OBJECTIVETo analyze the impact of depletion of the twin arginine translocation (TAT) system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity.

METHODSWe constructed a DeltatatC::SpR mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 DeltatatC::SpR strain as a donor.

RESULTSA P1-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type Y. enterocolitica strain and translocation of this enzyme was completely abolished by the DeltatatC::SpR mutation. In addition, the DeltatatC::SpR mutation showed a pleiotropic effect on the metabolism of Y. enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used.

CONCLUSIONUnlike other pathogenic bacteria studied, the TAT system of Y. enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O157:H7.

Drug Resistance, Microbial ; genetics ; Genes, Bacterial ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; metabolism ; Transduction, Genetic ; Virulence ; Yersinia enterocolitica ; enzymology ; genetics ; metabolism ; pathogenicity

OBJECTIVE: To detect potential variation in an ethnic Han Chinese family affected with late-onset lipid storage myopathy. METHODS: Next generation sequencing (NGS) was used to screen disease-related genes in the proband. Suspected mutation was validated with PCR and Sanger sequencing in two patients, their father, and 100 healthy controls. RESULTS: Heterozygous c.770A>G (p.Tyr257Cys) and c.1395dupT (p.Gly466Tryfs) mutation were detected in the two patients. Their father was found to be heterozygous for the c.770A>G (p.Tyr257Cys) mutation, while the c.1395dupT (p.Gly466Tryfs) variation was not reported previously and not found among the healthy controls. CONCLUSION Mutations of the ETFDH gene probably underlie the pathogenesis in this family. The novel c.1395dupT (p.Gly466Tryfs) has enriched the mutation spectrum of EDFDH gene.
Asian Continental Ancestry Group ; Electron-Transferring Flavoproteins ; genetics ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Iron-Sulfur Proteins ; genetics ; Lipid Metabolism, Inborn Errors ; genetics ; Muscular Dystrophies ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.
Animals ; Casein Kinase II ; Cell Line ; Cricetinae ; Hela Cells ; Humans ; Mice ; Oxidoreductases Acting on CH-NH Group Donors/genetics/*metabolism ; Phosphoamino Acids/metabolism ; Phosphorylation ; Protein Binding ; Protein-Serine-Threonine Kinases/*metabolism ; Recombinant Proteins/genetics/metabolism

BACKGROUNDThe critical roles of polyamines in cell growth and differentiation have made polyamine metabolic pathway a promising target for antitumor therapy. Recent studies have demonstrated in vitro that some antitumor polyamine analogues could be used as substrates and oxidized by purified recombinant human N(1)-acetylpolyamine oxidase (APAO, an enzyme that catabolizes natural polyamines), indicating a potential role of APAO in determining the sensitivity of cancer cells to specific antitumor analogues. This study evaluated, in vivo, the effect of APAO on cytotoxicity of antitumor polyamine analogue, N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPENS) and its mechanism when highly expressed in human lung cancer line A549.

METHODSA clone with high expression of APAO was obtained by transfecting A549 lung cancer cell line with pcDNA3.1/APAO plasmid and selecting with quantitative realtime PCR and APAO activity assay. Cell proliferation was determined by MTT method and apoptosis related events were evaluated by DNA fragmentation, sub-G1/flow cytometric assay, western blotting (for cytochrome C and Bax) and colorimetric assay (for casapse-3 activity).

RESULTSA clone highly expressing APAO was obtained. High expression of APAO in A549 cells inhibited accumulation of CPENS, decreased their sensitivity to the toxicity of CPENS and prevented CPENS induced apoptosis.

CONCLUSIONThese results indicate a new drug resisting, mechanism in the tumor cells. High expression of APAO can greatly decrease the sensitivity of tumor cells to the specific polyamine analogues by detoxifying those analogues and prevent analogue induced apoptosis.

Apoptosis ; drug effects ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; metabolism ; Polyamines ; metabolism ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify pathogenic mutation in a boy affected with riboflavin responsive-multiple acyl-CoA dehydrogenase deficiency (RR-MADD).

METHODSThe patient was initially diagnosed as primary carnitine deficiency (PCD) and has been treated with carnitine supplementation for 7 years. Clinical manifestations and characteristics of fibula muscle specimen were analyzed. Potential mutation in electron transfer flavoprotein dehydrogenase (ETFDH) gene (for the patient and his parents) and carnitine transfer protein gene (SLC22A5) (for the patient) was screened.

RESULTSElectronic microscopy of the muscle specimen has suggested lipid storage myopathy. Mutation analysis has found that the patient carried compound heterozygous mutations, c.250G>A and c.380T>C, in exon 3 of the ETFDH gene, whilst his father and mother were heterozygous for the c.380T>C and c.250G>A mutations, respectively. Screening of the SLC22A5 gene has yielded no clinically meaningful result. After the establishment of diagnosis of RR-MADD, the condition of the patient has improved greatly with supplementation of high doses of riboflavin along with continuous carnitine supplement.

CONCLUSIONThe c.250G>A (p.Ala84Thr) mutation of exon 3 of the ETFDH gene has been a hot spot in Southern Chinese population, whilst the c.380T>C (p.Leu127Pro) is rarely reported. Our case has suggested that therapeutic diagnosis cannot substitute genetic testing. The mechanism for having stabilized the patient with only carnitine supplementation for 7 years needs further investigation.

Adolescent ; Adult ; Base Sequence ; Child ; DNA Mutational Analysis ; Electron-Transferring Flavoproteins ; genetics ; metabolism ; Female ; Humans ; Iron-Sulfur Proteins ; genetics ; metabolism ; Male ; Molecular Sequence Data ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; enzymology ; genetics ; metabolism ; Muscle, Skeletal ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; metabolism ; Riboflavin ; metabolism ; Solute Carrier Family 22 Member 5

OBJECTIVETo evaluate the role of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and plasma homocysteine levels in Chinese patients with type 2 diabetes mellitus and diabetic retinopathy (DR).

METHODSMTHFR genetic C677T polymorphisms were determined by PCR-restriction fragment length polymorphism. Total plasma homocysteine levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection.

RESULTSThe frequencies of MTHFR T homogenetic type and CT heterogenetic type and allele T (28.18%, 41.82%, 49.09%) in type 2 diabetic patients with diabetic retinopathy were significantly higher than those in diabetic patients without retinopathy (18.37%,29.59%,33.16%) or the normal controls (17.54%, 28.07%, 31.58%). Howerver, there were no significant differences in the frequency of MTHFR genotype and allele between the type 2 diabetic patients without retinopathy and the normal controls. The presence of T allele appeared to have a strong association with the development of diabetic retinopathy. The odds ratio was 1.94 and the 95% confidence interval was 1.31-2.88. Moreover, the plasma homocysteine levels in patients with TT or CT genotype were markedly higher than those in patients with CC genotype.

CONCLUSIONMTHFR gene C677T mutation associated with a predisposition to increase of plasma homocysteine may represent a genetic risk factor for diabetic retinopathy in Chinese type 2 diabetes mellitus.

Adult ; Alleles ; DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Diabetes Mellitus, Type 2 ; blood ; complications ; genetics ; Diabetic Retinopathy ; blood ; etiology ; genetics ; Female ; Gene Frequency ; Genotype ; Homocysteine ; blood ; Humans ; Male ; Methylenetetrahydrofolate Reductase (NADPH2) ; Middle Aged ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; Point Mutation ; Polymorphism, Genetic

BACKGROUND: Late-onset multiple acyl-coA dehydrogenase deficiency (MADD) is an autosomal recessive inherited metabolic disorder. It is still unclear about the muscle magnetic resonance image (MRI) pattern of the distal lower limb pre- and post-treatment in patients with late-onset MADD. This study described the clinical and genetic findings in a cohort of patients with late-onset MADD, and aimed to characterize the MRI pattern of the lower limbs. METHODS: Clinical data were retrospectively collected from clinic centers of Peking University People's Hospital between February 2014 and February 2018. Muscle biopsy, blood acylcarnitines, and urine organic acids profiles, and genetic analysis were conducted to establish the diagnosis of MADD in 25 patients. Muscle MRI of the thigh and leg were performed in all patients before treatment. Eight patients received MRI re-examinations after treatment. RESULTS: All patients presented with muscle weakness or exercise intolerance associated with variants in the electron transfer flavoprotein dehydrogenase gene. Muscle MRI showed a sign of both edema-like change and fat infiltration selectively involving in the soleus (SO) but sparing of the gastrocnemius (GA) in the leg. Similar sign of selective involvement of the biceps femoris longus (BFL) but sparing of the semitendinosus (ST) was observed in the thigh. The sensitivity and specificity of the combination of either "SO+/GA-" sign or "BFL+/ST-" sign for the diagnosis of late-onset MADD were 80.0% and 83.5%, respectively. Logistic regression model supported the findings. The edema-like change in the SO and BFL muscles were quickly recovered at 1 month after treatment, and the clinical symptom was also relieved. CONCLUSIONS This study expands the clinical and genetic spectrums of late-onset MADD. Muscle MRI shows a distinct pattern in the lower limb of patients with late-onset MADD. The dynamic change of edema-like change in the affected muscles might be a potential biomarker of treatment response.
Adolescent ; Adult ; Biopsy ; methods ; Carnitine ; analogs & derivatives ; blood ; Electron-Transferring Flavoproteins ; genetics ; Female ; Hamstring Muscles ; diagnostic imaging ; metabolism ; pathology ; Humans ; Iron-Sulfur Proteins ; genetics ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; diagnostic imaging ; genetics ; pathology ; Muscle, Skeletal ; diagnostic imaging ; metabolism ; pathology ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; Retrospective Studies ; Young Adult