The aim of this study was to investigate the clinical features and DGUOK gene mutations of an infant with mitochondrial DNA depletion syndrome (MDS). The patient (more than 7 months old) manifested as hepatosplenomegaly, abnormal liver function, nystagmus and psychomotor retardation. Genetic DNA was extracted from peripheral blood samples of the patient and her parents. Targeted Exome Sequencing was performed to explore the genetic causes. Sanger sequencing was carried out to confirm the detected mutations. The sequencing results showed that the patient was a compound heterozygote for c.679G>A and c.817delT in the DGUOK gene. The former was a reportedly pathogenic missense mutation of maternal origin, while the latter, a frameshift mutation from the father, has not been described yet. The findings in this study expand the mutation spectrum of DGUOK gene, and provide molecular evidence for the etiologic diagnosis of the patient as well as for the genetic counseling and prenatal diagnosis in the family.
Female ; Humans ; Infant ; Mitochondrial Diseases ; genetics ; therapy ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; chemistry ; genetics

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

OBJECTIVETo construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.

METHODSThe gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining.

RESULTS AND CONCLUSIONWe established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

DNA Primers ; Escherichia coli O157 ; genetics ; Escherichia coli Proteins ; genetics ; Gene Deletion ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction

Glycerol is the main byproduct in ethanol production by Saccharomyces cerevisiae. In order to improve ethanol yield and the substrate conversion, a cassette about 4.5 kb for gene homologous recombination, gpd2Δ::PGK1(PT)-POS5-HyBR, was constructed and transformed into the haploid strain S. cerevisiae S1 (MATa) to replace the GPD2 gene by POS5 gene. The NADH kinase gene POS5 was successfully over expressed in the recombinant strain S. cerevisiae S3. Comparing with the parent strain, the recombinant strain S. cerevisiae S3 exhibited an 8% increase in ethanol production and a 33.64% decrease in glycerol production in the conical flask fermentation with an initiatory glucose concentration of 150 g/L. Overexpression of NADH kinase gene seems effective in reducing glycerol production and increasing ethanol yield.
Ethanol ; chemistry ; Fermentation ; Glycerol ; chemistry ; Industrial Microbiology ; Mitochondrial Proteins ; genetics ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism

OBJECTIVEThe minor allele T of rs113420705 (C/T) in caspase-3 gene (CASP3) has been found to significantly increase the risk of Kawasaki disease (KD) and complicate coronary artery lesions (CALs) in Japanese children. In this study, we have explored association of single nucleotide polymorphisms (SNPs) of CASP3 gene and clinic phenotypes of KD.

METHODSA total of 238 unrelated KD patients and 364 healthy controls with matched age, gender and ethnic origins were recruited. Genotypes of the 3 SNPs were determined with PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Allelic, genotypic and haplotypic frequencies were compared between patients and controls, patients with and without CALs, and patients resistant to and responsive to intravenous immunoglobulin (IVIG) treatment.

RESULTSThe T allele and T carriers of rs113420705 were significantly more common in KD patients than controls. A significant difference was also detected in haplotype distribution between patients and controls, where two haplotypes involving the T allele of rs113420705 showed higher frequencies in the patient group. Allelic and genotypic frequencies of the 3 SNPs were similar between patients with and without CALs and those resistant to and responsive to IVIG treatment.

CONCLUSIONOur results suggested that CASP3 probably plays an important role in KD. The T allele of rs113420705 may provide a useful marker for KD susceptibility, although no association between this SNP and clinical prognosis and treatment effect of KD has been found among the selected Chinese children patients.

Caspase 3 ; genetics ; Child ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; Humans ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome ; genetics ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymorphism, Single Nucleotide

NAD kinase catalyzes the phosphorylation of coenzyme I [NAD(H)] to form coenzyme II [NADP(H)], and NADPH is an important cofactor in L-isoleucine biosynthesis. In order to improve NADPH supply, ppnK, the gene encoding NAD kinase in Corynebacterium glutamicum was cloned and separately expressed in an L-isoleucine synthetic strain, Brevibacterium lactofermentum JHI3-156, by an inducible expression vector pDXW-8 and a constitutive expression vector pDXW-9. Compared with the control strain JHI3-156/pDXW-8, NAD kinase activity of the inducible ppnK-expressing strain JHI3-156/pDXW-8-ppnK was increased by 83.5%. NADP(H)/NAD(H) ratio was also increased by 63.8%. L-isoleucine biosynthesis was improved by 82.9%. Compared with the control strain JHI3-156/pDXW-9, NAD kinase activity of the constitutive ppnK-expressing strain JHI3-156/pDXW-9-ppnK was increased by 220%. NADP(H)/ NAD(H) ratio and NADPH concentration were increased by 134% and 21.7%, respectively. L-isoleucine biosynthesis was increased by 41.7%. These results demonstrate that NAD kinase can improve the coenzyme II supply and L-isoleucine biosynthesis, which would also be useful for biosynthesis of other amino acids.
Brevibacterium ; genetics ; metabolism ; Cloning, Molecular ; Corynebacterium glutamicum ; enzymology ; Isoleucine ; biosynthesis ; Metabolic Engineering ; NAD ; metabolism ; NADP ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.
Alkyl and Aryl Transferases ; genetics ; Antimalarials ; metabolism ; Artemisinins ; metabolism ; Enterococcus faecalis ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; methods ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sesquiterpenes ; metabolism ; Transformation, Bacterial

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*

AIMTo elucidate the effect of sphingosine kinase (SPK) on the hepatocyte growth factor (HGF)-induced migration of endothelial cells.

METHODSWe constructed recombinant adenoviral vectors, which contain SPK gene and its mutant respectively. These adenoviral vectors were packaged and amplified in 293 cells. And intracellular SPK activity was assayed via measurement of [32]P radioisotope labeled S1P; the effect of SPK activation on HGF-induced migration of endothelial cell was observed by Transwell technique.

RESULTSAdenoviral mediated expression of SPK gene increased in ECV 304 cells intracellular SPK activity, which in turn enhanced the HGF-induced migration. Whereas these activities were blocked by the dominant negative SPK gene.

CONCLUSIONThese findings show that SPK activation plays important roles in the regulation of HGF-induced migration of endothelial cells.

Adenoviridae ; metabolism ; Cell Line ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo explore the molecular mechanism of miR-124 suppressing the proliferation and invasion of gastric cancer cells.

METHODSSPHK1 3'UTR-luciferase vector was constructed and luciferase reporter gene assay was employed to examine the effect of miR-124 on luciferase activity. Human gastric cancer MGC-803 cells were transfected with miR-124 mimics, and then Western blot was performed to detect the expression of SPHK1 protein.

RESULTSLuciferase reporter vector system confirmed that SPHK1 was a target gene of miR-124. Western blot showed that the expression of SPHK1 protein was inhibited by miR-124. After transfection of miR-124 mimics or SPHK1 siRNA for 12 h, 24 h and 48 h, respectively, MTT assay showed that the A values of the three groups were significantly different (P < 0.05), and it was in a time-dependent manner. After transfection of miR-124 mimics or SPHK1 siRNA for 24 h, transwell invasion assay showed that the number of transmembrane cells was 54.6 ± 8.3 in the SPHK1 siRNA group and 47.8 ± 6.6 in the miR-124 mimics group, both were significantly lower than 100.6 ± 11.3 of the control group (P < 0.05), indicating that SPHK1 siRNA can slow down the invasion of MGC-803 cells.

CONCLUSIONmiR-124 can suppress the cell proliferation and invasion by targeting SPHK1 in gastric carcinoma.

Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Luciferases ; genetics ; metabolism ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Transfection