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Vascular smooth muscle cells(VSMCs)phenotype switching plays an essential role in the pathogenesis of various vascular diseases.The present study aims to investigate the role of receptor-interacting protein kinases 1(RIPK1)in VSMCs phenotypic switching induced by AngiotensinⅡ(AngⅡ).Expression of mRNA and protein of RIPK1,markers of VSMCs phenotypic switching and secretion,phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot.Meanwhile,EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively.At the same time,Necrostatin-1(Nec-1,an known RIPK1inhibitor)and RIPK1-specific small interference RNA(siRNA)were used to inhibit the expression of RIPK1.The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II.Moreover,the expression of RIPK1 and P65phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1or transfected with RIPK1-siRNA.Furthermore,the proliferation,secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1or its specific siRNA.The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II,which was possibly via up-regulating the NF-κB signaling pathway.

This study aims to investigate the role of calreticulin in(CRT)pressure overload induced cardiac hypertrophy.In our study,cardiac hypertrophy was induced by left ventricular pressure overload in male SD rats subjected to transverse aortic constriction(TAC)operation.Expression of gene and protein of calreticulin,markers of cardiac hypertrophy and endoplasmic reticulum stress(ERS)were measured with real-time qPCR and Western blot respectively.Meanwhile,atorvastatin(a known ERS inhibitor)and calreticulin-specific small interference ribonucleic acid(siRNA)were used to inhibit the expression of ERS and calreticulin respectively.The experimental data demonstrated that the gene and protein levels of calreticulin,hypertrophic and ERS markers were increased significantly in the heart tissues of TAC rat models after 4weeks.Moreover,atorvastatin administration improved the cardiac function and reduced the expression of calreticulin and ERS markers in TAC rats.In addition,cultured primary neonatal rat cardiomyocytes(NCMs)were treated with norepinephrine(NE),angiotensionⅡ(AngⅡ)or isoprenaline(ISO)to induce hypertrophic phenotype and ERS.The expression of hypertrophic markers was reduced in NCMs transfected with calreticulin-siRNA.The results suggested that calreticulin might be a promising target for the treatment of cardiac hypertrophy.

Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

L-Phenyllactic acid (L-PLA) is a novel antiseptic agent with broad and effective antimicrobial activity. In addition, L-PLA has been used for synthesis of poly(phenyllactic acid)s, which exhibits better mechanical properties than poly(lactic acid)s. However, the concentration and optical purity of L-PLA produced by native microbes was rather low. An NAD-dependent L-lactate dehydrogenase (L-nLDH) from Bacillus coagulans NL01 was confirmed to have a good ability to produce L-PLA from phenylpyruvic acid (PPA). In the present study, l-nLDH gene and formate dehydrogenase gene were heterologously coexpressed in Escherichia coli. Through two coupled reactions, 79.6mM l-PLA was produced from 82.8mM PPA in 40min and the enantiomeric excess value of L-PLA was high (>99%). Therefore, this process suggested a promising alternative for the production of chiral l-PLA., (Copyright © 2015. Published by Elsevier B.V.)

The aim of this study was to observe whether necroptosis is involved in the process of cardiac hypertrophy induced by pressure overload. SD rats underwent transverse abdominal aortic constriction (TAC) operation for establishing cardiac hypertrophy model. The structure and function of the left ventricle of rats were evaluated via echocardiography, left ventricular mass index, the expression of markers of cardiac hypertrophy and histological detection. Real-time PCR and Western blot were used to measure the gene and protein expression of receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3, the necroptosis markers) respectively. Four weeks after TAC operation, rat model for cardiac hypertrophy was established. The experimental data showed that the gene and protein expressions of RIPK1 and RIPK3 in the rat heart hypertrophic tissues after TAC for 4 weeks were increased significantly compared with those in the sham group. HE staining showed cardiomyocytes injury and hypertrophy in the hearts of TAC rat models. By transmission electron microscope, we observed that mitochondria of cardiomyocytes were damaged seriously in the TAC models. Treatment with losartan used, the selective antagonist of angiotensin II type I receptor could improve the cardiac function of TAC rats. Moreover, losartan treatment decreased the expression of RIPK1 and RIPK3 in heart tissues of TAC rats. The results suggest that necroptosis occurrs in the process of cardiac hypertrophy with pressure overload, and losartan could alleviate the cardiac hypertrophy and inhibit necroptosis.

The aim of this study is to construct specific shRNA expressing plasmids, and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation (H/R). RIPK1 and RIPK3 are the key kinases mediating the process of necroptosis. Using recombinant DNA technology, we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively. We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively, before we treated them with H/R stimulation. Then, we measured the relevant genes and proteins by real-time PCR and Western blot. Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury. The results showed that inhibition of ripk1 or ripk3 gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.

The aim of this study was to investigate whether the phosphatase and tensin homolog deleted on chromosome ten (PTEN) inhibitor dipotassium bisperoxo(pyridine-2-carboxyl) oxovanadate (BPV(pic)) attenuates early brain injury by modulating α-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA) receptor subunits after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in Sprague-Dawley rats. BPV(pic) treatment (0.2mg/kg) was evaluated for effects on neurological score, brain water content, Evans blue extravasation, hippocampal neuronal death and AMPA receptor subunits alterations after SAH. We found that BPV(pic) is effective in attenuating BBB disruption, lowering edema, reducing hippocampal neural death and improving neurological outcomes. In addition, the AMPA receptor subunit GluR1 protein expression at cytomembrane was downregulated, whereas the expression of GluR2 and GluR3 was upregulated after BPV(pic) treatment. Our results suggest that PTEN inhibited by BPV(pic) plays a neuroprotective role in SAH pathophysiology, possibly by alterations in glutamate AMPA receptor subunits., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient L-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for L-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett-Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l(-1)  L-lactic acid was obtained from 100 g l(-1) xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l(-1)  L-lactic acid was obtained in 36 h and the yield was 83.09 %.

Inulin-type oligosaccharides with different DP were prepared by size-exclusion chromatography and purity of each oligosaccharide was determined by HPLC equipped with cyclodextrin-bond column. The purities of obtained inulin-type oligosaccharides with different DP were more than 98% by one-step process. The DP and molecular weight were obtained through ESI-MS in negative mode. The characterization of the inulin-type oligosaccharides with different DP was studied by MS/MS spectra obtained by collision-induced dissociation of molecular ions ([M-H](-)). When the DP was lower, the fragment ions were formed through cross-ring cleavages of two bonds within the sugar ring and glycosidic cleavages. However, with the increase of DP, the ions resulting from glycosidic cleavages between two sugar residues were predominant.

Objectives: To investigate the effects of dietary supplementation of l-arginine (l-Arg) on shock in severely burned patients., Methods: This was a prospective, randomized, single blind, controlled study. Forty-seven severely burned patients due to various causes with a total burn surface area (TBSA) more than 50% each admitted in early postburn phase (within 10h postburn) were included in this study. All patients were treated by the traditional resuscitation program of our institute. After the nasogastric feeding tube was placed, they were randomly divided into three groups-(1) group A400 (n = 16): giving gastrointestinal feeding with 500 ml 5% GNS, containing l-Arg (400 mg/ kgday) at equal pace with fluid resuscitation; (2) group A200 (n = 16): giving gastrointestinal feeding with 500 ml 5% GNS containing l-Arg (200 mg/ kgday); (3) group C (n = 15): giving gastrointestinal feeding with 500 ml 5% GNS without any supplementation. The feeding started within 12h after burn and lasted for 72 h, the feeding rate was controlled by an enteral feeding pump. The following parameters were observed on days (PBD) 1-4: serum nitric oxide content (NO), mean arterial blood pressure (MAP), oxygenation index (PO2/FiO2), and arterial blood content of lactic acid (LA). Gastric mucosal blood flow was measured by laser Doppler flow-metry on PBD1 and PBD2., Results: (1) Enteral feeding of l-Arg did not change MAP of severely burned patients, with no difference in MAP between the l-Arg supplemented and control groups. (2) There were significant changes of the l-Arg supplemented groups (A400 and A200), with an increased gastric mucosa blood flow, oxygenation index, and a decreased LA content in arterial blood, compared with the control group. (3) The serous NO content was significantly decreased in the A400 group on PBD2-4 (P < 0.01), and in the A200 group on PBD4 (P < 0.05) compared with the control group., Conclusions: Enteral feeding with l-arginine supplementation on early stage of burn decreases NO production to a relatively normal level and exerts beneficial effects on the resuscitation of burned shock.

A method was established to analyze neutral chemical constituents in tobacco accurately by comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry (GC x GC-TOFMS). A DB-Petro column (50 m x 200 microm x 0.5 microm) was chosen as the column for the first dimension, and a DB-1701 column (2.3 m x 100 microm x 0.1 microm) was chosen as the column for the second dimension. The modulation period was set at 8 s, and the column pressure was 550 kPa. The initial temperatures of the two columns were set at 80 degrees C and 85 degrees C respectively and then increased with temperature programming. The contents of the neutral chemical constituents in different positions of tobacco leaves, product regions and varieties of tobacco were compared. The results showed that the total contents of the 24 neutral fractions in the middle leaves was the most, then in the upper leaves and the least in the lower leaves. These contents in the flue-cured tobacco produced by Brazil was the highest, followed by Zimbabwe, Yunyan85, Zhongyan101, NC89 and K326. In four kinds of tobacco, the total contents of the 24 neutral fractions in Oriental tobacco was the highest, followed by Burly tobacco, Flue-cured tobacco and Maryland tobacco.

A rapid and sensitive LC-MS/MS method was developed for the quantitative determination of sucrose esters (SEs) in Oriental tobacco samples. The sample preparation involved a 10-min sonication extraction procedure with acetone and five-fold dilution of the extract with methanol. The experiment was carried out in positive ion mode by ESI IT mass spectrometer. Because of lack of authentic standards of SEs, sucrose octa-acetate (internal standard, IS) was used as a surrogate to validate the proposed method. Matrix-matched standard calibration was used for quantification of IS in the spiked samples. Under optimized MS/MS conditions, an LOQ of 3.9 microg/g was achieved for IS, with an LOD of about 1.2 microg/g. Recoveries for IS were 95-97%. Among 19 monitored SEs, the contents of 11 SEs had RSDs lower than 13.7%. The method, with very little sample handling and good sensitivity, was applied to the rapid quantification of SEs in four Oriental tobacco samples. It appears that the sum of contents of the five SEs with MW 650, 664, and 678 Da occupied approximately 80% of the total content of SEs.

Sucrose esters (SEs) from oriental tobacco are normally characterized by gas chromatography/mass spectrometry (GC/MS) after a long saponification and derivatization procedure. To simplify the process, a rapid method has been developed by using liquid chromatography coupled with electrospray ion trap mass spectrometry (LC/ESI-MSn). Using the characteristic fragmentation behavior of abundant SEs identified by GC/MS after purification by gel permeation chromatography (GPC) from cuticular waxes of green oriental tobacco leaf, two types of SEs from green and cured oriental tobacco were identified by MSn analysis. The first is one of three types reported formerly and has 13 SE homologues. However, the presence of unsaturation in one of the acyl substituents of this first type gave rise to a new series with three homologues. The other was found to be a new type and had three homologues. The proposed method enables the rapid and sensitive characterization of SEs from oriental tobacco., (Copyright 2006 John Wiley & Sons, Ltd.)

Gas chromatography-mass spectrometry with electron ionization and positive-ion chemical ionization and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-TOF-MS) were applied for the characterization of the chemical composition of complex hydrocarbons in the non-polar neutral fraction of cigarette smoke condensates. Automated data processing by TOF-MS software combined with structured chromatograms and manual review of library hits were used to assign the components from GC x GC-TOF-MS analysis. The distributions of aliphatic hydrocarbons and aromatics were also investigated. Over 100 isoprenoid hydrocarbons were detected, including carotene degradation products, phytadiene isomers and carbocyclic diterpenoids. A total of 1800 hydrocarbons were tentatively identified, including aliphatic hydrocarbons, aromatics, and isoprenoid hydrocarbons. The identified hydrocarbons by GC x GC-TOF-MS were far more than those by GC-MS.

Cigarette smoke condensate is a complex chemical matrix. Analysis of nitrogen-containing compounds present therein is very difficult because of the limitation of the peak capacity of conventional one-dimensional chromatography. Extensive and laborious sample preparation is frequently required or selective detectors are frequently used. In this study, the basic fraction of mainstream cigarette smoke condensate has been investigated by using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS). Auto data processing by TOFMS software combined with manual identification was used to assign the components. 377 nitrogen-containing compounds, including 155 pyridine derivatives, 104 quinoline/isoquinoine derivatives, and 56 pyrazine derivatives were tentatively identified. By selecting appropriate unique masses and in the light of the component positions in the structured chromatogram, alkyl-substituted pyridines, pyrazines, and quinolines/isoquinolines were separately shown and further validated. The peaks of eight individual positional isomers of two-carbon-substituted pyridines and thirteen positional isomers of methyl-substituted quinolines/isoquinolines were further confirmed, based on linear incremental retention behavior in combination with TOFMS and the structured chromatogram of GC x GC.

Cigarette smoke condensate is a complex chemical matrix, and analysis of its components is very difficult because of the limitation of the peak capacity and sensitivity of conventional chromatography and the extensive and laborious sample preparation that is frequently required. In this study, the acidic fraction of mainstream cigarette smoke condensate has been investigated by using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS). Different column systems were tested and compared under proper GCxGC/TOFMS conditions. Auto data processing by TOFMS software combined with manual identification was used to assign the components. Over 1000 compounds, with S/N > or = 100, including 139 organic acids and over 150 phenols were tentatively identified by the developed method.

[reaction: see text] The title compound, 2, has been synthesized in 45% overall yield in six steps from 3-bromopyridine. The hexahydropyrrolo[3,2-f]pyrindine skeleton was constructed from key intermediate 5, via intramolecular azomethine ylide-alkene [3 + 2] cycloaddition. The present work constitutes a general method for rapid assembly of other related tricyclic nicotine analogues.