Site-saturation mutagenesis is a newly-developed technology in protein engineering.By manipulating the encoding genes,it can rapidly obtain the mutants of desired proteins whose target residues are substituted by 19 other common amino acids.Site-saturation mutagenesis could serve not only as a powerful tool in protein engineering,but also as an important method in exploring the structure-function relationship of proteins.Several techniques were summarized to achieve site-saturation mutagenesis and introduce their application status in the protein engineering.The problem and promising future of its application were also discussed.

Objective To explore the iodine nutrition level of people, prevalence of iodine deficiency disorders and the thyroid function of women of childbearing age in pasturing areas of Tibet. Methods Thirty families were selected respectively in pastoral Dangxiong county and agricultural Qushui county of Lhasa in 2009,drinking water and edible salt samples were collected to test iodine content; at least 50 people from each crowd of the following populations including children aged 8 - 10, women of childbearing age of 18 - 49 old and male adults aged 18 - 60 were randomly sampled and to measure their urinary iodine content and for thyroid palpation. Direct titrimetric method was used to test salt iodine(GB/T 13025.7-1999); As3+-Ce4+ oxidation reduction process to test water iodine (GB/T 5750.1-2006); As3+-Ce4+ catalytic spectrophotometry using ammonium persulfate digestion to test urine iodine(WS/T 107-2006), and goiter examination was based on Diagnostic and Classificatory Criteria of Endemic Goiter (WS 276-2007). Results The median of water iodine was 1.3 μg/L in pasturing area and 0.7 μg/L in agricultural areas, there was no statistical significant difference between them(Z =- 1.809, P ＞ 0.05).There was no iodized salt used in pastoral people, but iodized salt coverage rate was 90.0％(27/30) in agricultural residents. The median of urinary iodine among people of pasturing areas was 50.2 μg/L, lower than that of agricultural areas( 193.2 μg/L, Z =- 10.48, P ＜ 0.01 ). However, the goiter rate in pasturing area[1.0％(1/100)]was significantly lower than that of agricultural areas[18.0％(18/100) , x2 =16.8, P ＜ 0.01]. Serum level of FT4 and TT4 in pastoral population[(14.0 ± 2.0)pmol/L, (85.6 ± 17.5)nmol/L] was significantly lower than that of agricultural areas[(16.2 ± 6.3)pmol/L, (95.4 ± 21.1)nmoL/L, t =- 2.06, - 2.20, all P ＜ 0.05]. The thyroid dysfunction rate[5.9％ (2/34)]and subclinical hypothyroidism rate[2.9％ (1/34)]in pastoral population was significantly lower than that of agricultural areas[25.5％(12/47), 21.3％(10/47), x2 =5.328, 5.651, all P ＜ 0.05]. Conclusions Pastoral areas of iodine intake is significantly lower than the agricultural areas, urinary iodine levels reflect a serious iodine deficiency in pastoral people, but the blood biochemical and urinary iodine and goiter rate does not match,and shows hidden iodine hunger, which does not constitute a goiter epidemic.

The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq.HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles.The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR.RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line.Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs.The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line.The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (P＜0.001).Additionally,we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics,cell adhesion,invasion and migration.The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lncRNA UC001kfo.LncRNA UC001kfo may play a role in regulating cancer cell invasion and metastasis.It was suggested that mRNAs were differentially expressed in the HepG2 cell line after the down-regulation of lncRNA-UC001kfo.Some took part in the extracellular matrix,cell adhesion,motility,growth,and localization.The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

Tilianin was separated and authenticated from the seeds of Dracocephalum moldavia, a Uygur medicine, by chromatographic technique and spectroscopic method. The purity of tilianin is more than 98% determined by HPLC area normalization method. Thin layer chromatography (TLC) method was used to separate tilianin from D. moldavia by mixture of chloroform-methanol (5: 1) as a developing solvent on high performance silicagel precoated plate (SGF254) and using aluminium trichloride as a chromogenic agent for qualitative identification of D. moldavia. To establish a HPLC method for quantitative analysis of D. moldavia, tilianin was used as a Quantitative marker and separated on a C18 (4.6 mm x 250 mm, 5 μm) column with acetonitrile-01% formic acid (25: 75) as the mobile phase and detected at 330 nm. The calibration curve of tilianin displayed ideal linearity over the range of 0.617 2-123.44 μg x mL(-1) with a regression equation of Y = 33.773X - 0.824 8 (r = 1). The average recovery of tilianin was 101.0% with RSD of 3.7%. The RSD values of intra-day and inter-day precision were less than 2%. The content of tilianin in 4 batches of the authenticated semen of D. Moldavia was between 0.016 and 0.187 mg x g(-1). The qualitative and quantitative method established is suitable for the quality evaluation and assessment of semen of D. Moldavia.
Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Flavonoids ; chemistry ; isolation & purification ; standards ; Glycosides ; chemistry ; isolation & purification ; standards ; Lamiaceae ; chemistry ; Magnetic Resonance Spectroscopy ; Quality Control

OBJECTIVETo observe the effect of Yangjing Zhongyu Decoction (YZD) on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulose cells (GCs) cultured by excess androgen.

METHODSOvarian GCs from porcine follicles were isolated and cultured in vitro. Follicular stimulating hormone (FSH) or YZD was added in the GCs treated by excess testosterone propionate. Totally 48 h later mRNA and protein expression of PCNA, StAR, and FSHR were detected by RT-PCR and Western blot.

RESULTSExcess androgen inhibited mRNA and protein expression of PCNA, StAR, and FSHR of GCs. FSH and YZD could antagonize inhibition of excess androgens, and promote mRNA and protein expression of PCNA, StAR, and FSHR in GCs.

CONCLUSIONYZD could antagonize the inhibition of excess androgen on mRNA and protein expression of PCNA, StAR and FSHR in GCs. Thus, we inferred that YZD could improve the follicle dysplasia by promoting mRNA and protein expression of PCNA, StAR and FSHR in GCs.

Androgens ; pharmacology ; Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; Ovarian Follicle ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; RNA, Messenger ; genetics ; Receptors, FSH ; genetics ; metabolism ; Swine

AlM:To discuss the protective effect ofα-mangostin on retinal light damage in mice.METHODS:Totally 30 Balb/c mice, aged 6~8wk, were randomly divided into the control group, light-exposure group and α-mangostin group. Every group contained 10 mice. Mice of α-mangostin group were treated with alpha-mangostin at the dose of 30mg/( kg · d ) body weight by intragastric administration daily for 7d, and then exposed to white light at the 5th d. The light-exposure group and α-mangostin group were exposed to 5 000 ± 200lx white light-emmiting diodes (LEDs) for continuously 1h to establish the mice model of retinal light damage. Flash -electroretinograme was recorded 72h after light exposure. The changes in retinal morphology of mice were observed by light microscopy. Retinas were extracted to detect the malondialdhyde ( MDA ) content change of the retinal homogenate.RESULTS: Flash-electroretinogram ( F-ERG ) showed that retinal dysfunction was less severe in α-mangostin group than in light-exposure group ( P<0. 05 ). Light microscopy test showed that retina structural damage was less severe in α-mangostin group than in light-exposure group (P<0. 05). The level of MDA in retinal tissue of α-mangostin group was significantly lower when compared with light-exposure group (P<0. 05).CONCLUSlON: α-mangostin inhibits lipid peroxidation induced by light damage and protect retina against light damage.

Objective To investigate the prevalence and influencing factors related to abuse and negligence against the elderly in the rural areas. Methods 975 elderly over 60 years from 41counties in Anhui province were included. All participants completed an anonymous questionnaire including items as: educational background, marital condition, income, child-discipline, rude action to parents, daily activities, physical functions, having chronic illness, abuse and negligence against the elderly, etc. Results In the last year, rates of common physical abuse, serious physical abuse,emotional abuse, financial exploitation, negligence, overall abuse and negligence against the elderly were 4.5%, 1.5%, 26.9%, 4.9%, 7.2%, 29.9% respectively. Among the 281 victims, 80.4% reported that they were suffered more than 3 times of abuse and neglect episodes, and 34.9% reported that they were suffered more than 2 forms of abuse and negligence. The primary sadism was carried out by the daughter-in-law or son-in-law (43.2%) of the elderly. Low activity on daily life and having chronic illness were the risk factors causing common physical abuse while better education was the protective factor to it, Low ability in managing daily activity of living was the risk factor causing serions physical abuse. Less active on daily life and having rude action to parents were the risk factors to emotional abuse, but being strict with their children was the protective factor to emotional abuse. Less active on daily life, often beating their children and having rude action to parents were the risk factors related to financial exploitation. Less active on daily life, having rude action to parents and having bad physical functions were the risk factors causing negligence. Less active on daily life and having rude manner to parents were the risk factors of overall elderly abuse and negligence, but being strict with their children was protective factor to the abuse and negligence against the elderly. Conclusion High prevalence on abuse and negligence against the elderly was seen in the rural areas of China. Different forms on elderly abuse and negligence were affected by different factors that called for more attention to be paid to those elderly with lower ability in managing their daily life.

OBJECTIVETo establish the allele specific primer polymerase chain reaction sequence-based typing (PCR-SBT) and investigate the polymorphism of exon 3 of human leukocyte antigen( HLA)-DRB1.

METHODSThe fragment containing exons 2 and 3 of HLA-DRB1 gene was amplified by group specific primers. The amplified products were digested by restriction enzymes and directly sequenced in both directions. The genotype was assigned by using Assign 3.5 SBT software.

RESULTSThe exon 3 sequences of HLA-DRB1*08:09 and HLA-DRB1*12:02:01 were identified for the first time. There were 27 polymorphic sites in exon 3 among the twenty-five HLA-DRB1 alleles, which was 9.56% of all nucleotides of exon 3. The method could discriminate the HLA-DRB1*14:01:01/14:54 ambiguous samples, and the HLA-DRB1*14:01:01 was identified in the Chinese population.

CONCLUSIONThe PCR-SBT method for exon 3 of HLA-DRB1 from the present study was reliable and there were polymorphisms in exon 3 of HLA-DRB1.

Alleles ; Base Sequence ; DNA Primers ; genetics ; Evolution, Molecular ; Exons ; genetics ; Genotype ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; genetics

OBJECTIVETo analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband.

METHODSThe ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed.

RESULTSThe serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband.

CONCLUSIONA novel B112 allele of ABO group system with 559C>T was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

ABO Blood-Group System ; genetics ; Alleles ; Asian Continental Ancestry Group ; genetics ; Exons ; genetics ; immunology ; Female ; Genotype ; Humans ; Male ; Mutation, Missense ; genetics ; Pedigree ; Phenotype

OBJECTIVETo establish the eukaryotic cell expression system for the alpha-1,2 fucosyltransferase gene FUT1 293C to T and 658C to T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen.

METHODSGenomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified. The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography. Expressed protein was identified by SDS-PAGE and Western blotting.

RESULTSpcDNA3.1-FUT1 wildtype, pcDNA3.1-FUT1 293C to T and pcDNA3.1-FUT1 658C to T recombinant vectors were constructed, respectively. COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418. The FUT1 mRNA level of transfected cells with 293C to T and 658C to T recombinant vectors reached 97.10% and 104.74% of the wildtype FUT1 transfected cell. A specific protein band with about 46 000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6×His Tag antibody. The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction, while the enzyme activity of cell lysates from 293C to T and 658C to T were abolished.

CONCLUSIONThe results suggested that the 293C to T and 658C to T mutations of FUT1 gene did not affect the RNA and protein expression levels, but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.

Animals ; COS Cells ; Cercopithecus aethiops ; Fucosyltransferases ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Mutant Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics