Intention to adopt Electronic Animal Movement Permit (e-GTA) systems in Rio Grande do Sul, Brazil / Intenção de adoção de Guia de Trânsito Animal Eletrônica (e-GTA) no Rio Grande do Sul, Brasil

Control of livestock and their movement has long been recognized as a crucial for the prevention and control of diseases. In Brazil, the control of livestock movement established in 1934. Since 1995 is regulated based on the Animal Movement Permit (GTA). Since then, the process has been improved and updated on its legal framework and strategies, including the use of emerging technological alternatives, which made possible the launching of the electronic Animal Movement Permit (e-GTA) in 2011. From a broader perspective, the e-GTA is inserted in a global context of the development of Information and Communication Technology (ICT) which since the early 1980s enabled governments worldwide to drive policies for the development of electronic government systems (e-government). After that, at a global level, there was an expansion and improvement of e-government services; however, discrepancies among countries persisted. Nonetheless, the levels of adoption of e-government by citizens have been lower than those expected by government authorities, which has attracted the attention of researchers in the area. In this context, studies of this nature that used theoretical antecedents related to the adoption of ICT have exposed peculiarities of the process of adoption of e-government, demanding research efforts directed to the structuring of specific models for this area. In Brazil, studies of this nature are still at an early stage, although government actions aimed at e-government date back to the 1990s. The objective of this article was to investigate, in an exploratory way, influence factors on the adoption intention of e-government related to animal health, having the e-GTA as the object of research. The conceptual framework was defined based upon the e-Government Adoption Model (GAM) For this objective, an online questionnaire oriented to intention to adopt e-GTA was applied to equine owners of Rio Grande do Sul not users of this system...(AU)

O controle dos estoques e de movimentações de animais pecuários há muito tempo é reconhecido como um fator crucial para a prevenção e resposta para a introdução e disseminação de doenças animais ou zoonóticas. No Brasil, o controle de movimentação animal foi inicialmente estabelecido em 1934 e, desde 1995, é regulamentado com base na Guia de Trânsito Animal (GTA). Desde então, o processo foi aprimorado e atualizado em seu arcabouço legal e estratégias, inclusive no uso de alternativas tecnológicas emergentes, que possibilitaram o lançamento em 2011 da Guia de Trânsito Animal Eletrônica (e-GTA). Considerado desde uma perspectiva mais abrangente, o e-GTA está inserido em um contexto global de desenvolvimento da Tecnologia da Informação e Comunicação (TIC), que desde o início dos anos 80 permite que governos em todo o mundo conduzam políticas para o desenvolvimento de sistemas de governo eletrônico (governo eletrônico). Posteriormente, a disponibilidade do governo eletrônico tem estado em expansão de qualidade e de amplitude dos serviços oferecidos, embora este processo seja permeado por desigualdades entre os países. No entanto, os níveis de adoção do governo eletrônico pelos cidadãos têm sido inferiores aos esperados pelas autoridades governamentais, o que tem atraído a atenção dos pesquisadores da área. Nesse contexto, estudos dessa natureza, que utilizaram antecedentes teóricos relacionados à adoção das TIC expuseram idiossincrasias do processo de adoção do governo eletrônico, exigindo que esforços de pesquisa sejam direcionados à estruturação de modelos específicos para essa área. No Brasil, estudos dessa natureza ainda estão em estágio inicial, embora as ações governamentais direcionadas ao governo eletrônico datem dos anos 90. Dessa forma, o objetivo deste artigo foi investigar, de forma exploratória, fatores de influência na intenção de adoção do governo eletrônico relacionado à saúde animal, tendo o e-GTA como objeto de pesquisa...(AU)

Intention to adopt Electronic Animal Movement Permit (e-GTA) systems in Rio Grande do Sul, Brazil / Intenção de adoção de Guia de Trânsito Animal Eletrônica (e-GTA) no Rio Grande do Sul, Brasil

Control of livestock and their movement has long been recognized as a crucial for the prevention and control of diseases. In Brazil, the control of livestock movement established in 1934. Since 1995 is regulated based on the Animal Movement Permit (GTA). Since then, the process has been improved and updated on its legal framework and strategies, including the use of emerging technological alternatives, which made possible the launching of the electronic Animal Movement Permit (e-GTA) in 2011. From a broader perspective, the e-GTA is inserted in a global context of the development of Information and Communication Technology (ICT) which since the early 1980s enabled governments worldwide to drive policies for the development of electronic government systems (e-government). After that, at a global level, there was an expansion and improvement of e-government services; however, discrepancies among countries persisted. Nonetheless, the levels of adoption of e-government by citizens have been lower than those expected by government authorities, which has attracted the attention of researchers in the area. In this context, studies of this nature that used theoretical antecedents related to the adoption of ICT have exposed peculiarities of the process of adoption of e-government, demanding research efforts directed to the structuring of specific models for this area. In Brazil, studies of this nature are still at an early stage, although government actions aimed at e-government date back to the 1990s. The objective of this article was to investigate, in an exploratory way, influence factors on the adoption intention of e-government related to animal health, having the e-GTA as the object of research. The conceptual framework was defined based upon the e-Government Adoption Model (GAM) For this objective, an online questionnaire oriented to intention to adopt e-GTA was applied to equine owners of Rio Grande do Sul not users of this system. Results indicated that respondents positively evaluated the quality of their computers and cellphones, as well as their efficacy to operate them, while quality of the internet connection was considered regular. In this context, the availability of computer and cellphone resources and computer self-efficacy did not influence the intention to adopt e-GTA. Moreover, aspects related to compatibility of e-GTA with users lifestyle have a positive association with the intention to adopt e-GTA. Also the study indicated that public information campaigns could contribute to the improvement of adoption of the e-GTA. Additionally, the authors suggested that further investigations considering other livestock sectors and variables, as well as the continuity of use after the adoption, could contribute to the expansion of the knowledge on this field.(AU)

O controle dos estoques e de movimentações de animais pecuários há muito tempo é reconhecido como um fator crucial para a prevenção e resposta para a introdução e disseminação de doenças animais ou zoonóticas. No Brasil, o controle de movimentação animal foi inicialmente estabelecido em 1934 e, desde 1995, é regulamentado com base na Guia de Trânsito Animal (GTA). Desde então, o processo foi aprimorado e atualizado em seu arcabouço legal e estratégias, inclusive no uso de alternativas tecnológicas emergentes, que possibilitaram o lançamento em 2011 da Guia de Trânsito Animal Eletrônica (e-GTA). Considerado desde uma perspectiva mais abrangente, o e-GTA está inserido em um contexto global de desenvolvimento da Tecnologia da Informação e Comunicação (TIC), que desde o início dos anos 80 permite que governos em todo o mundo conduzam políticas para o desenvolvimento de sistemas de governo eletrônico (governo eletrônico). Posteriormente, a disponibilidade do governo eletrônico tem estado em expansão de qualidade e de amplitude dos serviços oferecidos, embora este processo seja permeado por desigualdades entre os países. No entanto, os níveis de adoção do governo eletrônico pelos cidadãos têm sido inferiores aos esperados pelas autoridades governamentais, o que tem atraído a atenção dos pesquisadores da área. Nesse contexto, estudos dessa natureza, que utilizaram antecedentes teóricos relacionados à adoção das TIC expuseram idiossincrasias do processo de adoção do governo eletrônico, exigindo que esforços de pesquisa sejam direcionados à estruturação de modelos específicos para essa área. No Brasil, estudos dessa natureza ainda estão em estágio inicial, embora as ações governamentais direcionadas ao governo eletrônico datem dos anos 90. Dessa forma, o objetivo deste artigo foi investigar, de forma exploratória, fatores de influência na intenção de adoção do governo eletrônico relacionado à saúde animal, tendo o e-GTA como objeto de pesquisa. A estrutura conceitual foi definida com base no Modelo de Adoção de Governo Eletrônico (GAM). Para esse objetivo, um questionário online dirigido à intenção de adoção do e-GTA foi aplicado a proprietários de equinos do Rio Grande do Sul que não eram usuários deste sistema. Os resultados indicaram que os entrevistados avaliaram positivamente a qualidade de seus computadores e telefones celulares, bem como sua eficácia em operá-los, enquanto a qualidade da conexão de internet foi considerada regular. Nesse contexto, a disponibilidade de recursos informáticos e de celulares, bem como a capacidade de operação dos mesmos não influenciaram a intenção de adoção do e-GTA. Além disso, aspectos relacionados à compatibilidade do e-GTA com o estilo de vida do usuário têm uma associação positiva com a intenção de adoção do e-GTA. Além disso, o estudo indicou que as campanhas de informação pública poderiam contribuir para o incremento na adoção efetiva do e-GTA. Finalmente, o estudo identificou oportunidades para o desenvolvimento de investigações adicionais, considerando distintas variáveis, setores produtivos e sistemas de governo eletrônico dirigidos à saúde animal, o que poderia contribuir para a expansão do conhecimento nesta área.(AU)

Mitochondrial involvement in memory impairment induced by scopolamine in rats.

OBJECTIVE: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action. METHODS: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function. RESULTS: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H2O2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly. CONCLUSION: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

Early modulation of the transcription factor Nrf2 in rodent striatal slices by quinolinic acid, a toxic metabolite of the kynurenine pathway.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor involved in the orchestration of antioxidant responses. Although its pharmacological activation has been largely hypothesized as a promising tool to ameliorate the progression of neurodegenerative events, the actual knowledge about its modulation in neurotoxic paradigms remains scarce. In this study, we investigated the early profile of Nrf2 modulation in striatal slices of rodents incubated in the presence of the toxic kynurenine pathway metabolite, quinolinic acid (QUIN). Tissue slices from rats and mice were obtained and used throughout the experiments in order to compare inter-species responses. Nuclear Nrf2 protein levels and oxidative damage to lipids were compared. Time- and concentration-response curves of all markers were explored. Nrf2 nuclear activation was corroborated through phase 2 antioxidant protein expression. The effects of QUIN on Nrf2 modulation and oxidative stress were also compared between slices of wild-type (Nrf2(+/+)) and Nrf2 knock-out (Nrf2(-/-)) mice. The possible involvement of the N-methyl-d-aspartate receptor (NMDAr) in the Nrf2 modulation and lipid peroxidation was further explored in mice striatal slices. In rat striatal slices, QUIN stimulated the Nrf2 nuclear translocation. This effect was accompanied by augmented lipid peroxidation. In the mouse striatum, QUIN per se exerted an induction of Nrf2 factor only at 1h of incubation, and a concentration-response effect on lipid peroxidation after 3h of incubation. QUIN stimulated the striatal content of phase 2 enzymes. Nrf2(-/-) mice were slightly more responsive than Nrf2(+/+) mice to the QUIN-induced oxidative damage, and completely unresponsive to the NMDAr antagonist MK-801 when tested against QUIN. Findings of this study indicate that: (1) Nrf2 is modulated in rodent striatal tissue in response to QUIN; (2) Nrf2(-/-) striatal tissue was moderately more vulnerable to oxidative damage than the Wt condition; and (3) early Nrf2 up-regulation reflects a compensatory response to the QUIN-induced oxidative stress in course as part of a general defense system, whereas Nrf2 down-regulation might contribute to more intense oxidative cell damage.

Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: cross talk between glycero- and sphingolipids in the kidney.

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the µM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction.

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Mitochondrial dysfunction in the hippocampus of rats caused by chronic oxidative stress.

The aim of this study was to analyze the effects of chronic oxidative stress on mitochondrial function and its relationship to progressive neurodegeneration in the hippocampus of rats chronically exposed to ozone. Animals were exposed to 0.25 ppm ozone for 7, 15, 30, or 60 days. Each group was tested for (1) protein oxidation and, manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx) and succinate dehydrogenase (SDH) activity using spectrophotometric techniques, (2) oxygen consumption, (3) cytochrome c, inducible nitric oxide synthase (iNOS), peroxisome proliferator-activated receptor γ Co-activator 1α (PGC-1α), B-cell lymphoma (Bcl-2), and Bax expression using Western blotting, (4) histology using hematoxylin and eosin staining, and (5) mitochondrial structure using electron microscopy. Our results showed increased levels of carbonyl protein and Mn-SOD activity after 30 days of ozone exposure and decreased GPx activity. The SDH activity decreased from 7 to 60 days of exposure. The oxygen consumption decreased at 60 days. Western blotting showed an increase in cytochrome c at 60 days of ozone exposure and an increase in iNOS up to 60 days of ozone exposure. The expression of PGC-1α was decreased after 15, 30, and 60 days compared to the earlier time Bcl-2 was increased at 60 days compared to earlier time points, and Bax was increased after 30 and 60 days of exposure compared to earlier time points. We observed cellular damage, and mitochondrial swelling with a loss of mitochondrial cristae after 60 days of exposure. These changes suggest that low doses of ozone caused mitochondrial abnormalities that may lead to cell damage.

Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

The antioxidant effect of the mesoionic compound SYD-1 in mitochondria.

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75µmolmg(-1) protein) inhibited the lipoperoxidation induced by Fe(3+)/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0µmolmg(-1) protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α'-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25-1.0µmolmg(-1) protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9-19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25-1.0µmolmg(-1) protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca(2+)/phosphate by approximately 30% (1.0µmolmg(-1) protein). When Ca(2+)/H2O2 were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0µmolmg(-1) of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.

Hypertonicity increases NO production to modulate the firing rate of magnocellular neurons of the supraoptic nucleus of rats.

Increases in plasma osmolality enhance nitric oxide (NO) levels in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and modulate the secretion of both vasopressin (VP) and oxytocin (OT). In this paper, we describe the effects of hypertonicity on the electrical properties of MNCs by focusing on the nitrergic modulation of their activity in this condition. Membrane potentials were measured using the patch clamp technique, in the presence of both glutamatergic and GABAergic neurotransmission blockers, in coronal brain slices of male Wistar rats. The recordings were first made under a control condition (295 mosm/kg H2O), then in the presence of a hypertonic stimulus (330 mosm/kg H2O) and, finally, with a hypertonic stimulus plus 500 µM L-Arginine or 100 µM N-nitro-L-Arginine methyl ester hydrochloride (L-NAME). Hypertonicity per se increased the firing frequency of the neurons. L-Arginine prevented the increase in fire frequency induced by hypertonic stimulus, and L-NAME (inhibitor of nitric oxide synthase) induced an additional increase in frequency when applied together with the hypertonic solution. Moreover, L-Arginine hyperpolarizes the resting potential and decreases the peak value of the after-hyperpolarization; both effects were blocked by L-NAME and hypertonicity and/or L-NAME reduced the time constant of the rising phase of the after-depolarization. These results demonstrate that an intrinsic nitrergic system is part of the mechanisms controlling the excitability of MNCs of the SON when the internal fluid homeostasis is disturbed.

Tumor cell death induced by the inhibition of mitochondrial electron transport: the effect of 3-hydroxybakuchiol.

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500µM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with ß-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and ß-oxidation of fatty acids.

Effect of biomolecules from human renal matrix of calcium oxalate monohydrate (CaOx) stones on in vitro calcium phosphate crystallization

PURPOSE: Investigate the activity of high and low molecular weight biomolecules present in the matrix of human calcium oxalate (CaOx) stones not only on the initial mineral phase formation of calcium and phosphate (CaP) but also on its growth and demineralization of the preformed mineral phase. MATERIALS AND METHODS: Surgically removed renal stones were analyzed by Fourier Transform Infra Red (FTIR) spectroscopy and only CaOx stones were extracted with 0.05M EGTA, 1 mM PMSF and 1 percent ß-mercaptoethanol. Renal CaOx stone extract was separated into > 10 kDa and < 10 kDa fractions by dialysis. Activity of both the fractions along with whole extract was studied on the three mineral phases of CaP assay system. RESULTS: It was interesting to observe that both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones exhibited different roles in the three mineral phases of CaP. Whole extract exhibited inhibitory activity in all the three assay systems; however, mixed (stimulatory and inhibitory) activity was exhibited by the > 10 kDa and < 10 kDa fractions. SDS-PAGE analysis showed bands of 66 kDa, 80 kDa, 42 kDa in whole EGTA extract lane and > 10 kDa fraction lane. CONCLUSION: Both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones have a significant influence on calcium and phosphate (CaP) crystallization.