To improve ethanol production in Saccharomyces cerevisiae,an integration plasmid pUPGKAT with PGK promoter(phosphoglycerate kinase promoter),adh1 gene(the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator(Cytochrome c transcription terminator) was constructed.Firstly,a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA.Subsequently,CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA.To integrate PGK-adh1-CYC1 into S.cerevisiae genome,pUPGKAT was digested by TthⅢⅠand the lin-earized plasmid was used to transform S.cerevisiae YS2-△adh2(adh2 disrupted strain) by lithium acetate method.The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed.Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.

OBJECTIVETo detect the methylenetetrahydrofolate reductase(MTHFR) gene C677T mutation with molecular beacon technique and assess the revant applicability.

METHODSA total of 228 samples were analyzed using molecular beacons which are oligonucleotide probes to become fluorescent upon hybridization. Wild-type molecular beacon and mutant beacon were designed to detect the genotypes of MTHFR gene.

RESULTSAnalysis of the 228 samples indicated that there were three genotypes including 41 homozygous mutants, 113 heterozygous individuals and 74 wild-type individuals. Every sample was identified clearly.

CONCLUSIONThe present method, a closed-tube PCR/hybridization assay, is a simple, high-throughput and fast procedure that is fully automated for detecting gene mutation.

DNA Mutational Analysis ; methods ; Fluorescent Dyes ; chemistry ; Genotype ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Oligonucleotide Probes ; chemistry ; genetics ; Point Mutation ; Sensitivity and Specificity

Obiective:To elucidate the phenotype and the genotype-phenotype correlations in Chinese patients with X-linked adrenoleukodystrophy(X-ALD).Methods:Clinical features of 40 Chinese patients with X-ALD were studied and mutation spectrums were investigated by polymerase chain reaction (PCR) and sequencing. Results:Among these patients, four were siblings from two unrelated families, the others were unrelated. There were 31 cases with childhood cerebral (CCALD), 8 cases with adolescent cerebral (ACALD) and 1 case with adrenomyeloneuropathy (AMN). Visual impairment, which presented in 12 cases (30%), was the most common initial symptom. Nine (69%) of 13 cases who had hydrocortisone and ACTH measured showed adrenal insufficiency. By follow-up date, 19 cases (47.5%) were dead. The interval from onset to death varied from 1 to 6 years and the average were 3.3 years. The mean age at death was 10.5 years. Eleven cases (27.5%) were in vegetable state. The mean interval from onset to apparently vegetable state was 2.8 years (range from 1 to 6 years). Four cases had progressive neurological disability. Four cases were lost follow-up. One case with CCALD and one case with ACALD progressed slowly. The courses of the disease of these two patients were 5 years and 15 years respectively. Thirty five mutations were identified in 40 cases. Most were located within exon 1-3 (40%, 16/40) and exon 6-8 (42%, 17/40). There is a distinct clustering of missense mutations in exon 6 (17%, 7/40). Five types of mutations were associated with CCALD, three with ACALD and a missense mutation was identified in the patients with AMN. The two patients with long disease courses had a missence mutation c.1559 T>A and a nonsense mutation c.1785 G>A respectively. The siblings with similar manifestations and onset age were observed in two families, whose mutations were c.887 A>G and c.1028 G>T. Conclusion:The phenotypes, disease severity and rate of neurodegeneration could not be predicted by the nature of mutations.

Objective To explore the clinical characteristics of cardiac involvement in children with mitochondriopathies.Methods The clinical data of 23 children with mitochondriopathies were reviewed.The changes of electrocardiography,echocardiography and heart enzymes were analyzed.Results In 15 cases of mitochondrial encephalomyopathy,lactic acidosis,and stroke-like episode(MELAS syndrome),electrocardiography was performed on 9 cases,6 of them showed abnormal electrocardiographic findings,including right bundle branch block,ST-T change,Wolff-Parkinson-White syndrome,et al.On echocardiographic examination in 9 MELAS syndrome ca-ses,only 1 case showed hypertrophy cardiomyopathy.Six cases had increased plasma creatine kinaseMB(CK-MB) mass and only one of 12 MELAS syndrome cases had increased cardiac troponin I(cTnI) level.In 8 cases of subacute necrotizing encephalomyopathy(Leigh syndrome),electrocardiography was performed on 5 cases,4 of them showed abnormal electrocardiographic findings,including sinus tachycardia,ST-T change and low voltage.Two cases showed normal electrocardiography.Three out of 6 cases with Leigh syndrome showed increased plasma CK-MB mass.The molecular genetic examinations were performed in 13 cases of MELAS syndrome and 6 cases of Leigh syndrome.The mitochondrial DNA nt 3243 A→G mutation was found in white blood cells of 9 MELAS syndrome cases,the mutation rate being 37%-60%.The mitochondrial DNA nt 8993 T→C mutation was found in white blood cells of 2 Leigh syndrome cases.Conclusion In children with mitochondriopathies,myocardiac involvement is comparatively common,and even cardiomyopathy can occur.

Traditional Chinese medicine (TCM) reference standards plays an important role in the quality control of Chinese herbal pieces. This paper overviewed the development of TCM reference standards. By analyzing the 2010 edition of Chinese pharmacopoeia, the application of TCM reference standards in the quality control of Chinese herbal pieces was summarized, and the problems exiting in the system were put forward. In the process of improving the quality control level of Chinese herbal pieces, various kinds of advanced methods and technology should be used to research the characteristic reference standards of Chinese herbal pieces, more and more reasonable reference standards should be introduced in the quality control system of Chinese herbal pieces. This article discussed the solutions in the aspect of TCM reference standards, and future development of quality control on Chinese herbal pieces is prospected.
Drugs, Chinese Herbal ; standards ; Humans ; Medicine, Chinese Traditional ; standards ; Quality Control ; Reference Standards

A(p.G888S)were detected in POLG1 gene.Sequence analysis of parental blood DNA revealed that her father carried L83P and her mother carried G888S.Conclusions The characteristics of clinical manifestation,electrophysiology,pathology and POLG1 gene mutation of the patient were highly consistent with Alpers syndrome.The prominent white matter change and increased immunological factors in CSF were first reported in Alpers syndrome.Alpers syndrome should be considered for those patients whose liver function were severely impaired after exposure to valproic acid.

Tumor hypoxia is the necessary process in the development of solid tumors, which is the key factor for drug resistance, recurrence, attack and shift of tumor. Hypoxic tumor cells have a certain extent of tolerance to radiation and chemotherapy. Tumor hypoxia is an important target for medication therapy. In the recent years, the bioreductive drugs targeted tumor hypoxia has made great process in the treatment of tumors. The latest advances of bioreductive drugs targeted hypoxia were reviewed in this paper.
Antineoplastic Agents ; therapeutic use ; Cell Hypoxia ; Humans ; Hypoxia ; Neoplasms ; drug therapy ; Quinones ; therapeutic use ; Reducing Agents ; therapeutic use

In order to explored the change of PML/PML-RARalpha protein during tetraarsenic tetrasulfide (As4S4) treatment, acute promyelocytic leukemia (APL) cells from a group of newly diagnosed APL patients were examined by indirect immunofluorescence staining with anit-PML monoclonal antibody. The results showed that all samples typically presented many microspeckle signals throughout the nucleus before treatment. The redistribution occurred as early as on the second day after As4S4 treatment, which revealed loss of microspeckles with the presentation of a few large speckles. Anti-PML staining also emerged in the perinuclear cytoplasm. At last, microspeckles and large speckles all disappeared. When the therapy was combining all-trans-retinoic acid (ATRA) with As4S4, similar results were obtained. However, APL cells from patients treated with ATRA alone performed totally different appearance, presenting microspeckles and large speckles at the same time, followed with entirely large speckles. The conclusion is that As4S4 makes redistribution of PML/PML-RARalpha protein in leukemic cells from APL patients during the treatment, which is quite different from that during the treatment of ATRA.
Adolescent ; Adult ; Aged ; Antineoplastic Agents ; therapeutic use ; Arsenicals ; therapeutic use ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; In Situ Nick-End Labeling ; Leukemia, Promyelocytic, Acute ; drug therapy ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; analysis ; Nuclear Proteins ; Oncogene Proteins, Fusion ; analysis ; Promyelocytic Leukemia Protein ; Transcription Factors ; analysis ; Tretinoin ; therapeutic use ; Tumor Suppressor Proteins

Objective To identify risk factors for a human orf disease outbreak in a village in Chongqing city. Methods Standardized case-definition was set and a case-finding program was conducted among all the residents of the village. All the patients were interviewed using a standardized questionnaire and collected fluids in the skin rash for laboratory testing. A retrospective cohort investigation was conducted among all the village residents who introduced the black goats to analyze the risk of orf infection, in relation to the mode and frequency of contacts to the infected goats. Results We found 18 cases (including 16 suspected cases and 2 confirmed cases) among the members of 10 families that introduced the black goats. Village residents who had ever used their legs to grip the goats were nearly five times as likely to develop orf disease as those who did not (RR=4.98, 95％CI: 1.34-75.27). Village residents who had ever washed and wiped the goats were three times as likely to develop orf disease as those who had not (RR = 3.09,95％CI: 0.98-45.38). The frequency of contacts with the infected goats was associated with the risk of developing orf disease in a dose-response fashion (x2 test for trends: P= 0.006).Frequently wearing long trousers when dealing with the goats appeared as a protective factor (RR=0.30,95％CI: 0.15-0.78). Conclusion This outbreak was caused by the introduced black goats which carried and infected by the orf virus. Direct physical contact with infected goats but without wearing protective clothing were risk factors for the development of human orf disease.

Objective To investigate the effects of fluoride on trabecular bone of the tibia and lumbar in BALB/c mice.Methods Totally 64 four-week-old BALB/c mice were randomly divided into 4 groups by weight,16 per group:control group (treated with distilled water) and 3 sodium fluoride (NaF) exposure groups (treated with NaF at 25,50 and 100 mg/L F-),respectively.At 12 weeks,mice were killed and blood,two hind limbs and lumbar were collected.Bone fluoride content and incidence rates of dental fluorosis were determined.Serum content of alkaline phosphatase (AKP) and acid phosphatase (ACP) were detected by micro enzyme labeled method.The ultrastructure of osteoblasts and osteoclasts in lumbar were observed via transmission electron microscope.The pathological changes of the trabecular bone of the tibia and the lumbar were observed under optical microscope,the percentage of trabecular area (％Tb.Ar) was measured with Image-Pro Plus (IPP) software.Results Bone fluoride contents of low,middle and high fluoride groups [(1 828.62 ± 102.93),(3 308.27 ± 185.63),(4 933.36 ± 301.16) mg/kg] were higher than that of the control group [(775.23 ± 92.56) mg/kg,all P ＜ 0.05].The incidences of dental fluorosis in the 4 groups were 0(0/16),47％(7/15),93％(14/15) and 100％(16/16),respectively;the difference was statistically significant (x2 =27.23,P ＜ 0.05).In middle and high fluoride groups,serum AKP [(18.30 ± 1.99),(24.50 ± 3.14) king unit/100 ml] and ACP [(11.97 ± 1.73),(11.31 ± 1.46) king unit/100 ml] were significantly higher than those of control [(14.63 ± 1.21),(9.07 ± 1.47) king unit/100 ml,respectively,all P ＜ 0.05].Under the electron microscope,osteoblast had developed organelles in each fluoride group,rough endoplasmic reticulum,Golgi body,and mitochondria were abundant,and nucleolus was obvious in the osteoblast.Osteoclast was rich in mitochondria,ruffled border clear and distributed phagocytic vacuoles in low fluoride group and middle fluoride group.Compared with the control group (17.03 ± 3.73),HE staining of tibia ％Tb.Ar in high fluoride group (28.79 ± 8.26) was significantly increased (P ＜ 0.05).The lumbar spine ％Tb.Ar in low,middle and high fluoride groups (15.87 ± 2.59,18.28 ± 0.89,21.99 ± 1.81) were higher than that of the control group (12.06 ± 1.76,all P ＜ 0.05].Conclusions BALB/c mice could be used as a model of skeletal fluorosis.Osteoblast and osteoclast are activated in BALB/c mice with skeletal fluorosis.Bone formation is more obvious than bone resorption and bone mass is increased.What is more,bone mass has increased more significantly in the lumbar spine of mice.