[Molecular detection of tomato yellow leaf curl virus (TYLCV)].

Tomato yellow leaf curl virus (TYLCV) is currently considered as one of the most devastating viruses in cultivated tomatoes (Solanum lycopersicum) worldwide. We reported here the development of a PCR-based method to quickly detect TYLCV using the primer pairs (TYLCV-F: 5'-ACG CAT GCC TCT AAT CCA GTG TA-3' and TYLCV-R: 5'-CCA ATA AGG CGT AAG CGT GTA GAC-3'), which was designed based on the genome sequence of TYLCV. A TYLCV-specific band of 543 bp was amplified from infected tomato plants. This protocol provides a rapid, reliable, and sensitive tool for molecular detection and identification of TYLCV in the industrial seedling and virus resistance breeding to facilitate safe and sustainable production of tomato.

Cholinergic drugs ameliorate endothelial dysfunction by decreasing O-GlcNAcylation via M3 AChR-AMPK-ER stress signaling.

AIMS: Obesity is associated with increased cardiovascular morbidity and mortality. It is accompanied by augmented O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of proteins via increasing hexosamine biosynthetic pathway (HBP) flux. However, the changes and regulation of the O-GlcNAc levels induced by obesity are unclear. MAIN METHODS: High fat diet (HFD) model was induced obesity in mice with or without the cholinergic drug pyridostigmine (PYR, 3 mg/kg/d) for 22 weeks and in vitro human umbilical vein endothelial cells (HUVECs) was treated with high glucose (HG, 30 mM) with or without acetylcholine (ACh). KEY FINDINGS: PYR significantly reduced body weight, blood glucose, and O-GlcNAcylation levels and attenuated vascular endothelial cells detachment in HFD-fed mice. HG addition induced endoplasmic reticulum (ER) stress and increased O-GlcNAcylation levels and apoptosis in HUVECs in a time-dependent manner. Additionally, HG decreased levels of phosphorylated AMP-activated protein kinase (AMPK). Interestingly, ACh significantly blocked damage to HUVECs induced by HG. Furthermore, the effects of ACh on HG-induced ER stress, O-GlcNAcylation, and apoptosis were prevented by treating HUVECs with 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, a selective M3 AChR antagonist) or compound C (Comp C, an AMPK inhibitor). Treatment with 5-aminoimidazole-4-carboxamide ribose (AICAR, an AMPK activator), 4-phenyl butyric acid (4-PBA, an ER stress inhibitor), and 6-diazo-5-oxonorleucine (DON, a GFAT antagonist) reproduced a similar effect with ACh. SIGNIFICANCE: Activation of cholinergic signaling ameliorated endothelium damage, reduced levels of ER stress, O-GlcNAcylation, and apoptosis in mice and HUVECs under obese conditions, which may function through M3 AChR-AMPK signaling.

Regulation of mitochondrial cristae remodelling by acetylcholine alleviates palmitate-induced cardiomyocyte hypertrophy.

Mitochondrial dysfunction is associated with obesity-induced cardiac remodelling. Recent research suggests that the cristae are the true bioenergetic components of cells. Acetylcholine (ACh), the major neurotransmitter of the vagus nerve, exerts cardio-protective effects against ischaemia. This study investigated the role of cristae remodelling in palmitate (PA)-induced neonatal rat cardiomyocyte hypertrophy and explored the beneficial effects of ACh. We found loose, fragmented and even lysed cristae in PA-treated neonatal cardiomyocytes along with declines in mitochondrial network and complex expression and overproduction of mitochondrial reactive oxygen species (ROS); these changes ultimately resulted in increased myocardial size. Overexpression of mitofilin by adenoviral infection partly improved cristae shape, mitochondrial network, and ATP content and attenuated cell hypertrophy. Interestingly, siRNA-mediated AMP-activated protein kinase (AMPK) silencing increased the number of cristae with a balloon-like morphology without disturbing mitofilin expression. Furthermore, AMPK knockdown abolished the effects of mitofilin overexpression on cristae remodelling and inhibited the interaction of mitofilin with sorting and assembly machinery 50 (Sam50) and coiled-coil helix coiled-coil helix domain-containing protein 3 (CHCHD3), two core components of the mitochondrial contact site and cristae organizing system (MICOS) complex. Intriguingly, ACh upregulated mitofilin expression and AMPK phosphorylation via the muscarinic ACh receptor (MAChR). Moreover, ACh enhanced protein-protein interactions between mitofilin and other components of the MICOS complex, thereby preventing PA-induced mitochondrial dysfunction and cardiomyocyte hypertrophy; however, these effects were abolished by AMPK silencing. Taken together, our data suggest that ACh improves cristae remodelling to defend against PA-induced myocardial hypertrophy, presumably by increasing mitofilin expression and activating AMPK to form the MICOS complex through MAChR. These results suggest new and promising therapeutic approaches targeting mitochondria to prevent lipotoxic cardiomyopathy.

Pyridostigmine alleviates cardiac dysfunction via improving mitochondrial cristae shape in a mouse model of metabolic syndrome.

Insulin resistance and autonomic imbalance are important pathological processes in metabolic syndrome-induced cardiac remodeling. Recent studies determined that disruption of mitochondrial cristae shape is associated with myocardial ischemia; however, the change in cristae shape in metabolic syndrome-induced cardiac remodeling remains unclear. This study determined the effect of pyridostigmine (PYR), which reversibly inhibits cholinesterase to improve autonomic imbalance, on high-fat diet (HFD)-induced cardiac insulin resistance and explored the potential effect on the shape of mitochondrial cristae. Feeding of a HFD for 22 weeks led to an irregular and even lysed cristae structure in cardiac mitochondria, which contributed to decreased mitochondrial content and ATP production and increased oxygen species production, ultimately impairing insulin signaling and lipid metabolism. Interestingly, PYR enhanced vagal activity by increasing acetylcholine production and exerted mito-protective effects by activating the LKB1/AMPK/ACC signal pathway. Specifically, PYR upregulated OPA1 and Mfn1/2 expression, promoted the formation of the mitofilin/CHCHD3/Sam50 complex, and decreased p-Drp1 and Fis1 expression, resulting in tight and parallel cristae and increasing cardiac mitochondrial complex subunit expression and ATP generation as well as decreasing release of cytochrome C from mitochondria and oxidative damage. Furthermore, PYR improved glucose and insulin tolerance and insulin-stimulated Akt phosphorylation, decreased lipid toxicity, and ultimately ameliorated HFD-induced cardiac remodeling and dysfunction. In conclusion, PYR prevented cardiac and insulin insensitivity and remodeling by stimulating vagal activity to regulate mitochondrial cristae shape and function in HFD-induced metabolic syndrome in mice. These results provide novel insights for the development of a therapeutic strategy for obesity-induced cardiac dysfunction that targets mitochondrial cristae.

Choline ameliorates cardiac hypertrophy by regulating metabolic remodelling and UPRmt through SIRT3-AMPK pathway.

AIMS: Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization, but the molecular events underlying the metabolic remodelling remain poorly understood. We explored metabolic remodelling and mitochondrial dysfunction in cardiac hypertrophy and investigated the cardioprotective effects of choline. METHODS AND RESULTS: The experiments were conducted using a model of ventricular hypertrophy by partially banding the abdominal aorta of Sprague Dawley rats. Cardiomyocyte size and cardiac fibrosis were significantly increased in hypertrophic hearts. In vitro cardiomyocyte hypertrophy was induced by exposing neonatal rat cardiomyocytes to angiotensin II (Ang II) (10-6 M, 24 h). Choline attenuated the mito-nuclear protein imbalance and activated the mitochondrial-unfolded protein response (UPRmt) in the heart, thereby preserving the ultrastructure and function of mitochondria in the context of cardiac hypertrophy. Moreover, choline inhibited myocardial metabolic dysfunction by promoting the expression of proteins involved in ketone body and fatty acid metabolism in response to pressure overload, accompanied by the activation of sirtuin 3/AMP-activated protein kinase (SIRT3-AMPK) signalling. In vitro analyses demonstrated that SIRT3 siRNA diminished choline-mediated activation of ketone body metabolism and UPRmt, as well as inhibition of hypertrophic signals. Intriguingly, serum from choline-treated abdominal aorta banding models (where ß-hydroxybutyrate was increased) attenuated Ang II-induced myocyte hypertrophy, which indicates that ß-hydroxybutyrate is important for the cardioprotective effects of choline. CONCLUSION: Choline attenuated cardiac dysfunction by modulating the expression of proteins involved in ketone body and fatty acid metabolism, and induction of UPRmt; this was likely mediated by activation of the SIRT3-AMPK pathway. Taken together, these results identify SIRT3-AMPK as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to cardiac stress. Choline treatment may represent a new therapeutic strategy for optimizing myocardial metabolism in the context of hypertrophy and heart failure.

[Effects of ligustrazine on the Ca2+ sensitivity in carotid artery smooth muscle of simulated weightlessness rats].

OBJECTIVE: To study the effects of Ligustrazine on the Ca2+ sensitivity in carotid artery smooth muscle of simulated weightless-ness rats. METHODS: Twenty-one female SD rats were divided into control group (CON), simulated weightlessness group (TS), simulated weightlessness plus Ligustrazine administration group (LTZ) (n=7). After 4 weeks, the contractile property and the Ca2+ sensitivity in carotid artery were measured. Furthermore, the effects of ML-7(a myosin light chain kinase specific inhibitor) on above mentioned parameters were recorded. RESULTS: Compared with CON group, the contractile force induced by phenylephrine (PHE) in TS group was increased by 25.14% (P<0.01). The force in LTZ group decreased by 13.46% (P<0.01) and increased by 8.30% compared with TS and CON group, respec-tively. After the artery rings were incubated with ML-7, the force in CON, TS and LTZ group were reduced by 8.84%, 16.24% (P<0.01) and 3.40%, respectively. Compared with CON group, the contractile force induced by KCl in TS group was increased by 40.46% (P<0.01). The force in LTZ group decreased by 18.80% and increased by 14.05% compared with TS and CON group, respectively. The force in CON, TS and LTZ group were reduced by 21.97% (P<0.05), 21.88% (P<0.01) and 10.84% by ML-7, respectively. Compared with CON group, the pD2[Ca2+] in TS group was increased by 10.03% (P<0.01). The pD2[Ca2+] in LTZ group was decreased by 7.01% (P<0.01) and increased by 2.32% compared with TS and CON group, respectively. The pD2[Ca2+] in CON, TS and LTZ group was reduced by 2.42%, 7.43% (P<0.01) and 2.51% by ML-7, respectively. CONCLUSIONS: The contraction of carotid artery is strength-ened in simulated weightlessness rats, it maybe results from the Ca2+ sensitivity of contractile proteins in smooth muscle increasing. Ligus-trazine can improve the contraction of carotid artery, reducing the Ca2+ sensitivity and inhibiting myosin light chain kinase may be involved in its mechanisms.

[Correlation between the activation of AP-1 signal transduction pathway and metastasis of colorectal carcinoma].

OBJECTIVE: To investigate the binding activity of activator protein-1 (AP-1) with DNA probe in the colorectal carcinoma (CRC) tissues and surrounding tissues and explore the correlation between the activation of AP-1 signal transduction pathway and metastasis of CRC. METHODS: The AP-1 DNA binding activities were investigated by electrophoretic mobility shift assay (EMSA) in CRC specimens (T), surrounding tissues including 2 cm (P(2)), 5 cm(P(5)) far away from primary tumor margin and distal resection margin of the specimens (N). The mRNA expression level of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9) were measured by quantitive reverse transcription polymerase chain reaction (Q- RT-PCR). RESULTS: The AP-1 DNA binding activity in T was significantly higher than those in P(2), P(5) and N (P< 0.05) tissues. There were significantly positive correlations between AP-1 DNA binding activity in tumor and invasive degree, lymphatic metastasis respectively (P< 0.01), but no correlation with histological classification and differentiation (P> 0.05). The transcription levels of VEGF and MMP-9 in CRC were significantly higher than those in P(5) and N (P< 0.01, P< 0.05) tissues. The transcription levels of VEGF and MMP-9 were significantly correlated with increasing AP-1 DNA binding activity (P< 0.01). CONCLUSIONS: AP-1 is significantly correlated to the invasion and metastasis in CRC. The activation of AP-1 signal transduction pathway might be involved in the angiogenesis and of degradation extracellular matrix during tumor metastasis.