Identification and expression analysis of citrate synthase 3 gene family members in apple / 生物工程学报

As one of the key enzymes in cell metabolism, the activity of citrate synthase 3 (CS3) regulates the substance and energy metabolism of organisms. The protein members of CS3 family were identified from the whole genome of apple, and bioinformatics analysis was performed and expression patterns were analyzed to provide a theoretical basis for studying the potential function of CS3 gene in apple. BLASTp was used to identify members of the apple CS3 family based on the GDR database, and the basic information of CS3 protein sequence, subcellular localization, domain composition, phylogenetic relationship and chromosome localization were analyzed by Pfam, SMART, MEGA5.0, clustalx.exe, ExPASy Proteomics Server, MEGAX, SOPMA, MEME, WoLF PSORT and other software. The tissue expression and inducible expression characteristics of 6 CS3 genes in apple were determined by acid content and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Apple CS3 gene family contains 6 members, and these CS3 proteins contain 473-608 amino acid residues, with isoelectric point distribution between 7.21 and 8.82. Subcellular localization results showed that CS3 protein was located in mitochondria and chloroplasts, respectively. Phylogenetic analysis divided them into 3 categories, and the number of genes in each subfamily was 2. Chromosome localization analysis showed that CS3 gene was distributed on different chromosomes of apple. The secondary structure of protein is mainly α-helix, followed by random curling, and the proportion of β-angle is the smallest. The 6 members were all expressed in different apple tissues. The overall expression trend from high to low was the highest relative expression content of MdCS3.4, followed by MdCS3.6, and the relative expression level of other members was in the order of MdCS3.3 > MdCS3.2 > MdCS3.1 > MdCS3.5. qRT-PCR results showed that MdCS3.1 and MdCS3.3 genes had the highest relative expression in the pulp of 'Chengji No. 1' with low acid content, and MdCS3.2 and MdCS3.3 genes in the pulp of 'Asda' with higher acid content had the highest relative expression. Therefore, in this study, the relative expression of CS3 gene in apple cultivars with different acid content in different apple varieties was detected, and its role in apple fruit acid synthesis was analyzed. The experimental results showed that the relative expression of CS3 gene in different apple varieties was different, which provided a reference for the subsequent study of the quality formation mechanism of apple.

Pathologically Elevated-Cyclic Stretch Suppressed Vascular Smooth Muscle Cell Mitochondrial Biogenesis by Down-regulating PGC1α Expression / 医用生物力学

Objective To investigate the effect of pathologically elevated-cyclic stretch induced by hypertension on mitochondrial biogenesis of vascular smooth muscle cells (VSMCs), and the role of PGC1α in this process. Methods The Flexcell-5000T stretch loading system in vitro was applied to VSMCs with a frequency of 1. 25 Hz and an amplitude of 5% or 15% to simulate the mechanical environment under normal physiological or hypertensive pathological conditions respectively. Western blotting and qPCR were used to detect the expression of PGC1α, citrate synthase and mitochondrial DNA (mtDNA) copy number in VSMCs under normal physiological or hypertensive pathological conditions. VSMCs were treated with PGC1α specific activator ZLN005 to promote PGC1α expression or specific interfering fragment siRNA to inhibit PGC1α expression in order to detect the effect on citrate synthase and mtDNA copy number. Results Compared with 5% physiological cyclic stretch, 15% pathologically elevated-cyclic stretch significantly suppressed the expression of PGC1α, citrate synthase and mtDNA copy number in VSMCs. Compared with control group, the protein expression of PGC1α was significantly decreased and increased respectively. When VSMCs transfected with PGC1α siRNA or incubated PGC1α activator ZLN005, the expression of citrate synthase and mtDNA copy number were also significantly down regulated and up-regulated in VSMCs accordingly. Under physiological cyclic stretch conditions, the protein level of PGC1α was significantly down-regulated by PGC1α siRNA, which also significantly down-regulated citrate synthase expression and mtDNA copy number. The protein expression of PGC1α was significantly up-regulated by ZLN005, which also enhanced the expression of citrate synthase and mtDNA copy number. Conclusions The pathological cyclic stretch induced by hypertension significantly down-regulated the expression of citrate synthase and mtDNA copy number via suppressing the expression of PGC1α, resulting in mitochondrial dysfunction of VSMCs. PGC1α may be a potential therapeutic target molecule to alleviate the progression of hypertension.

Bartonella henselae infection in diverse clinical conditions in a tertiary care hospital in north India

Background & objectives: Bartonella henselae causes infections which closely resemble febrile illness and chronic diseases such as tuberculosis and haematological malignancies. There are not many studies on Bartonella infections from India. The present study was undertaken to diagnose B. henselae infection in diverse clinical conditions in a tertiary care hospital in north India. Methods: A total of 145 patients including those with fever and lymphadenopathy, infective endocarditis and neuroretinitis were enrolled in the study. Whole blood, serum and lymph node aspirate and valvular vegetations if available, were obtained. Samples were plated on chocolate agar and brain-heart infusion agar containing five per cent fresh rabbit blood and were incubated at 35°C for at least four weeks in five per cent CO2with high humidity. Immunofluorescent antibody assay (IFA) was done for the detection of IgM antibodies in the serum using a commercial kit. Whole blood was used to perform polymerase chain reaction (PCR) for the citrate synthase gene (gltA). Results: IFA was positive in 11 of 140 (7.85%) patients and PCR was positive in 3 of 140 (2.14%) patients. Culture was negative in all the cases. A higher incidence of Bartonella infection was seen in patients with fever and lymphadenopathy (n=30), seven of whom were children. In ophthalmological conditions, four cases were IFA positive. Interpretation & conclusions: The present study shows that the threat of Bartonella infection is a reality in India. It is also an important treatable cause of fever and lymphadenopathy in children. Serology and PCR are useful tests for its diagnosis. Clinicians should consider Bartonella infection in the differential diagnosis of febrile illnesses and chronic diseases.

Respiratory muscle training improves diaphragm citrate synthase activity and hemodynamic function in rats with heart failure

Abstract INTRODUCTION: Enhanced respiratory muscle strength in patients with heart failure positively alters the clinical trajectory of heart failure. In an experimental model, respiratory muscle training in rats with heart failure has been shown to improve cardiopulmonary function through mechanisms yet to be entirely elucidated. OBJECTIVE: The present report aimed to evaluate the respiratory muscle training effects in diaphragm citrate synthase activity and hemodynamic function in rats with heart failure. METHODS: Wistar rats were divided into four experimental groups: sedentary sham (Sed-Sham, n=8), trained sham (RMT-Sham, n=8), sedentary heart failure (Sed-HF, n=7) and trained heart failure (RMT-HF, n=7). The animals were submitted to a RMT protocol performed 30 minutes a day, 5 days/week, for 6 weeks. RESULTS: In rats with heart failure, respiratory muscle training decreased pulmonary congestion and right ventricular hypertrophy. Deleterious alterations in left ventricular pressures, as well as left ventricular contractility and relaxation, were assuaged by respiratory muscle training in heart failure rats. Citrate synthase activity, which was significantly reduced in heart failure rats, was preserved by respiratory muscle training. Additionally, a negative correlation was found between citrate synthase and left ventricular end diastolic pressure and positive correlation was found between citrate synthase and left ventricular systolic pressure. CONCLUSION: Respiratory muscle training produces beneficial adaptations in the diaphragmatic musculature, which is linked to improvements in left ventricular hemodynamics and blood pressure in heart failure rats. The RMT-induced improvements in cardiac architecture and the oxidative capacity of the diaphragm may improve the clinical trajectory of patients with heart failure.

Brain Region Specificity of Mitochondrial Biogenesis and Bioenergetics Response to Nrf2 Knockdown: A Comparison Among Hippocampus, Prefrontal Cortex and Amygdala of Male Rat Brain

ABSTRACT Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been identified as the well-known coordinator of intracellular antioxidant defense system. Herein, we aimed to evaluate the effects of Nrf2 silencing on mitochondrial biogenesis markers peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM) and cytochrome c as well activities of two enzymes citrate synthase (CS) and malate dehydrogenase (MDH) in three brain regions hippocampus, amygdala, and prefrontal cortex of male Wistar rats. Small interfering RNA (siRNA) targeting Nrf2 was injected in dorsal third ventricle. Next, western blot analysis and biochemical assays were used to evaluation of protein level of mitochondrial biogenesis factors and CS and MDH enzymes activity, respectively. Based on findings, whilst Nrf2-silencing led to notably reduction in protein level of mitochondrial biogenesis upstream PGC-1α in three brain regions compared to the control rats, the level of NRF-1, TFAM and cytochrome c remained unchanged. Furthermore, although Nrf2 silencing increased CS activity, activity of MDH significantly decreased in hippocampus and prefrontal cortex areas. Interestingly, CS and MDH activities in amygdala did not change after Nrf2 knockdown. In conclusion, the present findings highlighted complexity of interaction of Nrf2 and mitochondrial functions in a brain region-specific manner. However, by outlining the exact interaction between Nrf2 and mitochondria, it would be possible to find a new therapeutic strategies for neurological disorders related to oxidative stress.

Enhancement of Exercise Capacity by Black Ginseng Extract in Rats / 한국실험동물학회지

This study was carried out to investigate an enhancing effect of black ginseng extract (BGE) on exercise capacity in an endurance exercising animal model. Fifty Sprague-Dawley rats were assigned to 5 experimental groups including non-training control, training control, and 3 treated groups (BGE at doses of 75, 150 and 300 mg/kg). The animals were treated with BGE for 6 weeks and their exercise ability in the maximal running distance test was determined using a treadmill every week. The blood lactic acid (LA) level and the activity of citrate synthase (CS) in the muscle were also measured after the exercise. The levels of glucose and glucose-6-phosphate (G-6-P) in the liver and muscle were determined using commercial assay kits. BGE treatments at the doses of 150 and 300 mg/kg significantly increased the exercise capacity compared with the non-training control or training control groups (P<0.05). The level of blood LA was decreased but the activity of CS was increased by the treatment of BGE at the dose of 300 mg/kg compared with the training control group. The level of G-6-P in the liver was elevated by the treatment of BGE at the dose of 300 mg/kg, compared to the training group. As compared with non-training control group, the treatments of BGE increased the levels of glucose and G-6-P in the liver and soleus muscle of rats. These results indicate that BGE have a potential for promoting exercise capacity by increasing CS activity in the muscle and decreasing LA in the serum of rats. These results also suggested that BGE can be used as a candidate supplement of health food products for promoting endurance exercise capacity in human athletes.

Enhancement of Exercise Capacity by Black Ginseng Extract in Rats / 한국실험동물학회지

This study was carried out to investigate an enhancing effect of black ginseng extract (BGE) on exercise capacity in an endurance exercising animal model. Fifty Sprague-Dawley rats were assigned to 5 experimental groups including non-training control, training control, and 3 treated groups (BGE at doses of 75, 150 and 300 mg/kg). The animals were treated with BGE for 6 weeks and their exercise ability in the maximal running distance test was determined using a treadmill every week. The blood lactic acid (LA) level and the activity of citrate synthase (CS) in the muscle were also measured after the exercise. The levels of glucose and glucose-6-phosphate (G-6-P) in the liver and muscle were determined using commercial assay kits. BGE treatments at the doses of 150 and 300 mg/kg significantly increased the exercise capacity compared with the non-training control or training control groups (P<0.05). The level of blood LA was decreased but the activity of CS was increased by the treatment of BGE at the dose of 300 mg/kg compared with the training control group. The level of G-6-P in the liver was elevated by the treatment of BGE at the dose of 300 mg/kg, compared to the training group. As compared with non-training control group, the treatments of BGE increased the levels of glucose and G-6-P in the liver and soleus muscle of rats. These results indicate that BGE have a potential for promoting exercise capacity by increasing CS activity in the muscle and decreasing LA in the serum of rats. These results also suggested that BGE can be used as a candidate supplement of health food products for promoting endurance exercise capacity in human athletes.

Adaptations of myoglobin in rat skeletal muscles to endurance running training. Effects of intensity, duration and period of training / 体力科学

A study was conducted to clarify the effects of running intensity and duration of endurance training on myoglobin concentration ( [Mb] ) in rat skeletal muscles, and to clarify its temporal changes during the training. One hundred five male Wistar rats were divided into a training group and an untrained group. The training was carried out at 5 times a week for 12 weeks when the animals were 4 to 16 weeks of age. The training intensities were set at 20, 30 and 40 m/min with a duration of 60 min. The training duration was varied to 30, 60, 90 and 120 min when the rats were trained at 30 m/min. The temporal changes in the [Mb] were examined after the first, third and ninth week of training, during which the rats were trained at 40 m/min for 60 min per session. Three muscles (soleus: Sol, plantaris: P1, gastrocnemius-surface/deep: Gas-S, Gas-D) were analyzed for the [Mb] and citrate synthase activity (CS activity) . With regard to the intensity of training, the [Mb] increased with exercise intensity in Sol, Gas-D and P1, but not in Gas-S. P1 showed a greater increase of the [Mb] than Sol or Gas-D. On the other hand, CS activity in red muscle (Sol and Gas-D) increased even at low intensity, whereas white muscle (fast-twitch muscle: Pl and Gas-S) showed a significant increase in CS activity at an intensity of 40m/min. As to the duration of training, the [Mb] increased with the duration of running at 30 m/min of intensity, and showed the maximal adaptation with 90-min duration in all muscles except for Gas-S. Changes in CS activity according to the duration of running were similar to those for the [Mb] in all muscles. Finally, the [Mb] increased significantly with prolongation of the training period (after the 1 st, 3 rd and 9 th weeks training) in all muscles except Gas-S. However, the adaptive response of Mb tended to be delayed as compared with CS activity. These results suggest that <I>1) </I>the response of Mb to training stimuli can depend on the muscle specificity (fiber type composition or the initial [Mb] ), and level of motor unit recruitment in usual, <I>2) </I> Mb synthesis can be enhanced by an increase of training intensity, <I>3) </I> a training duration of 90 min can bring out the Mb adaptation maximally and <I>4) </I> the adaptive response of Mb would need more time as compared with CS activity.

Lipoic acid and diabetes—Part III: Metabolic role of acetyl dihydrolipoic acid.

Rat liver lipoyl transacetylase catalyzes the formation of acetyl dihydrolipoic acid from acetyl coenzyme A and dihydrolipoic acid. In an earlier paper the formation of acetyl dihydrolipoic from pyruvate and dihydrolipoic acid catalyzed by pyruvate dehydrogenase has been reported. Acetyl dihydrolipoic acid is a substrate for citrate synthase, acetyl coenzyme A carboxylase and fatty acid synthetase. The Vmax. for citrate synthase with acetyl dihydrolipoic acid was identical to acetyl coenzyme A (approximately 1 μmol citrate formed/min/mg protein) while the apparent Km was approximately 4 times higher with acetyl dihydrolipoic acid as the substrate. This may be due to the fact that synthetic acetyl dihydrolipoic acid is a mixture of 4 possible isomers and only one of them may be the substrate for the enzymatic reaction. While dihydrolipoic acid can replace coenzyme A in the activation of succinate catalyzed by succinyl coenzyme A synthetase, the transfer of coenzyme A between succinate and acetoacetyl dihydrolipoic acid catalyzed by succinyl coenzyme A: 3 oxo-acid coenzyme A transferase does not occur.