OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

Glycogen storage disease type V (GSD-V) is the most common disorder of muscle glycogenosis with characteristic clinical and laboratory findings. A 32-yr-old woman complained of exercise intolerance and myoglobulinuria since early adolescence. She reported several episodes of second-wind phenomenon. Physical examination did not show any neurological abnormality, including fixed muscle weakness or atrophy. Serum creatine kinase level was 1,161 IU/L at rest. The result of the non-ischemic forearm exercise test was compatible with GSD-V. Mutation analysis identified the compound heterozygous mutations of the PYGM, p.D510fs and p.F710del, which has not yet been reported in Korea. The present case recognizes that detail clinical and laboratory analysis is the first step in the diagnosis of GSD-V.
Adult ; Base Sequence ; Creatine Kinase/blood ; Exons ; Female ; Frameshift Mutation ; Gene Deletion ; Genotype ; Glycogen Phosphorylase, Muscle Form/genetics ; Glycogen Storage Disease Type V/*diagnosis/genetics/pathology ; Humans ; Pedigree ; Sequence Analysis, DNA

Antiporters ; genetics ; Base Sequence ; Female ; Glycogen Storage Disease Type I ; diagnosis ; genetics ; Humans ; Infant ; Monosaccharide Transport Proteins ; genetics ; Mutation ; Polymerase Chain Reaction ; Sequence Analysis, DNA

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3beta) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3beta kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.
Alzheimer Disease ; Amyloid ; Catalytic Domain ; Down-Regulation ; Glycogen Synthase Kinase 3 ; Negotiating ; Neurofibrillary Tangles ; Phosphorylation* ; Phosphotransferases ; Protein Isoforms ; Protein Phosphatase 2* ; Spectrum Analysis ; tau Proteins*

A beta-glucan synthase gene was isolated from the genomic DNA of polypore mushroom Sparassis crispa, which reportedly produces unusually high amount of soluble beta-1,3-glucan (beta-glucan). Sequencing and subsequent open reading frame analysis of the isolated gene revealed that the gene (5,502 bp) consisted of 10 exons separated by nine introns. The predicted mRNA encoded a beta-glucan synthase protein, consisting of 1,576 amino acid residues. Comparison of the predicted protein sequence with multiple fungal beta-glucan synthases estimated that the isolated gene contained a complete N-terminus but was lacking approximately 70 amino acid residues in the C-terminus. Fungal beta-glucan synthases are integral membrane proteins, containing the two catalytic and two transmembrane domains. The lacking C-terminal part of S. crispa beta-glucan synthase was estimated to include catalytically insignificant transmembrane alpha-helices and loops. Sequence analysis of 101 fungal beta-glucan synthases, obtained from public databases, revealed that the beta-glucan synthases with various fungal origins were categorized into corresponding fungal groups in the classification system. Interestingly, mushrooms belonging to the class Agaricomycetes were found to contain two distinct types (Type I and II) of beta-glucan synthases with the type-specific sequence signatures in the loop regions. S. crispa beta-glucan synthase in this study belonged to Type II family, meaning Type I beta-glucan synthase is expected to be discovered in S. crispa. The high productivity of soluble beta-glucan was not explained but detailed biochemical studies on the catalytic loop domain in the S. crispa beta-glucan synthase will provide better explanations.
Agaricales ; Cell Wall ; Classification ; Clone Cells* ; Cloning, Organism* ; DNA ; Efficiency ; Exons ; Glycogen Synthase ; Humans ; Introns ; Membrane Proteins ; Open Reading Frames ; RNA, Messenger ; Sequence Analysis

OBJECTIVETo reveal the molecular genetic pathogenesis of the glycogen storage disease type III (GSDIII) and to provide a prerequisite for prenatal gene diagnosis in future.

METHODAll the coding regions as well as the border areas between exons and introns of the AGL gene and the parental relevant mutation sites were directly sequenced, so that to affirm the origin of the mutation. Then, detected novel heterozygous mutation was confirmed by cloning sequencing. Finally, definite diagnoses of the novel mutation were performed by a series of identification methods, including screening for the 100 normal controls by DHPLC in order to count the mutational frequency, analyze the conservative of the mutant amino acid sequence from 11 kinds of species and comprise the difference of the tertiary structure between the mutant protein and the normal one.

RESULTThe patient had compound heterozygous mutations, the c.100C>T (p.R34X) nonsense mutation and c. 1176_1178 del TCA deletion mutation. The p.R34X has been reported abroad, but the 1176_1178 del TCA/p.His392fs mutation is a novel one. The proband's father is heterozygous with the p.R34X mutation while his mother carries the c.1176_1178 del TCA mutation. The result from searching the dbSNP database, HGMD database and papers published in recent years showed that the c.1176_1178 del TCA is a novel mutation, but not an SNP. Conservative analysis results in 11 species indicate that the amino acid of the mutation site is highly conserved in the stage of evolution. Comparison results between the mutant protein and the normal one demonstrate that the deletion mutation results in the obvious variation of the spatial conformation of AGL protein.

CONCLUSIONThe "c.1176_1178 del TCA (p.392delHis)" mutation is a novel pathogenic mutation. This mutation and the c.100C>T (p.R34X) is the cause that the proband suffer from the GSDIIIa disease. These two mutations are inherited from mother and father respectively. The methods from this paper can be used for further prenatal gene diagnosis.

Adult ; Amino Acid Sequence ; Base Sequence ; Case-Control Studies ; Child, Preschool ; DNA Mutational Analysis ; Exons ; Female ; Glycogen Debranching Enzyme System ; chemistry ; genetics ; Glycogen Storage Disease Type III ; diagnosis ; genetics ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Protein Conformation ; Sequence Alignment

OBJECTIVEGlycogen storage disease type Ib (GSDIb) is caused by a deficiency of glucose-6-phosphate translocase (G6PT) activity due to SLC37A4 gene mutations. Most GSDIb patients have recurrent infections and inflammatory bowel disease, with poor prognosis. Detection of SLC37A4 gene mutations is of great significance for the diagnosis, subtyping and outcome prediction of GSD patients. This study aims to analyze SLC37A4 gene mutations in Chinese GSDIb patients and to investigate the relationship between its genotypes and clinical manifestations.

METHODSAll exons and their flanking introns of SLC37A4 gene in 28 Chinese children with a primary diagnosis of GSDIb were screened by PCR combined with direct DNA sequencing to detect SLC37A4 gene mutations.

RESULTSFive SLC37A4 gene mutations were detected in 7 (25%) of the 28 children, i.e., p.Gly149Glu (9/13, 69%), p.Gly115Arg (1/13, 8%), p.Pro191Leu (1/13, 8%), c.959-960 insT (1/13, 8%) and c.870+5G>A (1/13, 8%).

CONCLUSIONSIn this study, c.959-960 insT is a novel mutation and p.Gly149Glu is the most common mutation. p.Gly149Glu may be associated with severe infections in children with GSDIb.

Antiporters ; genetics ; Child, Preschool ; Female ; Glycogen Storage Disease Type I ; complications ; genetics ; Humans ; Infant ; Male ; Monosaccharide Transport Proteins ; genetics ; Mutation ; Sequence Analysis, DNA

OBJECTIVETo investigate the effect of aqueous solution of Biophytum sensitivum leaf extract (BSEt) on normal and streptozotocin (STZ)-nicotinamide-induced diabetic rats.

METHODSDiabetes was induced in adult male Wistar rats by the administration of STZ-nicotinamide (40, 110 mg/kg b.w., respectively) intraperitoneally. BSEt (200 mg/kg) was administered to diabetic rats for 28 days. The effect of extract on blood glucose, plasma insulin, total haemoglobin, glycosylated haemoglobin, liver glycogen and carbohydrate metabolism regulating enzymes of liver was studied in diabetic rats.

RESULTSBSEt significantly reduced the blood glucose and glycosylated haemoglobin levels and significantly increased the total haemoglobin, plasma insulin and liver glycogen levels in diabetic rats. It also increased the hexokinase activity and decreased glucose-6-phosphatase, fructose-1, 6-bisphosphatase activities in diabetic rats.

CONCLUSIONSThe results of our study suggest that BSEt possesses a promising effect on STZ-nicotinamide-induced diabetes.

Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; drug therapy ; Enzymes ; analysis ; Glycated Hemoglobin A ; analysis ; Glycogen ; analysis ; Hypoglycemic Agents ; isolation & purification ; therapeutic use ; Insulin ; blood ; Liver ; chemistry ; enzymology ; Male ; Niacinamide ; toxicity ; Oxalidaceae ; chemistry ; Plant Extracts ; isolation & purification ; therapeutic use ; Plant Leaves ; chemistry ; Plasma ; chemistry ; Rats, Wistar ; Streptozocin ; toxicity ; Treatment Outcome

Diabetes is a global threat threatening human health in the world, with an increasing incidence rate in recent years. The disorder of glucose metabolism is one of the major factors. As relevant glucose metabolic enzymes such as alpha-glucosidase, glucose-6-phosphatase (G-6-P), glycogen phosphorylase (GP) and glycogen synthase kinase-3 (GSK-3) get involved in and control the process of glucose metabolism, the regulation of the activity of glucose metabolic enzymes is of significance to the treatment of diabetes. Traditional Chinese medicines (TCMs) have been widely researched because of their low toxicology and high efficiency, and many extracts and components from TCMs have been proven to be regulators of glucose metabolic enzymes. Compared with anti-diabetic western medicines, anti-diabetic TCMs feature safety, reliability and low price. This essay summarizes the anti-diabetic effect of TCMs on regulating glucose metabolic enzymes.
Animals ; Diabetes Mellitus ; drug therapy ; enzymology ; metabolism ; Drugs, Chinese Herbal ; analysis ; therapeutic use ; Enzyme Activators ; analysis ; therapeutic use ; Enzyme Inhibitors ; analysis ; therapeutic use ; Glucose ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Humans ; Hypoglycemic Agents ; analysis ; therapeutic use ; alpha-Glucosidases ; metabolism

OBJECTIVETo investigate the mutation of glucose-6-phosphatase gene (G6PC gene) in a patient with glycogen storage disease Ⅰa.

METHODSPCR was used to amplify all five exons of G6PC gene. The PCR products were directly sequenced to detect the mutations.

RESULTSA heterozygous 743G>A mutation was found in the patient and his mother, resulting in the substitution of glycine (G) by arginine (R) in codon 222(G222R) in the putative membrane-spanning domain in human G6Pase, but not in his father and his sister.

CONCLUSIONSG222R mutation in G6PC gene was first identified in a patient with glycogen storage disease Ⅰa in mainland China.

Child, Preschool ; Glucose-6-Phosphatase ; genetics ; Glycogen Storage Disease Type I ; genetics ; Humans ; Male ; Mutation ; Sequence Analysis, DNA