Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Expansion of T regulatory lymphocytes by murine bone marrow dendritic cells previously stimulated with Anisakis simplex larval antigens

BACKGROUND Anisakis simplex antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) in mice. OBJECTIVES To study the capacity of DCs stimulated with A. simplex excretory-secretory (ES) or crude extract (CE) to generate Tregs. To investigate in vitro effects of antigens on the metabolic activity of splenocytes induced by LPS or CpG. METHODS Phenotypic and functional characterization of T cells co-cultured with A. simplex-pulsed DCs was performed by flow cytometry. Lymphocyte mitochondrial respiratory activity was estimated by the Alamar Blue® Assay. FINDINGS In C57BL/6J, CD4+CD25-Foxp3+ and CD8+CD25-Foxp3+ populations increased by CE-stimulated-DCs. In BALB/c, CE-stimulated-DCs caused the expansion of CD4+CD25+Foxp3+IL-10+ and CD8+CD25+Foxp3+IL-10+. IFN-γ expression raised in BALB/c CD4+CD25+ and CD4+CD25- for CE and ES, respectively. ES-stimulated-DCs increased CD4+CD25+ Foxp3+ and CD8+CD25- Foxp3+ expression in T cells. The association of ES or CE with LPS produced the increase in splenocyte activity in C57BL/6J. The association of CE with CpG decreased the proliferation caused by CpG in C57BL/6J. MAIN CONCLUSIONS A. simplex increase the frequency of Tregs, which in turn produce IL-10 and IFN-γ. The host genetic base is essential in the development of anti-Anisakis immune responses (Th2, Th1, Treg).

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Comparação da qualidade seminal de caprinos das raças Canindé e Alpina Britânica no Nordeste brasileiro / Comparison of the seminal quality of Canindé and British Alpine goats in the Brazilian Northeast

O estudo objetivou avaliar a qualidade seminal de caprinos das raças Canindé (autóctone) e Alpina Britânica (exótica) no Nordeste brasileiro. O experimento foi realizado nos períodos de julho a setembro dos anos de 2015 e 2016. As coletas seminais foram realizadas com auxílio de vagina artificial, de machos das raças Canindé (n = 4) e Alpina Britânica (n = 7). Após a coleta, o sêmen foi avaliado quanto a: volume (ml), concentração (sptz/ml), motilidade (%) e vigor (1-5). Em seguida, diluído em ACP-101c e criopreservado em máquina TK3000TM. Posteriormente, foram analisados os parâmetros cinéticos, através do software SCA®, e a atividade mitocondrial. No sêmen fresco, as duas raças apresentaram valores de motilidade e concentração dentro do preconizado para a espécie. Entretanto, os valores de volume e circunferência escrotal foram superiores na raça Alpina Britânica (0,73 ml ± 0,32; 27,35 cm ± 2,09) do que na raça Canindé (0,36 ml ± 0,07; 23,25 cm ± 0,95) (p < 0,05). Já no sêmen descongelado, os parâmetros motilidade total - MT (36,96% ± 11,16 vs. 20,50% ± 14,15), motilidade progressiva - MP (26,67% ± 11,13 vs. 11,29% ± 9,42), vigor (2,91 ± 0,90 vs. 1,88 ± 0,67), velocidade curvilinear - VCL (78,12 µm/s ± 12,41 vs. 59,28 µm/s ± 15,57), velocidade linear - VSL (49,23 µm/s ± 9,87 vs. 29,9 µm/s ± 9,48), velocidade média da trajetória - VAP (66,08 µm/s ± 12,77 vs. 45,70 µm/s ± 12,20), linearidade - LIN (62,97% ± 6,96 vs. 49,54%±9,50), retilinearidade - STR (74,63% ± 6,44 vs. 65,77% ± 8,92), e oscilação - WOB (84,32% ± 5,94 vs. 74,42% ± 7,31) foram superiores para a raça Canindé em detrimento à Alpina Britânica (p < 0,05). Portanto, o sêmen fresco das duas raças podem ser utilizados em biotécnicas reprodutivas. Já o sêmen pós-descongelação da raça Canindé apresentou melhor qualidade, provavelmente pelos animais estarem mais adaptados às condições adversas da região Nordeste, sendo recomendado para programas de inseminação artificial.(AU)

The objective of this study was to evaluate the sperm quality of Canindé (native) and British Alpine (exotic) goats in the Northeast of Brazil. The experiment was carried out from July to September of the years 2015 and 2016. Sperm collections of Canindé (n = 4) and British Alpine (n = 7) males were performed using artificial vagina. After collection, the sperm was evaluated for volume (ml), concentration (sptz/ml), motility (%), and vigor (1-5). Then diluted in ACP-101c and cryopreserved in TK3000TM machine. Subsequently, kinetic parameters were analyzed through SCA TM software and mitochondrial activity. In fresh sperm, the two races presented values of motility and concentration within the recommended for the specie. However, volume and scrotal circumference values were higher in the British Alpine breed (0.73 mL ± 0.32; 27.35 cm ± 2.09) than in the Canindé breed (0.36 mL ± 0.07; 23 , 25 cm ± 0.95) (p <0.05). In the thawed sperm, the parameters total motility - TM (36.96% ± 11.16 vs. 20.50% ± 14.15), progressive motility - PM (26.67% ± 11.13 vs. 11.29 % ± 9.42), vigor (2.91 ± 0.90 vs. 1.88 ± 0.67), curvilinear velocity - VCL (78.12 µm/s ± 12.41 vs. 59.28 µm/s ± 15.57), linear velocity - VSL (49.23 µm/s ± 9.87 vs. 29.9 µm/s ± 9.48), mean velocity of the trajectory - VAP (66.08 µm/s ± 12.77 vs. 45.70 µm/s ± 12.20), linearity - LIN (62.97% ± 6.96 vs. 49.54% ± 9.50), rectilinearity - STR (74.63% ± 6.44 vs. 65.77% ± 8.92), and oscillation -WOB (84.32% ± 5.94 vs. 74.42% ± 7.31) were higher for Canindé breed than for British Alpine ( p < 0.05). Therefore, fresh sperm from both breeds can be used in reproductive biotechniques. On the other hand, the post-thawed sperm of the Canindé breed showed better quality, probably because the animals were more adapted to the adverse conditions of the Northeast region and are recommended for artificial insemination programs.(AU)

Bacillus sp. SAB E-41-derived extract shows antiaging properties via ctt1-mediated oxidative stress tolerance response in yeast Schizosaccharomyces pombe

Objective: To analyze potential activation of oxidative stress tolerance systems by SAB E-41 bacterial extract in promoting the life span of yeast Schizosaccharomyces pombe. Methods: In vitro analysis was done to assess antioxidant activity of SAB E-41 bacterial extract. Antiaging property of the particular extract was then assayed through spot test and chronological life span assays. Furthermore, sty1 mitogen-activated protein kinase, pap1 transcriptional factor of oxidative stress response and its downstream genes, ctt1 were evaluated via real time PCR. The protein level of ctt1 was then observed via Western Blot analysis. In addition, accumulation of reactive oxygen species and mitochondrial activity were conducted to understand the effect of SAB E-41 upon oxidative stress response systems in vivo. Results: The IC

Bacillus sp. SAB E-41-derived extract shows antiaging properties via ctt1-mediated oxidative stress tolerance response in yeast Schizosaccharomyces pombe

Objective: To analyze potential activation of oxidative stress tolerance systems by SAB E-41 bacterial extract in promoting the life span of yeast Schizosaccharomyces pombe. Methods: In vitro analysis was done to assess antioxidant activity of SAB E-41 bacterial extract. Antiaging property of the particular extract was then assayed through spot test and chronological life span assays. Furthermore, sty1 mitogen-activated protein kinase, pap1 transcriptional factor of oxidative stress response and its downstream genes, ctt1 were evaluated via real time PCR. The protein level of ctt1 was then observed via Western Blot analysis. In addition, accumulation of reactive oxygen species and mitochondrial activity were conducted to understand the effect of SAB E-41 upon oxidative stress response systems in vivo. Results: The IC50 values of corresponding extract for antioxidant (DPPH; ABTS) and antiglycation were 402.40, 358.13 and 683.55 μg/mL, respectively. In addition, SAB E-41 extract (750 μg/mL) exhibited antiaging properties, which could be attributed to significant up-regulation of oxidative stress response genes, sty1, pap1 and ctt1. Interestingly, SAB E-41 extract could enhance stress tolerance phenotype of Schizosaccharomyces pombe against H2O2-induced oxidative stress. These results were supported by increasing mitochondrial activity and reactive oxygen species intracellular levels. Conclusions: SAB E-41 extract could promote yeast life span likely via up-regulation of oxidative stress responses in yeast. Our results suggest that adaptive response via up-regulation of oxidative stress transcriptional factors, and its downstream gene, ctt1, as well as mitochondrial activity contributes in combating oxidative stress thus promoting yeast life span.

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation / Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.(AU)

O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de congelação-descongelação, a fim de melhorar o protocolo.(AU)

Effect of resveratrol on mitochondrial activity in differentiated mature adipocytes / Efecto del resveratrol sobre la actividad mitocondrial en adipocitos maduros diferenciados

Obesity has reached epidemic proportions, the World Health Organization (WHO) estimates that there are more than 1,000 million overweight adults world-wide. Furthermore, obesity is characterized as an overgrowth of white adipose tissue as a result of adipocyte hypertrophy and hyperplasia. Mitochondria is considered the source of energy within the adipocyte, since it contains the molecular machinery, and it is involved in a large number of metabolic pathways, besides the transformation of chemical energy into adenosine triphosphate. Mitochondria shortage and adipocyte dysfunction result in an excessive accumulation of triacylglycerol in the cytoplasm, which determines an imbalance between energy production and energy expenditure. Resveratrol (RSV) is a polyphenol found in different plants and its effects have been associated with mitochondrial biogenesis. An adipogenesis in vitro model (3T3-L1 preadipocytes) was used, and these cells were differentiated into mature adipocytes. Subsequently the effect of RSV on the adipocytes morphology, the lipid content and mitochondrial activity was evaluated using microscopic and flow cytometry techniques. The effect of RSV on differentiated mature adipocytes, was characterized by the decrease in lipid content and the consequently declination of the mitochondrial activity. 3T3-L1 preadipocytes retained the differentiation ability until passage 18. The RSV at doses of 25 and 50 µM for 48 hours in differentiated mature adipocytes promoted the decreased in lipid content probably due to an increase in mitochondrial activity in the early hours of RSV exposure, causing the consequently declination of mitochondrial activity at the end of 48 hours.

La obesidad ha tomado dimensiones epidémicas globales y la Organización Mundial de la Salud estima que hay más de 1,000 millones de adultos con sobrepeso. Así mismo, la obesidad se ha caracterizado como la expansión del tejido adiposo blanco condicionada por la hipertrofia e/o hiperplasia de los adipocitos. La mitocondria es considerada la fuente de energía dentro del adipocito, debido a que contiene la maquinaria molecular que dirige, a través de diversas vías metabólicas, la transformación de la energía química en adenosíntrifosfato. La escasez de mitocondrias así como su disfunción en el adipocito, resulta en una acumulación excesiva de triacilgliceroles en el citoplasma, lo que condiciona un desequilibrio entre producción de energía y gasto energético. El resveratrol (RSV) es un polifenol que se encuentra en diferentes grupos de plantas y sus efectos se han asociado con la inducción de genes para la biogénesis mitocondrial. Se empleó un modelo de adipogénesis (in vitro) materializado por una línea celular de preadipocitos 3T3-L1, mismos que se diferenciaron a adipocitos maduros. Posteriormente se evaluó el efecto del RSV sobre la morfología, contenido lipídico y actividad mitocondrial en los adipocitos maduros diferenciados a través de las técnicas: microscopía invertida, confocal y citometría de flujo. El efecto del RSV sobre los adipocitos maduros diferenciados, se caracterizó por la disminución del contenido lipídico y consecuentemente de la actividad mitocondrial. Los preadipocitos 3T3-L1 conservaron la capacidad de diferenciación hasta el pase 18. Por otra parte, el resveratrol a dosis de 25 y 50 µM durante 48 horas en adipocitos maduros diferenciados, promueve una disminución en el contenido lipídico probablemente debido a un aumento de la actividad mitocondrial en las primeras horas de exposición al tratamiento, provocando la disminución de la actividad mitocondrial al término de 48 horas.

The beneficial effect of long-term supplementation of vitamin C on renal mitochondrial disturbances in streptozotocin-induced diabetic rats.

Background: Oxidative stress induces renal dysfunction in diabetes, in which renal mitochondrial disturbance was implicated. Vitamin C (VC) supplementation may ameliorate the renal dysfunction in diabetics. However, it is not clear whether VC supplementation is effective for renal mitochondrial disturbances in diabetes. Objective: Investigate whether long-term continuous VC supplementation could ameliorate the renal mitochondrial disturbances in streptozotocin (STZ)-induced diabetic rats. Methods: Thirty-five male Sprague-Dawley rats were used, and diabetes was induced by an injection of STZ. The rats were divided into three groups: control rats (CON), STZ-induced diabetic rats (STZ), and diabetic rats supplemented by vitamin C (STZ-VC). The CON and STZ rats were given tap water, while STZ-VC rats received VC (1 g/L) every day for eight, 24 and 52 weeks. The kidney was isolated and homogenized. Oxygen comsumption (Vo2) was measured in mitochondria homogenate using an oxygen consumption monitor. Based on Vo2 tracings, the respiration control index (RCI) and P/O ratio (= ADP/ O ratio) were measured at week 8, 24 and 52. Results: At week eight, using either glutamate plus malate (for site I) or succinate (for site II) as substrates, both RCI and P/O ratio were not significantly different among three groups. The P/O ratio in STZ and STZ-VC rats increased from eight to 52 weeks after VC supplementation. At week 24, the P/O ratio at site II was normalized in STZ-VC rat. The increased P/O ratio (only site I) and the increased RCI (only site II) of STZ-VC rats were slower than those of STZ rats. Conclusion: Short-term VC supplementation might not influence the renal mitochondrial activity. The long-term VC supplementation could ameliorate the mitochondrial disturbances induced in STZ-induced diabetic rats.

Protective effect of GSTT on myocardium injuried by ischemia reperfusion / 中国药理学通报

AIM To investigate the protective effect of GSTT on myocardial injury induced by ischemia reperfusion. METHODS The content of lactic dehydrogenase(LDH),creatine kinase(CK),methylenedioxyamphetamine(MDA),superoxide dismutase(SOD),tumor necrosis factor alpha(TNF ?) and interleukine 6(IL 6) were determined in medium after reperfusion by simulating model of ischemia reperfusion; The content of nitric oxide(NO) was determined in medium by TNF ? injury myocardium,and mitochondrial activity was determined by MTT after TNF ? stimulated. RESULTS Compared with the model control,in the GSTT, the content of LDH,CK,MDA,TNF ? and IL 6 in medium decreased and activity of SOD increased after reperfusoin,the level of NO in medium decreased and OD 490 increased after TNF ? injuried. CONCLUSION It is suggested that GSTT can mitigate injury of ischemia reperfusion on myocardium, and has a direct protective effect on injuried myocardium.