The metabolic changes in diabetics result in progressive retinopathy and influence corneal metabolism. Changes in corneal autofluorescence were demonstrated originating from mitochondrial flavoproteins and influenced by the metabolic changes in cornea in diabetics. The corneal autofluorescence was determined to evaluate its correlation with diabetic retinopathy using fluorophotometer in 25 healthy controls, 25 diabetic mellitus(DM) pationts without retinopathy, 25 background diabetic retinopathy(BDR)patients, 25 preproliferative diabetic retinopathy (PPDR) patients, and 25 proliferative diabetic retinopathy(PDR) patients. The mean values(mean +/- standard deviation in ng fluorescein/ml) were 13.9 +/- 1.9, 18.7 +/- 3.1, 19.6 +/- 2.3, 20.2 +/- 4.0, 24.3 +/- 4.2, respectively. The means of coreal autofluorescence values in diabetics were significantly higher than that of the healthy controls(p<0.001). The mean values in DM patients without retinopathy, BDR patients and PPDR patients did not differ significantly(p>0.05), but the mean value in PDR patients was significantly higher than those of the other 4 groups(p<0.001). These results indicate that measurement of corneal autofluorescence can play a supplementary role in diagnosing diabetic retinopathy.
Cornea ; Diabetic Retinopathy* ; Flavoproteins ; Humans ; Metabolism

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).
Animals ; Cloning, Molecular ; DNA Primers ; Escherichia coli/genetics/metabolism ; Flavoproteins/chemistry/genetics ; Kinetics ; Membrane Glycoproteins/chemistry/*genetics/isolation & purification ; *NADPH Oxidase ; Neutrophils/physiology ; Polymerase Chain Reaction/methods ; Recombinant Fusion Proteins/chemistry/isolation & purification/metabolism ; Recombinant Proteins/chemistry/isolation & purification ; Restriction Mapping ; Sequence Deletion

Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Flavoproteins are involved in a wide array of biological processes, such as photosynthesis, DNA repair and natural product biosynthesis. It should be noted that 5%-10% of flavoproteins have a covalently linked flavin prosthetic group. Such covalent linkages benefit the holoenzyme in several ways including improving the stability and catalytic potency. During the past decade, significant progress has been made in covalent flavoproteins, especially with respect to enzyme-dependent biogenesis and discovery of novel linkage types. The present review gives a condensed overview of investigations published from March 2009 to December 2021, with emphasis on the discovery, biogenesis and their catalytic role in natural product biosynthesis.
Flavoproteins/metabolism* ; Flavin-Adenine Dinucleotide/metabolism* ; Flavin Mononucleotide/metabolism* ; Riboflavin ; Biological Products

Short-chain acyl-CoA dehydrogenase deficiency (SCADD; OMIM # 201470) is an autosomal recessive inborn error of mitochondrial fatty acid beta-oxidation, presenting with a variety of clinical signs and symptoms. Developmental delay, hypertonia or hypotonia, ketotic hypoglycemia, and epilepsy are most frequently reported. In general, patients diagnosed through newborn screening have shown normal growth and development in contrast to those diagnosed as a result of clinically initiated evaluations. Here, the case of an asymptomatic Korean newborn with SCADD identified by tandem mass spectrometry is reported. The patient showed an elevated concentration of butyrylcarnitine detected on newborn screening. Urinary excretion of ethylmalonic acid was elevated by urine organic acid analysis. To confirm the diagnosis of SCADD, a direct sequencing analysis of 10 coding exons and the exon-intron boundaries of the ACADS gene were performed. Genetic analysis of ACADS showed the following novel compound heterozygous missense mutations: c.277C>A (p.Leu93Ile) on exon3 and c.682G>A (p.Glu288Lys) on exon6. These results will provide further evidence of mutational heterogeneity for SCADD.
Acyl-CoA Dehydrogenase ; Butyryl-CoA Dehydrogenase ; Carnitine ; Clinical Coding ; Databases, Genetic ; Epilepsy ; Exons ; Growth and Development ; Humans ; Hypoglycemia ; Infant, Newborn ; Malonates ; Mass Screening ; Muscle Hypotonia ; Population Characteristics ; Tandem Mass Spectrometry

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive mitochondrial disorder of fatty acid β-oxidation, and is associated with mutations in the acyl-CoA dehydrogenase (ACADS) gene. Recent advances in spectrometric screening for inborn errors of metabolism have helped detect several metabolic disorders, including SCADD, without symptoms in the neonate period. This allows immediate initiation of treatment and monitoring, so they remain largely symptomless metabolic disease. Here, we report a 15-month-old asymptomatic male, who was diagnosed with SCADD by newborn screening. Spectrometric screening for inborn errors of metabolism 72 hours after birth revealed an elevated butyrylcarnitine (C4) concentration of 2.25 µmol/L (normal, <0.99 µmol/L). Urinary excretion of ethylmalonic acid was also elevated, as detected by urine organic acid analysis. To confirm the diagnosis of SCADD, direct sequencing analysis of 10 coding exons and the exon-intron boundaries of the ACADS gene were performed. Subsequent sequence analysis revealed compound heterozygous missense mutations c.164C>T (p.Pro55Leu) and c.1031A>G (p.Glu344Gly) on exons 2 and 9, respectively. The patient is now growing up, unretarded by symptoms such as seizure and developmental delay.
Acyl-CoA Dehydrogenase* ; Butyryl-CoA Dehydrogenase ; Clinical Coding ; Diagnosis ; Exons ; Humans ; Infant ; Infant, Newborn* ; Male ; Mass Screening ; Metabolic Diseases ; Metabolism, Inborn Errors ; Mitochondrial Diseases ; Mutation, Missense ; Neonatal Screening ; Parturition ; Seizures ; Sequence Analysis

OBJECTIVE: To analyze the clinical features and genetic variants in four neonates with very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. METHODS: Neonates with a tetradecenoylcarnitine (C14:1) concentration at above 0.4 μmol/L in newborn screening were recalled for re-testing. Four neonates were diagnosed with VLCAD deficiency by MS-MS and genetic testing, and their clinical features and genotypes were analyzed. RESULTS: All cases had elevated blood C14:1, and the values of first recalls were all lower than the initial test. In 2 cases, the C14:1 had dropped to the normal range. 1 case has remained at above 1 μmol/L after the reduction, and the remainder one case was slightly decreased. In total eight variants of the ADACVL genes were detected among the four neonates, which included 5 missense variants and 3 novel variants (p.Met344Val, p.Ala416Val, c.1077+6T>A). No neonate showed salient clinical manifestations. CONCLUSION Above findings have enriched the spectrum of ADACVL gene mutations and provided a valuable reference for the screening and diagnosis of VLCAD deficiency.
Acyl-CoA Dehydrogenase/genetics* ; Acyl-CoA Dehydrogenase, Long-Chain ; Congenital Bone Marrow Failure Syndromes ; Genetic Testing ; Humans ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; Mitochondrial Diseases ; Muscular Diseases ; Tandem Mass Spectrometry

OBJECTIVE: To explore the clinical features and variations of ACADVL gene in 9 neonates with very long chain acyl-coenzyme A dehydrogenase deficiency (VLCADD). METHODS: VLCADD was suspected based on the results of neonatal screening by tandem mass spectrometry (MS-MS), with tetradecenoylcarnitine ± tetradecenoylcarnitine/octanoylcarnitine (C14: 1 ± C14: 1/C8) as the mark indexes. Infants with positive outcome were confirmed by sequencing of the ACADVL gene. RESULTS: Among 9 VLCADD cases, one case lost during follow-up, the observed phenotypes comprised 2 with severe early-onset form, 1 with hepatic form and 5 with late-onset form. Optimal outcome was acquired for all patients except the 2 early-onset cases. In total 16 ACADVL variations were detected among the 9 infants, which included 8 novel variations (c.96-105del GCCCGGCCCT, c.541C>T, c.863T>G, c.878+1G>C, c.895A>G, c.1238T>C, c.1276G>A, and c.1505T>A) and 11 missense variations. There were 9 genotypic combinations, including 1 homozygote and 8 compound heterozygotes. Except for two patients carrying null variations, all had a good outcome. CONCLUSION VLCADD is relatively rare in southern China, for which late-onset form is common. Carriers of null variations of the ACADVL gene may have relatively poorer clinical outcome. Above results will provide valuable information for the diagnosis and management of VLCADD.
Acyl-CoA Dehydrogenase, Long-Chain ; deficiency ; genetics ; Carnitine ; China ; Humans ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; genetics ; Mitochondrial Diseases ; genetics ; Muscular Diseases ; genetics ; Neonatal Screening

OBJECTIVE: To explore the molecular mechanisms of colonic epithelial aging related proteins and aged colonic epithelial susceptibility to tumor. METHODS: The proteins of normal human colonic epithelial tissue from young and old people were separated by 2-dimensional gel electrophoresis (2DGE), respectively. Then gels were stained by silver, scanned by imagescanner and analyzed with PDQuest software. The differentially expressed protein spots of colonic epithelium between the old and the young groups were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: Well-resolved and reproducible 2DGE maps of normal human colonic epithelium from the young and the old were acquired. Nineteen more than 2 fold differentially expressed protein spots were identified representing 17 different proteins by MALDI-TOF-MS. The functions of these proteins involve in metabolism, energy generation, transportation, antioxidation, translation and protein folding. CONCLUSION Seventeen aging related proteins of human colonic epithelium identified indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium. These data provided useful clues for elucidating the mechanisms of colonic epithelial aging and aged colonic epithelial susceptibility to cancer.
Aging ; metabolism ; Cells, Cultured ; Cellular Senescence ; genetics ; Chloride Channels ; biosynthesis ; genetics ; Colon ; cytology ; Electron-Transferring Flavoproteins ; biosynthesis ; genetics ; Epithelial Cells ; cytology ; Humans ; Intestinal Mucosa ; cytology ; Proteins ; metabolism

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.
Adenoviruses, Human ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Flavoproteins ; chemistry ; genetics ; metabolism ; Humans ; Phototropins ; chemistry ; genetics ; metabolism ; Singlet Oxygen ; chemistry ; Staining and Labeling ; Transfection

Acceleration ; Acyl-CoA Dehydrogenase, Long-Chain ; genetics ; Animals ; Aorta ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Expression ; Heart ; Heme Oxygenase (Decyclizing) ; genetics ; Male ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley