos, calcio y magnesio en el cerebro induce la agregación de proteínas en las mitocondrias del cerebro

OBJECTIVE: Alzheimer s disease and Parkinson s disease are two of several neurodegenerative disorders that affect the elderly. Although their aetiology remains uncertain, studies suggest that elevated aluminium or other metal ions in the brain directly influence the development of the histological abnormalities normally associated with these diseases; other investigations suggest that metal-ion-induced-dysfunction of mitochondria might be a critical factor. METHODS: In this study, the impact of elevated aluminum (Al3+), ferric (Fe3+), calcium (Ca2+) and magnesium (Mg2+) ions on brain histology and on the protein composition of brain mitochondria were evaluated. Rabbits were injected intra-cerebrally with 1.4% solutions of either aluminium chloride (AlCl3), ferric chloride (FeCl2), calcium chloride (CaCl2) or magnesium chloride (MgCl2) and sacrificed 10 days later. RESULTS: Histological analysis revealed that Al3+ but not the other ions induced neurofibrillary degeneration within the midbrain and medulla. Alternatively, SDS-PAGE revealed that Fe3+, Ca2+ and Mg2+ but not Al3+ induced alterations to the distribution of brain mitochondrial proteins. Both Fe3+ and Ca2+ triggered decreased concentration of three low molecular weight proteins (~7-14 kd) but Ca precipitated their total absence. Both ions led to increased concentration of a high molecular weight protein (~ 110 kd). In contrast, Mg2+ led to the total absence of the protein of lowest molecular weight (~7 kd) and increased concentration of a ~36 kd protein. CONCLUSION: These results suggest that elevation of some metal ions in the brain induces protein aggregation with the nature of the aggregation being highly ion dependent. The results also point toward major differences between the histopathological effect of Al3+ and other ions.

OBJETIVO: La enfermedad de Alzheimer y la enfermedad de Parkinson son dos de los varios trastornos neurodegenerativos que afectan al anciano. Aunque su etiologia sigue siendo incierta, los estudios sugieren que el aumento de los iones de aluminio, influyen directamente sobre el desarrollo de las anormalidades histológicas asociadas normalmente con estas enfermedades. Otras investigaciones sugieren que la disfunción de las mitocondrias, inducida por iones metálicos, pudiera ser un factor critico. MÉTODOS: Este estudio evalúa el impacto del aumento de los iones de aluminio (Al3+), los iones férricos (Fe3+), y los iones de calcio (Ca2+) y magnesio (Mg2+) sobre la histologia del cerebro y la composición proteica de las mitocondrias del cerebro. Un número de conejos recibieron inyecciones intracerebrales de soluciones al 1.4% de soluciones de cloruro de aluminio (AlCl3), cloruro ferroso (FeCl3), cloruro de calcio (CaCl2), o cloruro de magnesio (MgCl2), y fueron sacrificados después de 10 días. RESULTADOS: El análisis histológico reveló que el Al3+ indujo una degeneración neurofibrilar dentro del mesencéfalo y la médula, Sin embargo, esto no ocurrió con los otros iones. Alternativamente, la técnica de electroforesis SDS-PAGE reveló que los iones Fe3+, Ca2+ y Mg2+, a diferencia del ión Al3+, inducían alteraciones de la distribución de las proteínas mitocondriales cerebrales. Tanto el Fe3+ como el Ca2+ desencadenaron una disminución de la concentración de tres proteínas de bajo peso molecular (~7-14 kd) pero Ca2+ precipitó su ausencia total. Ambos iones condujeron a un aumento de una proteína de peso molecular alto (~ 110 kd). En cambio, Mg2+ llevó a la ausencia total de la proteína de más bajo peso molecular (~7 kd) y al aumento de la concentración de una proteína de ~36 kd. CONCLUSIÓN: Estos resultados parecen sugerir que la elevación de algunos iones de metal en el cerebro induce la agregación de la proteína, siendo la naturaleza de la agregación altamente dependiente de los iones. Los resultados también apuntan a grandes diferencias entre el efecto histopatológico del Al3+ y otros iones.

Early electron transfer in cytochrome c oxidase occurs by a chymotrypsin type relay / La transferencia temprana de electrones en el citocromo c oxidasa ocurre por un relé tipo quimotripsina

Evidence suggests that when ferrocytochrome c (the substrate) reduces cytochrome c oxidase (COX), electrons from the former enter the latter via Trp-104. What is still to be determined is the method by which electrons are transferred from ferrocytochrome c to Trp-104 and the method by which electrons arriving at Trp-104 are moved on to cua, the first of the enzyme's four redox centres to be reduced. To shed light on this process, we used the computer to create and analyse an enzyme-substrate complex formed from the published structure of the two proteins. It was found that the front haem edge of ferrocytochrome c was in close proximity to Trp-104 of COX and that inclusive of Trp-104, only nine amino acid residues from COX lie along a broad channel stretching from Trp-104 to the enzyme's CuA centre. Six of the nine residues, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 and Glu-198, had the ideal chemical properties and were properly aligned to facilitate electron transfer. Here we propose that the reduction of Trp-104 and the subsequent reduction of CuA occur by a hydride/hydrogen ion relay system similar to that seen at the active site of chymotrypsin.

La evidencia sugiere que cuando el ferrocitocromo C (el sustrato) reduce el citocromo c oxidasa (COX), los electrones del primero entran a este último vía Trp-104. Lo que queda aún por determinar es el método por el cual los electrones son transferidos del ferrocitocromo c al Trp-104, así como el método mediante el cual los electrones que llegan al Trp-104 son trasladados al cua, el primero de los cuatro centros de reducción-oxidación (redox) de la enzima a ser reducidos. A fin de arrojar luz sobre este proceso, usamos la computadora para crear y analizar un complejo de sustrato enzimático formado a partir de la estructura publicada de las dos proteínas. Se halló que el borde frontal del hemo del ferrocitocromo c estaba muy cercano al Trp-104 del COX, incluyendo el Trp-104. Sólo nueve residuos de aminoácidos de COX se encuentran a lo largo de un ancho canal que se extiende desde Trp-104 hasta el centro CuA de la enzima. Seis de los nueve residuos, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 y Glu-198, poseían las propiedades químicas ideales y estaban alineados adecuadamente para facilitar la transferencia de electrones. En este trabajo proponemos que la reducción de Trp-104 y la subsiguiente reducción de CuA ocurre mediante un sistema de relé iónico hidrógeno-hidruro, similar al que se observa en el sitio activo de la quimotripsina.

The effects of ingested aluminium on brain cytochrome oxidase activity / Efectos de la ingestión de aluminio sobre la actividad de la oxidasa citocrómica del cerebro

Aluminium has a unique combination of physical and chemical properties which has enabled man to put this metal to very wide and varied use. However, prolonged exposure to aluminium ions may lead to adverse health effects. In this study, we evaluated the effects of dietary aluminium on the protein composition and the intrinsic activity of cytochrome oxidase (COX) for brain mitochondria. New Zealand white rabbits were maintained on a diet of commercial rabbit pellets and distilled water for a period of 12 weeks. For the experimental group, AlCl3, 330mg/kg/L was added to the drinking water. When compared to the control, mitochondria isolated from the brains of the AlCl3 fed rabbits showed no change in Km but an approximate 35% decrease in both the low and high affinity Vmax values. Also, whereas the protein composition of the mitochondria from both sources appeared to be normal, isolation of highly purified COX proved to be difficult and for the AICI3 fed rabbits, a number of the enzyme's low molecular weight subunits were absent. These results appear to confirm a relationship between long term aluminium consumption and low brain COX activity; they further suggest that an altered COX structure may be the cause of the low enzymic activity.

El aluminio posee una combinación única de las propiedades físicas y químicas que ha permitido al ser humano hacer un uso amplio y variado de este metal. Sin embargo, un número de estudios recientes, sugiere que la exposición prolongada a los iones de aluminio puede tener efectos nocivos sobre la salud. En el presente estudio, evaluamos los efectos del aluminio dietético sobre la composición proteínica y la actividad intrínseca de la oxidasa citocrómica (COX) para la mitocondria cerebral. Conejos blancos de Nueva Zelanda, fueron mantenidos con una dieta de alimento para conejos y agua destilada por un período de 12 semanas. Para el grupo experimental AlCl3, 330mg/kg/L fueron añadidos al agua potable. En comparación con el grupo de control, las mitocondrias aisladas de los cerebros de los conejos alimentados con AlCl3 no mostraron cambios en Km pero hubo una disminución de aproximadamente 35% tanto en los valores Vmax de baja y alta afinidad. Por otro lado, mientras que la composición proteica de las mitocondrias de ambas fuentes parecía ser normal, resultó difícil aislar el COX altamente purificado y un número de enzimas de subunidades de bajo peso molecular MMMM estuvieron ausentes. Estos resultados parecen confirmar una relación entre el consumo de aluminio a largo plazo y la baja actividad del COX del cerebro. Asimismo, sugieren que una alteración de la estructura del COX puede ser la causa de una baja actividad enzimática.

Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach.

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach / Investigación de las especifidades de la unión de substrato para la citocromo-c-oxidasa: un enfoque asistido por computadora

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

La deficiencia de la citocromo-c-oxidasa (COX) se halla asociada con un número de enfermedades, pero los detalles del mecanismo de acción - especialmente la interacción con su substrato, el ferrocitocromo c - no está aún claro. Se sabe que la transferencia de electrones del ferrocitocromo c a la COX, es facilitada por la formación de los complejos enzima-substrato (ES), los cuales son estabilizados por puentes intermoleculares de sal. No obstante, la identidad de los residuos que participan en los puentes sigue sin estar clara. Recurriendo a las estructuras publicadas de dos proteínas, se emplearon simulaciones por computadora a fin de obtener un modelo de sus interacciones, en un intento por identificar los residuos que toman parte en los puentes de sal. El proceso de simulación fue guiado principalmente por estudios de reticulación, que proponen que el Lys-13 del citocromo c está pareado con el Asp-18 de la COX. El complejo enzima-sustrato inicial creado mediante la manipulación asistida por computadora de las dos estructuras, exhibía tres puentes de sal. Tras aplicar los procedimientos de minimización de la energía, el número de puentes de sal aumentó a siete y hubo veinticuatro enlaces intermoleculares de hidrógeno. Los puentes de sal emanaron de: Glu-119 y Asp-221 de la subunidad I; Glu-114, Asp-115 y Asp-158 de la subunidad II y Asp-73 y Glu-78 de la subunidad VIb. Estos fueron pareados con Lys-87, 8, 25, 27, 13, 22 y 100 respectivamente del citocromo c. Estos resultados sugieren que las subunidades I, II y VIb juegan un papel directo en la unión del substrato. Los resultados también sugieren que los enlaces de hidrógeno contribuyen significativamente a la estabilidad del complejo-ES.

Early electron transfer in cytochrome c oxidase occurs by a chymotrypsin type relay.

Evidence suggests that when ferrocytochrome c (the substrate) reduces cytochrome c oxidase (COX), electrons from the former enter the latter via Trp-104. What is still to be determined is the method by which electrons are transferred from ferrocytochrome c to Trp-104 and the method by which electrons arriving at Trp-104 are moved on to Cu(A), the first of the enzyme's four redox centres to be reduced. To shed light on this process, we used the computer to create and analyse an enzyme-substrate complex formed from the published structure of the two proteins. It was found that the front haem edge of ferrocytochrome c was in close proximity to Trp-104 of COX and that inclusive of Trp-104, only nine amino acid residues from COX lie along a broad channel stretching from Trp-104 to the enzyme's Cu(A) centre. Six of the nine residues, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 and Glu-198, had the ideal chemical properties and were properly aligned to facilitate electron transfer. Here we propose that the reduction of Trp-104 and the subsequent reduction of Cu(A) occur by a hydride/hydrogen ion relay system similar to that seen at the active site of chymotrypsin.

The effects of ingested aluminium on brain cytochrome oxidase activity.

Aluminium has a unique combination of physical and chemical properties which has enabled man to put this metal to very wide and varied use. However prolonged exposure to aluminium ions may lead to adverse health effects. In this study, we evaluated the effects of dietary aluminium on the protein composition and the intrinsic activity of cytochrome oxidase (COX) for brain mitochondria. New Zealand white rabbits were maintained on a diet of commercial rabbit pellets and distilled water for a period of 12 weeks. For the experimental group, AlCl3, 330 mg/kg/L was added to the drinking water. When compared to the control, mitochondria isolated from the brains of the AICl3 fed rabbits showed no change in Km but an approximate 35% decrease in both the low and high affinity Vmax values. Also, whereas the protein composition of the mitochondria from both sources appeared to be normal, isolation of highly purified COX proved to be difficult and for the AlCl3 fed rabbits, a number of the enzyme's low molecular weight subunits were absent. These results appear to confirm a relationship between long term aluminium consumption and low brain COX activity; they further suggest that an altered COX structure may be the cause of the low enzymic activity.

Marginally low copper causes lesions of the midbrain in animal models: the implications for man

Serum copper levels must be maintained between very strict limits for the maintenance of good health. High levels have recently been linked to Alzheimer's disease while low levels during pregnancy cause enzootic ataxia (swayback disease) in offspring. In this study, we investigated the prolonged effect of serum copper that was maintained at and around 0.5 ppm, the level presently regarded as safe. Pregnant sheep and rabbits in the last trimester (1-4 weeks) of pregnancy were treated with the copper chelator ammonium tetrathiomolybdate (ATM). Treatment was continued until the young were one month old at which time the animals were sacrificed Serum copper levels of the parents and offspring were monitored by atomic absorption. The difference spectra (400-630 nm) was examined and SDS PAGE was used to evaluate the protein composition of the brain mitochondria. The anatomy of the midbrain was also studied. Although the young sheep and rabbits from the ATM-treated mothers showed no visible signs of disability or swayback disease, the midbrain of those young animals with serum copper between 0.3-0.9 ppm showed evidence of vacuolation, cavitation and chromatolysis. In contrast, the difference spectra and the protein composition of the brain mitochondria from these animals were all normal. These results suggest that although animals may appear normal and exhibit some normal biochemical markers, serum copper in the region of 0.5 ppm may not be safe for some breeds of sheep or rabbits. It is possible that a similar situation applies to man.

Los niveles séricos cúpricos tienen que ser mantenidos dentro de límites muy estrictos, si se quiere tener una buena salud. Los altos niveles de cobre han sido asociados recientemente con la enfermedad de Alzheimer, mientras que los niveles bajos durante el embarazo causan ataxia enzoótica (swayback) enla descendencia. En este estudio investigamos el efecto prolongado del cobre sérico mantenido a 0.5 ppm ó alrededor de 0.5 ppm – el nivel considerado seguro actualmente. Ovejas y conejas preñadas, en el último trimestre (1-4 semanas) de gestación, fueron tratadas con el quelante del cobre conocido como tetratiomolibdato de amonio (TM). El tratamiento continuó hasta que las crías tuvieron un mes,momento en el que los animales fueron sacrificados. Los niveles séricos cúpricos de los progenitores y la progenie fueron monitoreados mediante absorción atómica. Se examinaron los espectros de diferencia (400– 630 nm). Se usó la técnica de SDS-PAGE para evaluar la composición proteica de lasmitocondrias cerebrales. También se estudió la anatomía del mesencéfalo. Aunque la anatomía de las crías de ovejas y conejas madres tratadas con TM no mostraron señales visibles de discapacidad o enfermedad swayback, el mesencéfalo de estas crías con cobre sérico entre 0.3–0.9 ppm, mostróevidencias de vacuolación, cavitación y cromatolisis. En contraste con ello, los espectros de diferencia y la composición proteica de las mitocondrias del cerebro de estos animales, fueron todos normales. Estos resultados sugieren que aunque los animales puedan parecer normales y presentar marcas bioquímicas normales, el cobre sérico en el rango de 0.5 ppm, puede no ser seguro para algunas crías de ovejas y conejos. Es posible que una situación similar se aplique al ser humano.

Marginally low copper causes lesions of the midbrain in animal models: the implications for man.

Serum copper levels must be maintained between very strict limits for the maintenance of good health. High levels have recently been linked to Alzheimer's disease while low levels during pregnancy cause enzootic ataxia (swayback disease) in offspring. In this study, we investigated the prolonged effect of serum copper that was maintained at and around 0.5 ppm, the level presently regarded as safe. Pregnant sheep and rabbits in the last trimester (1-4 weeks) of pregnancy were treated with the copper chelator ammonium tetrathiomolybdate (ATM). Treatment was continued until the young were one month old at which time the animals were sacrificed Serum copper levels of the parents and offspring were monitored by atomic absorption. The difference spectra (400-630 nm) was examined and SDS PAGE was used to evaluate the protein composition of the brain mitochondria. The anatomy of the midbrain was also studied. Although the young sheep and rabbits from the ATM-treated mothers showed no visible signs of disability or swayback disease, the midbrain of those young animals with serum copper between 0.3-0.9 ppm showed evidence of vacuolation, cavitation and chromatolysis. In contrast, the difference spectra and the protein composition of the brain mitochondria from these animals were all normal. These results suggest that although animals may appear normal and exhibit some normal biochemical markers, serum copper in the region of 0.5 ppm may not be safe for some breeds of sheep or rabbits. It is possible that a similar situation applies to man.

The control of hypertension by use of coconut water and mauby: two tropical food drinks.

In this study, the authors investigated the effect of regular consumption of two tropical food drinks, coconut (Cocos nucifera) water and mauby (Colubrina arborescens), on the control of hypertension. Twenty-eight hypertensive subjects were assigned to four equal groups and their systolic and diastolic blood pressures recorded for two weeks before and then for another two weeks while receiving one of four interventions. One group (the control) received bottled drinking water, the second group received coconut water, the third received mauby and the fourth group, a mixture of coconut water and mauby. Significant decreases in the mean systolic blood pressure were observed for 71%, 40% and 43% respectively of the groups receiving the coconut water, mauby and the mixture (p < or = 0.05). For these groups, the respective proportions showing significant decreases in the mean diastolic pressure were 29%, 40% and 57%. For the group receiving the mixture, the largest decreases in mean systolic and mean diastolic pressure were 24 mmHg and 15 mmHg respectively; these were approximately double the largest values seen with the single interventions.

The control of hypertension by use of coconut water and mauby: two tropical food drinks / El Control de la Hipertensión Mediante el uso del Agua de Coco y el Mauby: Dos Bedidas Tropicales Naturales

In this study, the authors investigated the effect of regular consumption of two tropical food drinks, coconut (Cocos nucifera) water and mauby (Colubrina arborescens), on the control of hypertension. Twenty-eight hypertensive subjects were assigned to four equal groups and their systolic and diastolic blood pressures recorded for two weeks before and then for another two weeks while receiving one of four interventions. One group (the control) received bottled drinking water, the second group received coconut water, the third received mauby and the fourth group, a mixture of coconut water and mauby. Significant decreases in the mean systolic blood pressure were observed for 71, 40 and 43 respectively of the groups receiving the coconut water, mauby and the mixture (p < or = 0.05). For these groups, the respective proportions showing significant decreases in the mean diastolic pressure were 29, 40 and 57. For the group receiving the mixture, the largest decreases in mean systolic and mean diastolic pressure were 24 mmHg and 15 mmHg respectively; these were approximately double the largest values seen with the single interventions

En el presente estudio, los autores investigaron el efecto del consume regular de dos bebidas tropicales naturales: el agua de coco (Cocos nucifera) y el mauby (Colubrina arborescens) sobre el control de la hipertensión. A veintiocho sujetos hipertensos asignados a cuatro grupos iguales, se les hizo un registro de su presión sanguínea sistólica y diastólica por espacio de dos semanas antes, y luego otras dos semanas mientras que recibían una de cuatro intervenciones. Un grupo (de control) recibió agua mineral embotellada; un segundo grupo recibió agua de coco; un tercer grupo recibió mauby; y finalmente el cuarto grupo recibió una bebida mixta de agua de coco y mauby. Se observaron disminuciones significativas de la presión sistólica media en el 71%, el 40% y el 43% respectivamente, de los grupos que recibieron el agua de coco, el mauby, y la bebida combinada (p ≤ 0.05). Para estos grupos, las proporciones respectivas que mostraron disminuciones significativas de la presión diastólica media fueron 29%, 40% y 57%. Para el grupo que recibió la bebida mixta, las disminuciones mayores en las presiones medias sistólica y diastólica fueron 24 mmHg y 15 mmHg respectivamente - aproximadamente el doble de los valores observados en cada una de las intervenciones.

Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme turnover.

We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the 'traditional' oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady-state reducing conditions. Whereas for the 'oxidized' complex cytochrome c became bound exclusively to subunit II of the enzyme, for the 'steady-state' complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV. For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady-state spectral analysis (400-630 nm) combined with stopped-flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer. Additionally, stopped-flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55x10(6) m(-1) x s(-1) while that for the steady-state was 0.27x10(6) m(-1) x s(-1). These values were approximately 25-50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.

Understanding the mechanism of action of cytochrome c oxidase in normal and disease states: shark heart enzyme, a potential model.

Cytochrome c oxidase, the final member of the electron transport chain, is crucial to respiration and also contributes to the synthesis of cellular ATP. The total absence of this enzyme is incompatible with life and its deficiency or malfunction leads to a number of serious disease states. Understanding the mechanism of action of this enzyme, which is an important prerequisite to unravelling its role in the pathogenesis of disease states, is hampered by the lack of suitable enzyme models. The bovine enzyme, which is commonly used, is enormously complex and the bacterial enzymes, which are structurally simple, appear to follow a different mechanism of action. The hammer head shark is a seasonal resident of the warm waters of the Caribbean Sea. The work presented here indicates that, like the bovine enzyme, the enzyme of the heart of this shark (i) possesses thirteen subunits and two substrate binding sites and (ii) exhibits biphasic kinetics. The work also confirms that, unlike the bovine enzyme which is dimeric, the shark enzyme functions as a monomer. Given this latter simplifying feature, in conjunction with its kinetic and structural similarities to the more complex mammalian varieties, we propose that shark heart cytochrome c oxidase replace the bovine and bacterial forms as the enzyme of choice for model studies.