Aspectos ecoepidemiológicos, detección natural e identificación molecular de Leishmania spp. en Lutzomyia reburra, Lutzomyia barrettoi majuscula y Lutzomyia trapidoi / Eco-epidemiological aspects, natural detection and molecular identification of Leishmania spp. in Lutzomyia reburra, Lutzomyia barrettoi majuscula and Lutzomyia trapidoi

Resumen Introducción. La provincia de Pichincha, Ecuador, es un área endémica de leishmaniasis cutánea, en donde se han determinado como vectores los flebotomíneos antropofílicos con infección natural por Leishmania spp. Sin embargo, no se ha evaluado el papel en la transmisión de las especies zoofílicas. Objetivo. Evaluar la infección natural por Leishmania en dos especies de flebotomíneos zoofílicos, Lutzomyia reburra y Lu. barrettoi majuscula, y en una antropofílica, Lu. trapidoi, así como la endofagia y la sinantropía de estas especies en el noroccidente de Pichincha. Materiales y métodos. Los flebotomíneos se recolectaron en trampas de luz CDC colocadas en diferentes hábitats y altitudes en sitios que son focos de leishmaniasis cutánea. La infección con Leishmania spp. se detectó en el ADN genómico de hembras de las especies de flebotomíneos de interés. Se amplificó el gen espaciador interno de la transcripción del ARN ribosómico, unidad I (ITS1), y los genes de las topoisomerasas mitocondrial II (mtTOPOII) y nuclear II (TopoII). Se determinaron los porcentajes de positividad para Leishmania a escala espaciotemporal, la proporción de endofagia y el índice de sinantropía. Resultados. Se determinó la presencia de infección natural por Le. amazonensis en Lu. reburra (9,5 %) y Lu. b. majuscula (23,8 %); en Lu. trapidoi se detectaron Le. amazonensis, Le. brazilienis y Le. naiffilainsoni. Los flebotomíneos eran asinantrópicos y con baja endofagia. Conclusión. Se registró por primera vez la presencia de infección natural en Lu. reburra y Lu. barrettoi majuscula por Le. amazonensis, y se demostró la importancia de los flebotomíneos zoofílicos en el mantenimiento del ciclo de transmisión de Leishmania spp. en focos endémicos.

Abstract Introduction: The province of Pichincha in Ecuador is an endemic area of cutaneous leishmaniasis, where anthropophilic sand flies with natural infection by Leishmania, have been reported as vectors. However, the role in transmission of zoophilic species has not been evaluated. Objective: To evaluate natural infection by Leishmania in two zoophilic phlebotomine sand fly species, Lutzomyia reburra and Lu. barrettoi majuscula, and one anthropophilic species, Lu. trapidoi, as well as the endophagy and synanthropism of these species in the northwest of Pichincha. Materials and methods: Phlebotomines were collected using CDC light traps in different habitats and altitudes with presence of cutaneous leishmaniasis. Leishmania infection was detected using genomic DNA from females of the collected sand flies. We amplified the internal transcribed spacer gene of ribosomal RNA I (ITS1), the mitochondrial topoisomerase II gene (mtTOPOII), and the nuclear topoisomerase II gene (TopoII). Percentages of positivity for Leishmania, at spatio-temporal scale, proportion of endophagy and synanthropism index were calculated. Results: Natural infection was determined for Le. amazonensis in Lu. reburra (9.5%) and Lu. b. majuscula (23.8%), while in Lu. trapidoi we detected Le. amazonensis, Le. brazilienis and Le. naiffilainsoni. Phlebotomines were asynanthropic and with low endophagy. Conclusion: Natural infection with Le. amazonensis was recorded for the first time in Lu. reburra and Lu. b. majuscula, demonstrating the importance of zoophilic phlebotomines in the maintenance of the Leishmania transmission cycle in endemic foci.

Differential expression on mitochondrial tryparedoxin peroxidase (mTcTXNPx) in Trypanosoma cruzi after ferrocenyl diamine hydrochlorides treatments

Abstract Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5 kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58 kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5 kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.

Molecular characterization of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenée) (Lepidoptera: Crambidae) based on mitochondrial marker cytochrome oxidase I and their phylogenetic relationship.

Shoot and fruit borer, Leucinodes orbonalis is an important insect pest infesting brinjal or eggplant in India. Molecular characterization of nine different populations belonging to various brinjal growing regions was done using Cytochorome C Oxidase I (COI) gene. Nucleotide analysis of genetic diversity and phylogenetic analysis of the COI indicate that the L. orbonalis from different geographical regions are homogenous. The results showed less nucleotide diversity (π = 0.007895) and overall mean distance (0.008±0.003). Topologies of neighbour-joining (NJ) trees indicate all the populations belong to single major clade. Therefore, it is inferred that there was no significant molecular diversity within L. orbonalis of different geographical locations of India with respect to COI.

Adherence to standard precautions from the standpoint of the Health Belief Model: the practice of recapping needles / Adesão às precauções padrão sob o prisma do Modelo de Crenças em Saúde: a prática de reencapar agulhas

The aim of this study was to applythe Health Belief Model to explain the adherence to the recommendation not to recap needles by dentists and dental assistants of the public health system in a municipality in the State of São Paulo. A questionnaire validated and adapted for the oral health area was used, which included variables related to the frequency of recapping and health beliefs using Likert-type scales. The relationship between beliefs and adherence to the recommendation not to recap needles was obtained by regression analysis. Of all the professionals in this study (n=79), the majority (83.5%) reported recapping needles at least once in the last month. Through regression analysis, it was observed that the relationship between the beliefs described by the model and the attitude whether or not to follow the recommendation not to recap needles was explained by a lower perception of psychological barriers and a greater perception of stimuli not to recap needles. The conclusion reached is that the acceptance of recommendations to prevent working accidents with biological material was explained by some dimensions of the Health Belief Model, enabling discussion about reformulation of training offered to professionals of the public health system.

Objetivou-se neste estudo aplicar o Modelo de Crenças em Saúde a fim de explicar a adesão à recomendação de não reencapar agulhas por cirurgiões-dentistas e auxiliares de saúde bucal da rede pública de um município paulista. Utilizou-se um questionário validado e adaptado para a área de saúde bucal, que contemplava variáveis relativas à frequência do reencape e crenças em saúde, por meio de escalas tipo Likert. A relação entre as crenças e a adesão à recomendação de não reencapar agulhas foi obtida por meio da análise de regressão. Da amostra de profissionais obtida por adesão ao estudo (n = 79), a maioria (83,5%) relatou ter reencapado agulhas pelo menos alguma vez no último mês. Por meio da análise de regressão, foi observado que a relação entre as crenças descritas pelo modelo e a atitude de aderir ou não à recomendação de não reencapar agulhas foi explicada por uma menor percepção de barreiras psicológicas e por uma maior percepção de estímulos para não reencapar agulhas. Conclui-se que a aceitação das recomendações para prevenir acidentes do trabalho com material biológico foi explicado por algumas dimensões do Modelo de Crenças em Saúde, possibilitando a discussão sobre a reformulação de capacitações oferecidas para profissionais do sistema público de saúde.

Suppression of miR-181 a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

BACKGROUND: Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice. RESULTS: Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice. CONCLUSIONS: These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.

Two Human Cases of Diphyllobothrium nihonkaiense Infection in Korea

Diphyllobothrium latum and Diphyllobothrium nihonkaiense are the 2 reported main causes of human diphyllobothriasis in the Republic of Korea. However, the differentiation of these 2 species based on morphologic features alone is difficult. The authors used nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene to diagnose Diphyllobothrium spp. Two patients visited the emergency room at Kyungpook National University Hospital on 3 April and 12 April 2013, respectively, with fragments of parasites found while defecating. The parasites were identified as Diphyllobothrium spp. based on morphologic characteristics, and subsequent cox1 gene sequencing showed 99.9% similarity (1,478/1,480 bp) with D. nihonkaiense. Our findings support the hypothesis that D. nihonkaiense is a dominant species in Korea.

Molecular Identification of Haemadipsa rjukjuana (Hirudiniformes: Haemadipsidae) in Gageo Island, Korea

There are 60 species of blood-feeding land leeches, 50 species belonging to the family Haemadipsidae and 10 species belonging to the family Xerobdellidae. Despite recent papers on the land leeches, their taxonomic identification is not fully understood, especially at a species level. In Korea, there have been no historical records of the terrestrial leeches, but recently an unrecorded blood-feeding land leech was discovered at Gageo-do (Island), Korea. Molecular analysis was used to identify the species of 29 leeches collected from Mt. Dock-Sil in Gageo-do. Conventional PCR was conducted using nuclear 18S rRNA and mitochondrial cytochrome c oxidase subunit 1 (CO1) genetic marker. The 18S rRNA sequences revealed that the leeches share 99.9% identity with Haemadipsa rjukjuana (inhabiting Taiwan), and the CO1 sequences revealed that the leeches are very close to H. rjukjuana (inhabiting Taiwan). The CO1 sequences were separated into 2 categories, 1 with 94.6% and the other with 94.3% similarity to the H. rjukjuana L00115A (inhabiting Taiwan). This new finding of the land leech is the first record in Korea. In addition, the north range of the distribution of the blood-feeding leech (Hirudiniformes: Haemadipisidae) should be reconsidered including Korea.

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.

Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.

Prophylactic treatment with Bacopa monnieri leaf powder mitigates paraquat- induced oxidative perturbations and lethality in Drosophila melanogaster.

Environmental exposure to the oxidant-producing herbicide, paraquat (PQ) (1, 1’-dimethyl-4, 4’-bipyridinium dichloride) has long been implicated as a risk factor in Parkinson’s disease (PD). PQ-induced oxidative stress has been exploited as a model to screen putative neuroprotective compounds employing Drosophila. In the present study, we investigated the prophylactic efficacy of Bacopa monnieri (BM) against PQ-induced oxidative stress, mitochondrial dysfunctions and lethality. Exposure of adult male flies (Oregon K) to PQ alone (40 mM in 5% sucrose) resulted in 50% mortality at 48 h. Prophylaxis (7 days) with BM extract (0.1%) offered significant protection (40%) against PQ-induced mortality. Further, oxidative impairments and mitochondrial dysfunctions were monitored among Drosophila exposed to PQ (20, 40 mM) for 24 h. Significant induction of oxidative stress was observed in terms of enhanced malondialdehyde and hydroperoxide levels, and elevated activities of antioxidant enzymes (catalase and SOD). Mitochondrial dysfunctions included of significant reduction in the activities of succinate dehydrogenase (23%), complex I-III (26%), and complex II-III (30%) enzymes. Interestingly, prophylaxis with BM extract prevented the oxidative stress induction by PQ and restored the activity of ETC complexes, suggesting clearly its specific effect on the mitochondria. While the precise mechanism of action of BM needs further investigations, it may be related to its ability to enhance antioxidant defences and thus mitigate PQ-induced oxidative stress in Drosophila.

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.

Primate numts and reticulate evolution of capped and golden leaf monkeys (Primates: Colobinae).

A recent phylogenetic study of langurs and leaf monkeys of South Asia suggested a reticulate evolution of capped and golden leaf monkeys through ancient hybridization between Semnopithecus and Trachypithecus .To test this hybridization scenario, I analysed nuclear copies of the mitochondrial cytochrome b gene (numts) from capped,golden and Phayre's leaf monkeys. These numts were aligned with mitochondrial cytochrome b sequences of various species belonging to the genera Semnopithecus and Trachypithecus .In the phylogenetic tree derived from this alignment,the numts fell into three distinct clades (A,B and C) suggesting three independent integration events.Clade A was basal to Semnopithecus, and clades B and C were basal to Trachypithecus. Among the numts in clades A and C were sequences derived from species not represented in their respective sister mitochondrial groups.This unusual placement of certain numts is taken as additional support for the hybridization scenario. Based on the molecular dating of these integration events,hybridization is estimated to have occurred around 7.1 to 3.4 million years ago.Capped and golden leaf monkeys might have to be assigned to a new genus to reconcile their unique evolutionary history. Additionally, northeast India appears to be a 'hot spot' for lineages that might have evolved through reticulate evolution.

Tratamiento y post-tratamiento con lonidamina en la línea celular de carcinoma colónico humano HT-29 / Treatment and post-treatment with lonidamine in human colon carcinoma HT-29 cell line

Lonidamina (1-[ 2,4-diclorofenil metil]-1H indazol-3-ácido carboxílico), (lnd), es una droga antineoplásica cuyo mecanismo de acción se ejerce sobre el metabolismo intermedio de la glucosa. Los efectos de la lnd sobre el crecimiento celular y el metabolismo celular se investigaron en las células HT- 29, línea celular de carcinoma colónico humano, que requiere altas concentraciones de glucosa para su crecimiento indiferenciado en cultivo. La lnd en dosis de 0.2 mM disminuyó significativamente el crecimiento celular y la formación de colonias en agar; con la interrupción del tratamiento se observó el restablecimiento del crecimiento celular en 24 horas. El tratamiento con lnd produce la redistribución de los glicoconjugados y el receptor de la manosa, sin afectar en forma drástica la síntesis de glucógeno ni la de proteínas. Estas posiblemente sean las causas de la rápida reversibilidad del tratamiento.

Lonidamine (1-[ 2,4-dichlorophenyl methyl]-1H indazole-3-carboxylic acid), lnd, is an antitumoral drug acting on mitochondria and glucose metabolism. Cell growth and metabolic effects of lnd and drug post-treatment effect were investigated in undifferentiated HT-29 human colonic carcinoma cell line which requires high glucose medium concentration for growth. 0.2 mM lnd significantly decreased cell spreading and growth in monolayer or agar cell culture. After drug treatment cell growth was reestablished to control value within 24 h. Ind modified glycoconjugates and mannose-receptor distribution (analyzed by confocal microscopy), while glucose-glycogen and protein synthesis were not affected, these being the possible reasons for the fast reversible effect.

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.

Effect of T3 treatment on glutathione redox pool and its metabolizing enzymes in mitochondrial and post-mitochondrial fractions of adult rat testes.

T3 (3,3', 5-triiodo-L-thyronine; 20 microg/100 g body weight/day in 0.01 N NaOH, i.p for 1, 3 and 5 days) treatment modulated reduced (GSH) and oxidized (GSSG) glutathione contents along with the activities of its metabolizing enzymes (such as glutathione peroxidase, glutathione reductase and glutathione S-transferase) in the testis of Wistar rats. However, the magnitude and nature of changes in the above biochemical parameters in response to T3 treatment were noticed to be different between mitochondrial and post-mitochondrial fractions. This was accompanied with elevated levels of lipid hydroperoxide and ascorbic acid in the crude homogenate of testis. The level of hydrogen peroxide in the post-mitochondrial fractions of testes did not change on first day, decreased on 3rd day and increased on 5th day of the hormone treatment when compared to respective controls. Nevertheless, its content in mitochondria was significantly elevated in response to all the three durations of the hormone treatment having the highest induction on 3rd day. The changes observed in the levels of GSH and GSSG and its metabolizing enzymes in response to T3 treatment reflect an alteration in the redox state of testis, which may be a causative factor for the impairment of testicular physiology as a consequence of oxidative stress.

Formal TCA cycle description based on elementary actions.

Many databases propose their own structure and format to provide data describing biological processes. This heterogeneity contributes to the difficulty of large systematic and automatic functional comparisons. To overcome these problems, we have used the BioPsi formal description scheme which allows multi-level representations of biological process information. Applied to the description of the tricarboxylic acid cycle (TCA), we show that BioPsi allows the formal integration of functional information existing in current databases and make them available for further automated analysis. In addition such a formal TCA cycle process description leads to a more accurate biological process annotation which takes in account the biological context. This enables us to perform an automated comparison of the TCA cycles for seven different species based on processes rather than protein sequences. From current databases, BioPsi is able to unravel information that are already known by the biologists but are not available for automated analysis tools and simulation software, because of the lack of formal process descriptions. This use of the BioPsi description scheme to describe the TCA cycle was a key step of the MitoScop project that aims to describe and simulate mitochondrial metabolism in silico.

Respiratory-chain enzyme activities in isolated mitochondria of lymphocytes from patients with Parkinson's disease: preliminary study.

BACKGROUND: Evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that promote neural cell death in stroke and in Parkinson's disease. The sites of mitochondrial ROS production are not established but are generally believed to be located within the electron transport chain. AIMS: We studied the mitochondrial respiratory chain enzymes function from human circulating lymphocytes. SETTING AND DESIGN: Open study. MATERIALS AND METHODS: Forty patients with Parkinson's disease (PD) with 30 age-matched control subjects were selected in this study. The patients had received no treatment before the study was conducted. STATISTICAL ANALYSIS: The data from patients and controls were compared using two-tailed student's t-test and values were expressed as means +/- standard deviation (SD). RESULTS: Respiratory complex I + III and IV activities were significantly lower (P < 0.001) in patients than in control subjects. CONCLUSIONS: The use of lymphocytes for investigating the respiratory chain enzymes provides an easy, noninvasive method to assess mitochondrial function in patients with PD. Furthermore, our study supports the hypothesis that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 æmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 æmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18 percent, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.