OBJECTIVEIn this report are reviewed two unrelated patients with typical normokalemic periodic paralysis (normoKPP) features and the results of screening the SCN4A gene for the disease-related mutation.

METHODSTwo sporadic cases with normoKPP were screened for previously known mutations in SCN4A gene (T704M, A1156T, M1360V, I1495F, M1592V) that lead to hyperKPP; denaturing high performance liquid chromatography (DHPLC) was used. Then the rest exons of SCN4A gene were screened by DHPLC, and sequence analysis was performed on those with DHPLC chromatogram variation when compared with unaffected control.

RESULTSTwo cases and one patient's father were detected with V781I, which was proved to be a singular missense mutation in SCN4A gene.

CONCLUSIONThe mutation V781I exists in Chinese patients with normoKPP and may be responsible for normoKPP.

Adult ; Amino Acid Sequence ; Base Sequence ; Child ; Chromatography, High Pressure Liquid ; methods ; DNA ; analysis ; genetics ; Exons ; Female ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; NAV1.4 Voltage-Gated Sodium Channel ; Paralyses, Familial Periodic ; genetics ; Point Mutation ; Sequence Analysis, DNA ; Sodium Channels ; genetics

OBJECTIVETo investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification.

METHODSSH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR).

RESULTSIn high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride.

CONCLUSIONFluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

Cell Line, Tumor ; Fluoride Poisoning ; metabolism ; Fluorides ; toxicity ; Humans ; Neuroblastoma ; metabolism ; pathology ; Protein Processing, Post-Translational ; drug effects ; Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Nicotinic ; biosynthesis ; genetics

OBJECTIVETo compare the painless effect of four anesthetic methods during opening pulp cavity and undergoing pulpectomy for acute or chronic pulpitis.

METHODS80 teeth of 80 patients were randomly allocated into four groups. Each group had 20 teeth. Anesthetic methods applied four different groups included block anesthesia of nerve, supraperiosteal infiltration, periodontal membrane injection and intrapulpal injection. Anesthesia doses were recorded and the pierced points, the zones of pain, the time of anesthesia action, the time of anesthesia persistence and the degrees of anesthesia were evaluated with four levels synthetic evaluation standard of anesthesia.

RESULTSCompared with periodontal membrane injection and intrapulpal injection, block anesthesia of nerve and supraperiosteal infiltration had the later time of anesthesia action and the longer time of anesthesia persistence (P<0.05). In four anesthetic methods, block anesthesia of nerve had the best painless effect (P<0.05).

CONCLUSIONFour anesthetic methods have their own superiorities, and we should select proper anesthetic methods in clinical work.

Adult ; Anesthesia, Dental ; Anesthetics ; Anesthetics, Local ; Bicuspid ; Female ; Humans ; Injections ; Lidocaine ; Male ; Mandibular Nerve ; Nerve Block ; Periodontal Ligament ; Pulpectomy ; Pulpitis

OBJECTIVETo establish a new method for the determination of fangchinoline and tetrandrine in Stephania tetrandra and Fengtongan capsule by noanqueous capillary electrophoresis.

METHODSeparation was carried out in an uncoated fused capillary (50 cm x 75 microm i.d.) with a running buffer containing 50 mmol x L(-1) ammonium acetate, 1.0% acetic acid and 20% acetonitrile in methanol. A separation voltage of 20 kV and a UV detector wavelength at 214 nm were adopted. Sample was introduced from the anode.

RESULTThe calibration ranges were 1.00, 500 mg x L(-1) for both analytes. Under the optimum conditions, the relative standard deviation (RSD, n = 6) for the migration time of each analyte were 0.09%, 1.9% (intra-day) and 0.63%, 1.9% (inter-day); The RSD for the peak area of each analyte were 0.45%, 5.9% (intra-day) and 2.3%, 5.6% (inter-day), respectively. The contents of the analytes were determined easily with average recoveries 102% for fangchinoline and 105% for tetrandrine in S. tetrandra and 94.6% for fangchinoline and 98.7% for tetrandrine in Fengtongan capsules, respectively.

CONCLUSIONThe proposed method is simple, rapid, accurate and higher repeatable, and can be used to control of the quality of S. tetrandra and Fengtongan capsules.

Benzylisoquinolines ; analysis ; Calibration ; Capillary Electrochromatography ; methods ; Capsules ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results ; Stephania tetrandra ; chemistry

Acrylamide ; toxicity ; Animals ; Behavior, Animal ; drug effects ; Blotting, Western ; Cytoskeletal Proteins ; blood ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Gait Ataxia ; blood ; chemically induced ; Male ; Motor Activity ; drug effects ; Neurotoxicity Syndromes ; blood ; etiology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the efficiency of multiplex ligation-dependent probe amplification (MLPA) combined with short tandem repeat (STR) linkage analysis for the prenatal diagnosis for Duchenne muscular dystrophy (DMD).

METHODSGender of the fetus was first determined by the presence of Y chromosome sex-determining gene (SRY). Subsequently, combined MLPA and STR linkage analysis were applied for the probands, pregnant women and fetuses in 45 affected families.

RESULTSAmong the 45 families, 31 SRY-positive fetuses were identified, among whom six were diagnosed with DMD. For 14 SRY-negative fetuses, four were diagnosed as carriers. The remainders were normal.

CONCLUSIONMLPA can detect mutations in the exons of dystrophin gene, whilst STR linkage analysis can determine whether the fetus has inherited the maternal X chromosome bearing the mutant gene. As the result, the method can detect affected fetuses in which no exonic mutations are detected with MLPA. By combining the two methods, the diagnostic rate for DMD can be greatly improved.

Dystrophin ; genetics ; Exons ; Female ; Genetic Linkage ; Heterozygote ; Humans ; Male ; Microsatellite Repeats ; Multiplex Polymerase Chain Reaction ; Muscular Dystrophy, Duchenne ; diagnosis ; genetics ; Mutation ; Pregnancy ; Prenatal Diagnosis

OBJECTIVETo determine the feasibility and accuracy of detecting numerical chromosomal abnormalities by high-flux sequencing analysis of free fetal DNA from maternal plasma.

METHODSHigh-flux sequencing was applied to analyze fetal chromosome sequence copy numbers in 153 pregnant women. Fetal karyotyping was also carried out on amniocentesis samples.

RESULTSSix cases were detected with fetal chromosomal abnormalities by high-flux sequencing analysis, among which five were confirmed by karyotyping to be chromosomal aneuploidies (47,XYY; 45,X; 47,XY,+18; 47,XY,+21 and 47,XY,+13), 1 case was confirmed to be structural rearrangement, i.e., 46,XY,der(13;21)(q10;q10),+21. Furthermore, 3 chromosomal polymorphisms (one 46,XY,21p+ and two 46,XY,Yqh-) were identified. The two methods yielded similar results on fetal chromosome copy number detection.

CONCLUSIONHigh-flux sequencing analysis of free DNA derived from maternal plasma is efficient for detecting fetal chromosomal aneuploidies, and is non-invasive, highly sensitive and specific. It therefore has a broad application in antenatal diagnosis.

Adult ; Amniocentesis ; methods ; Aneuploidy ; Chromosome Disorders ; diagnosis ; genetics ; DNA ; chemistry ; genetics ; Female ; Fetus ; Humans ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult

OBJECTIVEMutation screening was performed to a Chinese family with hypokalaemic periodic paraiysis(HOKPP) for locating the corresponding mutations of gene and for specifying the clinical features associated with mutations.

METHODSThe cilnical features of patients from HOKPP family were summurized. Techniques of target exon PCR and direct sequencing were used to screen the mutation in CACNA1S and SCN4A genes in all numbers of the family.

RESULTSTwo patients of the family showed the typical features of HOKPP: the age of disease onset is during the childhood, acetazolamide is effective to patients treated. A heterozygous point mutation 3716 (G>A) causing R1239H was found in exon 30 of CACNA1S gene of the patients, but not found in normal members of the family.

CONCLUSIONThe mutant R1239H in CACNA1S gene exists in Chinese patients with familial hypokalaemic periodic paralysis.

Adolescent ; Adult ; Base Sequence ; Calcium Channels ; genetics ; China ; DNA Mutational Analysis ; Family Health ; Female ; Humans ; Hypokalemic Periodic Paralysis ; genetics ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo study the protective impact of tea polyphenols (TP) on the injury of fibrinolytic functions induced by high-methionine dietary in rats.

METHODS50 male Wistar rats were divided by stratified based on body weight into 5 groups with 10 in each group: namely control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group. The rats in model group and TP groups were fed with 3% methionine dietary, control group rats with routine diet. In addition, rats in low-dose, medium-dose and high-dose TP groups were treated with TP at 50, 100 and 200 mg/kg dosage respectively by gavages every day, control group and model group rats were given with same amount distilled water. The animals were sacrificed after 8 weeks. The levels of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in plasma were determined by ELISA assays, mRNA levels of t-PA and PAI-1 in aortic arch were detected by RT-PCR, t-PA and PAI-1 expression in aortic arch were detected by immunohistochemistry strept-avidin-biotin complex (SABC).

RESULTSAfter experiment, the t-PA expression of aortic arch in control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group were 133.03 ± 10.14, 95.46 ± 11.08, 111.97 ± 11.91, 130.23 ± 10.80, 139.39 ± 9.41 (F = 14.15, P < 0.01), respectively, and the PAI-1 expression were 90.91 ± 8.67, 166.76 ± 12.18, 139.63 ± 12.71, 134.66 ± 13.19, 109.49 ± 10.82 (F = 31.44, P < 0.01). The t-PA concentration of plasma were (10.69 ± 1.26), (6.13 ± 0.92), (8.56 ± 1.19), (9.69 ± 0.92), (11.97 ± 1.08) ng/ml, respectively (F = 41.98, P < 0.01), and the PAI-1 concentration of plasma were (6.31 ± 0.81), (16.98 ± 1.27), (11.39 ± 0.82), (8.46 ± 0.67), (8.08 ± 0.91) ng/ml, respectively (F = 207.74, P < 0.01). The mRNA levels of t-PA in aortic arch were 1.12 ± 0.02, 0.75 ± 0.14, 1.01 ± 0.09, 0.95 ± 0.08, 1.05 ± 0.13 (F = 5.77, P < 0.05), and the mRNA levels of PAI-1 in aortic arch were 1.25 ± 0.11, 1.74 ± 0.06, 1.23 ± 0.05, 1.09 ± 0.14, 1.23 ± 0.04 (F = 23.56, P < 0.01).

CONCLUSIONThe results indicate that TP seems to have regulatory function on transcription and protein levels of t-PA and PAI-1, in addition to maintaining the balance between PAI-1 and t-PA and healing the injury of fibrinolytic functions in rats induced by high-methionine dietary.

Animals ; Diet ; Fibrinolysis ; drug effects ; Male ; Methionine ; adverse effects ; Plasminogen Activator Inhibitor 1 ; blood ; Polyphenols ; pharmacology ; Rats ; Rats, Wistar ; Tea ; chemistry ; Tissue Plasminogen Activator ; blood

OBJECTIVETo clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells.

METHODSThe entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope.

RESULTSThe entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation.

CONCLUSIONSThe entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

Cell Line, Tumor ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Receptors, Purinergic P2 ; genetics ; physiology ; Receptors, Purinergic P2X7 ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection