The present study aimed to investigate the protective effect of S-propargyl-cysteine (SPRC) on atherosclerosis progression in mice. A mouse model of vulnerable atherosclerotic plaque was created in ApoE^-/- mice by carotid artery tandem stenosis (TS) combined with a Western diet. Macrophotography, lipid profiles, and inflammatory markers were measured to evaluate the antiatherosclerotic effects of SPRC compared to atorvastatin as a control. Histopathological analysis was performed to assess the plaque stability. To explore the protective mechanism of SPRC, human umbilical vein endothelial cells (HUVECs) were cultured in vitro and challenged with oxidized low-density lipoprotein (ox-LDL). Cell viability was determined with a Cell Counting Kit-8 (CCK-8). Endothelial nitric oxide synthase (eNOS) phosphorylation and mRNA expression were detected by Western blot and RT-qPCR respectively. The results showed that the lesion area quantified by en face photographs of the aortic arch and carotid artery was significantly less, plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced, plaque collagen content was increased and matrix metalloproteinase-9 (MMP-9) was decreased in 80 mg/kg per day SPRC-treated mice compared with model mice. These findings support the role of SPRC in plaque stabilization. In vitro studies revealed that 100 μmol/L SPRC increased the cell viability and the phosphorylation level of eNOS after ox-LDL challenge. These results suggest that SPRC delays the progression of atherosclerosis and enhances plaque stability. The protective effect may be at least partially related to the increased phosphorylation of eNOS in endothelial cells.
Animals ; Humans ; Mice ; Atherosclerosis ; Cholesterol/metabolism* ; Cysteine/pharmacology* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Plaque, Atherosclerotic/pathology*

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.
Cinnamomum camphora/enzymology* ; Alkyl and Aryl Transferases/chemistry*

Male infertility caused by idiopathic oligoasthenospermia (OAT) is known as idiopathic male infertility. Glutathione S-transferase (GST) and fluoride may play important roles in idiopathic male infertility, but their effects are still unknown. Our study examined the relationship between GST polymorphisms and fluoride-induced toxicity in idiopathic male infertility and determined the underlying mechanism. Sperm, blood, and urine samples were collected from 560 males. Fluoride levels were measured by a highly selective electrode method, and GST genotypes were identified using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Semen parameters, DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and oxidative stress (OS) biomarkers were statistically assessed at the P < 0.05 level. Compared with healthy fertile group, semen parameters, fluoride levels, OS biomarkers, sex hormone levels, and MMP and DFI levels were lower in the idiopathic male infertility group. For glutathione S-transferase M1 (GSTM1[-]) and glutathione S-transferase T1 (GSTT1[-]) or glutathione S-transferase P1 (GSTP1) mutant genotypes, levels of semen fluoride, OS, MMP, and DFI were considerably higher, and the mean levels of sperm parameters and testosterone were statistically significant in GSTM1(+), GSTT1(+), and GSTP1 wild-type genotypes. Both semen and blood fluoride levels were associated with oxidative stress in idiopathic male infertility patients. Elevated fluoride in semen with the genotypes listed above was linked to reproductive quality in idiopathic male infertility patients. In conclusion, GST polymorphisms and fluorine may have an indicative relationship between reproductive quality and sex hormone levels, and OS participates in the development of idiopathic male infertility.
Humans ; Male ; Fluorides/adverse effects* ; Semen ; Polymorphism, Genetic ; Glutathione Transferase/genetics* ; Glutathione S-Transferase pi/genetics* ; Infertility, Male/genetics* ; Genotype ; Biomarkers ; Genetic Predisposition to Disease ; Case-Control Studies

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.
Gynostemma ; Phylogeny ; Alkyl and Aryl Transferases ; Chloroplasts

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 μmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 μmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 μmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 μmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 μmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(P<0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 μmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 μmol·m~(-2)·s~(-1)) and strong light(160 μmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 μmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.
Farnesyl-Diphosphate Farnesyltransferase/metabolism* ; Ginsenosides ; Panax/metabolism* ; Plant Roots/metabolism* ; Sand ; Squalene Monooxygenase

OBJECTIVE: To assess the association of polymorphisms of glutathione S-transferase P1 (GSTP1) and phospholipase C epsilon-1 (PLCE1) genes with the susceptibility of primary esophageal cancer and their interaction with environmental factors. METHODS: 162 patients with primary esophageal cancer and 162 healthy controls were recruited in this cross-sectional study. Basic information such as gender, age, history of smoking and alcohol consumption and family history of esophageal cancer were collected. Single nucleotide polymorphisms at A105G locus of GSTP1 gene and rs3765524, rs2274223 and rs3781264 loci of PLCE1 gene were detected. A logistic regression model was established to analyze the risk factors of esophageal cancer and the interaction among the factors. RESULTS: The proportions of individuals with smoking history, family history of esophageal cancer and hot diet in esophageal cancer group were higher than those in the control group (P<0.05). Conditional Logistic regression analysis showed that smoking, family history of esophageal cancer and GG genotype at the rs2274223 locus of PLCE1 gene were the risk factors for esophageal cancer (P<0.05), and AG/GG genotypes at the A105G locus of GSTP1 gene were the protective factors for esophageal cancer (P<0.05). In the two-factor interaction model, both AA genotype at A105G locus of GSTP1 gene and GG genotype at rs2274223 locus of PLCE1 gene had an interaction with smoking, and the risk of esophageal cancer has increased by 83.6% and 85.7%, respectively (P<0.05). AA genotype at A105G locus of GSTP1 gene, GG genotype at rs2274223 locus of PLCE1 gene and smoking constituted the best three-factor interaction model, and the risk of esophageal cancer has increased by 244.0% (P<0.05). Four-factor interaction model analysis showed that the risk of esophageal cancer among individuals with AA genotype at A105G locus of GSTP1 gene, GG genotype at rs2274223 locus of PLCE1 gene, smoking and family history of esophageal cancer has increased by 264.4% (P<0.05). CONCLUSION The AG and GG genotypes at the A105G locus of GSTP1 gene are protective factors for esophageal cancer, and the GG genotype at rs2274223 locus of PLCE1 gene is a risk factor, both of them may interact with smoking and affect the susceptibility to esophageal cancer.
Humans ; Genetic Predisposition to Disease ; Glutathione Transferase/genetics* ; Cross-Sectional Studies ; Case-Control Studies ; Esophageal Neoplasms/genetics* ; Polymorphism, Single Nucleotide ; Genotype ; Risk Factors ; Glutathione S-Transferase pi/genetics*

Genome mining for the search and discovery of two new globin-like enzymes, TriB fromFusarium poae and TutaA from Schizophyllum commne, are involved in the synthesis of two linear terpenes tricinonoic acid (1) and 2-butenedioic acid (3). Both in vivo heterologous biosynthesis and in vitro biochemical assays showed that these two enzymes catalyzed the C-C double bond cleavage of a cyclic sesquiterpene precursor (-)-germacrene D (7) and a linear diterpene backbone schizostain (2), respectively. Our work presents an unusual formation mechanism of linear terpenes from fungi and expands the functional skills of globin-like enzymes in the synthesis of terpene compounds.
Terpenes/chemistry* ; Alkyl and Aryl Transferases ; Globins ; Diterpenes

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.
Amino Acid Sequence ; Cloning, Molecular ; Geranyltranstransferase/genetics* ; Pogostemon ; Transcription Factors/genetics*

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.
Fermentation ; Geranyltranstransferase/genetics* ; Monoterpenes/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*