Bacterial Proteins/metabolism* ; Operon/genetics* ; Phosphotransferases/metabolism* ; Quorum Sensing/genetics* ; Vibrio cholerae/genetics*

The purpose of the present study was to investigate the effect and potential mechanism of knockdown of sphingosine kinase-1 (SPHK1) on the proliferation, cell cycle and apoptosis of non-small cell lung cancer (NSCLC) cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect SPHK1 mRNA expression in human healthy lung fibroblasts (MRC-5 cells) and four NSCLC cell lines. Then, A549 and H1299 cells were transfected with SPHK1-shRNA and corresponding negative control. CCK-8, Annexin V-FITC/PI dual staining and cell cycle assay were performed to evaluate cell proliferation, apoptosis and cell cycle distribution, respectively. JC-1 mitochondrial membrane potential measurement kit was adopted to measure mitochondrial membrane potential. Western blot was used to detect the protein expression levels of cell cycle and mitochondrial apoptotic pathway-related proteins, as well as MEK/ERK signaling pathway. The results showed that the mRNA expression of SPHK1 in NSCLC cells was higher than that in MRC-5 cells. SPHK1-shRNA significantly inhibited the proliferation of A549 and H1299 cells, blocked the cell cycle in G0/G1 phase, and promoted cell apoptosis through the mitochondrial pathway. Compared with the control group, the expression of p-MEK and p-ERK proteins in the SPHK1-shRNA group was significantly down-regulated. Moreover, MEK/ERK inhibitor could dramatically suppress cell proliferation and promote cell apoptosis. These results suggest that SPHK1 knockdown can inhibit the proliferation of NSCLC cells and might promote mitochondrial apoptotic pathway by inhibiting MEK/ERK signaling pathway.
Apoptosis ; Carcinoma, Non-Small-Cell Lung/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Gene Knockdown Techniques ; Humans ; Lung Neoplasms/genetics* ; Phosphotransferases (Alcohol Group Acceptor)/genetics*

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
Adipose Tissue/cytology* ; Animals ; Cytokines ; Encephalomyelitis, Autoimmune, Experimental/therapy* ; Interleukin-17 ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred C57BL ; Phosphotransferases (Alcohol Group Acceptor)/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Th17 Cells/cytology* ; Transfection

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
Cytidine Monophosphate ; metabolism ; Escherichia coli ; genetics ; Industrial Microbiology ; methods ; Phosphotransferases (Phosphate Group Acceptor) ; metabolism ; Uridine Kinase

OBJECTIVE: To explore the molecular etiology for a Chinese family with mitochondrial DNA depletion syndrome. METHODS: Genomic DNA was extracted from peripheral blood samples of the patient and her parents.Targeted capture and next-generation sequencing was carried out to detect potential variants. Suspected variant was validated by Sanger sequencing. RESULTS: A novel homozygous frameshift variant c.505_508delTATC was identified in the patient, for which both his mother and father were carriers. CONCLUSION The frameshift variant c.505_508delTATC probably underlies the mitochondrial DNA depletion syndrome in this patient. The result also enriched the variant spectrum of DGUOK gene.
Asian Continental Ancestry Group ; genetics ; DNA, Mitochondrial ; genetics ; Female ; Frameshift Mutation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Syndrome

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
Cinnamomum camphora/genetics* ; Cloning, Molecular ; Genes, Plant ; Phosphotransferases (Phosphate Group Acceptor)/genetics* ; Phylogeny ; Sequence Alignment

OBJECTIVE: To identify potential mutation of PMM2 gene in an infant with congenital disorders of glycosylation type 1a (CDG-1a). METHODS: Genomic DNA was extracted from peripheral blood sample of the patient. All coding exons (exons 1-8) and splicing sites of the PMM2 gene were amplified with PCR. Potential variants were detected by direct sequencing of the PCR products and comparing the results against the ESP and SNP human gene databases. A protein BLAST system was employed to analyze cross-species conservation of the variants amino acid. A PubMed BLAST CD-search system was employed to identify functional domains damaged by variants of the PMM2 gene. Impact of potential variants was analyzed using software including PolyPhen-2 SIFT and Mutation Taster. Whole exome sequencing was used to identify additional variants of the PMM2 gene which may explain the condition of the patient. RESULTS: The child was found to carry compound heterozygous variants (c.458_462delTAAGA and c.395T>C) of the PMM2 gene, which were inherited respectively from his father and mother. The c.458_462delTAAGA has not been reported previously and may result in disruption of 10 functional domains within the PMM2 protein. The c.395T>C mutation has been recorded by a SNP database with frequency unknown. Both mutations were predicted as "probably damaging". Whole exome sequencing has identified no additional disease-causing variant which can explain the patient's condition. CONCLUSION The patient's condition may be attributed to the compound heterozygous variants c.458_462delTAAGA and c.395T>C of the PMM2 gene. Above results has facilitated molecular diagnosis for the patient.
Congenital Disorders of Glycosylation ; genetics ; Exons ; Humans ; Infant ; Mutation ; Phosphotransferases (Phosphomutases) ; genetics

OBJECTIVE: To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC). METHODS: GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). RESULTS: Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. CONCLUSIONS GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.
Adenoviridae ; Animals ; Apoptosis ; Carcinoma, Hepatocellular/therapy* ; Cell Line, Tumor ; Female ; Liver Neoplasms/therapy* ; Membrane Proteins/genetics* ; Mice ; Mice, Inbred BALB C ; Oncolytic Virotherapy/methods* ; Phosphotransferases (Alcohol Group Acceptor)/genetics* ; Promoter Regions, Genetic

PURPOSE: Kawasaki disease (KD) is an acute systemic vasculitis. Both the etiology of KD and the erythema of Bacille Calmette-Guérin (BCG) injection sites observed in the disease are poorly understood. We investigated the association between KD and single nucleotide polymorphisms (SNPs) in two candidate genes: inositol 1,4,5-triphosphate 3-kinase (ITPKC), a well-studied KD-associated gene, and solute carrier 11a1 (SLC11A1), which is associated with the hypersensitive reaction to the BCG strain in Koreans. MATERIALS AND METHODS: Associations between KD and SNPs in two genes were evaluated. Potential associations between BCG injection site erythema and SNPs in two genes were also evaluated. Gene-gene interactions between ITPKC and SLC11A1 in KD and BCG injection site erythema were also analyzed. RESULTS: Three tagging SNPs in ITPKC and five tagging SNPs in SLC11A1 were genotyped in 299 KD patients and 210 control children. SNP rs28493229 in ITPKC was associated with KD and coronary artery complications. SNP rs77624405 in SLC11A1 was associated with KD. Comparisons of KD patients with and without BCG injection site erythema revealed that SNP rs17235409 in SLC11A1 was associated with erythema; no erythema-associated SNPs in ITPKC were identified. Interactions between ITPKC rs28493229_GG and SLC11A1 rs17235409_GA and between ITPKC rs10420685_GG and SLC11A1 rs17235409_AA were strongly associated with BCG injection site erythema. CONCLUSION: This study identified several important polymorphisms in the ITPKC and SLC11A1 genes in Koreans. The genetic variants identified in this study affected KD and erythema of BCG injection sites independently and through gene-gene interactions. Also, the effects of the polymorphisms were age-dependent.
Asian Continental Ancestry Group/genetics ; BCG Vaccine/administration & dosage ; Case-Control Studies ; Cation Transport Proteins/genetics ; Child ; Child, Preschool ; Epistasis, Genetic ; Erythema/complications ; Female ; Genetic Association Studies ; Genetic Predisposition to Disease ; Humans ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome/genetics ; Mutation Rate ; Phosphotransferases (Alcohol Group Acceptor)/genetics ; Polymorphism, Single Nucleotide/genetics ; Republic of Korea

PURPOSE: Although it is well known thatmortality and morbidity due to cardiovascular diseases are higher in salt-sensitive subjects than in salt-resistant subjects, their underlying mechanisms related to obesity remain unclear. Here, we focused on salt-sensitive gene variants unrelated to monogenic obesity that interacted with sodium intake in humans. METHODS: This review was written based on the modified 3(rd) step of Khans' systematic review. Instead of the literature, subject genes were based on candidate genes screened from our preliminary Genome-Wide Association Study (GWAS). Finally, literature related to five genes strongly associated with salt sensitivity were analyzed to elucidate the mechanism of obesity. RESULTS: Salt sensitivity is a measure of how blood pressure responds to salt intake, and people are either salt-sensitive or salt-resistant. Otherwise, dietary sodium restriction may not be beneficial for everyone since salt sensitivity may be associated with inherited susceptibility. According to our previous GWAS studies, 10 candidate genes and 11 single nucleotide polymorphisms (SNPs) associated with salt sensitivity were suggested, including angiotensin converting enzyme (ACE), α-adducin1 (ADD1), angiotensinogen (AGT), cytochrome P450 family 11-subfamily β-2 (CYP11β-2), epithelial sodium channel (ENaC), G-protein b3 subunit (GNB3), G protein-coupled receptor kinases type 4 (GRK4 A142V, GRK4 A486V), 11β-hydroxysteroid dehydrogenase type-2 (HSD 11β-2), neural precursor cell-expressed developmentally down regulated 4 like (NEDD4L), and solute carrier family 12(sodium/chloride transporters)-member 3 (SLC 12A3). We found that polymorphisms of salt-sensitive genes such as ACE, CYP11β-2, GRK4, SLC12A3, and GNB3 may be positively associated with human obesity. CONCLUSION: Despite gender, ethnic, and age differences in genetics studies, hypertensive obese children and adults who are carriers of specific salt-sensitive genes are recommended to reduce their sodium intake. We believe that our findings can contribute to the prevention of early-onset of chronic diseases in obese children by facilitating personalized diet-management of obesity from childhood to adulthood.
Adult ; Angiotensinogen ; Blood Pressure ; Cardiovascular Diseases ; Child ; Chronic Disease ; Cytochrome P-450 Enzyme System ; Epithelial Sodium Channels ; Genetics ; Genome-Wide Association Study ; GTP-Binding Proteins ; Humans ; Hypertension ; Obesity* ; Oxidoreductases ; Peptidyl-Dipeptidase A ; Phosphotransferases ; Polymorphism, Single Nucleotide ; Sodium ; Sodium, Dietary