This paper reported a new type of amylase (neoamylase) secreted by a Bacillus strain ZX99. The enzyme was a kind of ectoenzyme that could catalyze starch into isomalto-oligosaccharide effectively, but could not act on pullulan as substrate. The strain Bacillus ZX99 was mutated by ultraviolet ray and a mutant strain BS3.232 was screened. The activity of the neoamylase produced from BS3.232 increased by 60% over that from ZX99 under the same conditions. The results of thin-layer chromatography of products from starch and pullulan catalyzed by the enzyme demonstrated that the enzyme was different from neopullulanase and can be used to produce isomaltooligosaccharide from starch, including isomaltose, panose, isomaltotriose, isomaltotetose.

Background Studies showed that macophenolic acid (MPA)down-regulates and inhibits the expression and secretion of tissue growth factor and inflammatory factor,and further impacts the proliferation and inflammation process.Pterygium is an inflammatory and proliferative lesion.Whether MPA has an inhibitory effect on pterygium is unclear.Objective This study was to investigate the antifibrotic effects of macophenolic acid on pterygium fibroblasts(PFBs) in vitro and discuss its mechanism.Methods Pterygium tissue was obtained from pterygium patient during the surgery.PFBs were cultured using explants and identified with vimentin immunohistochemisty.0,0.125,0.250,0.500,1.000 μmol/L MPA were added into the culture medium,respectively,and the cells were cultured in the medium without MPA as the control group.MTT colorimetry was used to find the optimization effective concentration of MPA and evaluate their inhibitory effect on PFBs,and BrdU fluorescence staining was used to assess the growth statue of PFBs.Expressions of nuclear factor-κB(NF-κB),p65 and inhibitor of NF-κB-α(IκB-α) in the cells were detected by Western blot.Results The cells was spindle in shape 3 days after cultured and showed the vortex and radial arrangement with the positive response to vimentin.With the increase of MPA,the proliferative value of PFBs (A560)showed gradually decline,with a significant difference among the five groups (F =42.874,P＜0.01).In addition,the proliferative value of PFBs (A560) significantly lowed as the prolong of MPA active time(F=26.038,P＜0.01).BrdU fluorescence staining showed a significant decrease of DNA synthesis of PFBs with the elevation of MPA dose among the five groups(F=175.279,P＜0.05),and the A560of PFBs DNA synthesis in different concentrations of MPA groups was lower than that of the control group (all at P＜0.05).No apoptotic and necrotic cell was found after MPA action by DAPI staining.The expression level of p65 in the PFBs was 0.886±0.072 and 1.542±0.124 in the MPA group and the control group,indicating a declined value in the MPA group(P＜0.05).However,the expression value of IκB-α in the cytoplasm PFBs was significantly higher in the MPA group compared with the control group(2.141 ±0.305 vs.1.559±0.267) (P＜0.05).Conclusions MPA has an inhibitory effect on the growth of PFBs,which probably is related to the arresting of NF-κB pathway.

Animals ; Carps ; genetics ; Cloning, Molecular ; Fish Proteins ; genetics ; Hydroxylation ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Lakes

OBJECTIVETo evaluate the impact of platelet membrane glycoprotein (GP)-specific autoantibodies on high-dose dexamethasone therapy in patients with idiopathic thrombocytopenic purpura (ITP).

METHODSModified direct monoclonal antibody immobilization of platelet antigen assay (MAIPA) was used to detect platelet GPIIb/IIIa and/or GPI b specific autoantibodies. All patients received oral dexamethasone 40 mg/d for 4 days.

RESULTSThe response rate of high-dose dexamethasone in GPIIb/IIIa and/or GPIb specific autoantibody-negative patients was significantly different from that of antibody-positive patients (P<0.05). The response rate of GPIIb/IIIa specific autoantibody-positive patients was lower than that of antibody-negative patients (P<0.05). GPIb specific autoantibody had no significant impact on the efficacy of high-dose dexamethasone (P>0.05).

CONCLUSIONPlatelet membrane GPIIb/IIIa-specific autoantibody can be a potential negative indicator for ITP patients'response to high-dose oral dexamethasone.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Autoantibodies ; blood ; Dexamethasone ; administration & dosage ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; drug therapy ; immunology ; Treatment Outcome ; Young Adult

Bronchiolitis ; diagnosis ; microbiology ; pathology ; Female ; Humans ; Infant ; Leukotriene D4 ; analysis ; Male ; Nasopharynx ; chemistry ; microbiology ; pathology

OBJECTIVETo evaluate application of the sponge of demineralized bone matrix (SDBM) in tissue engineering of bone.

METHODSSDBM was prepared from long bone of rabbits. Bone marrow cells were flushed from the bone shaft of femurs of a two-month-old New Zealand white rabbit. After the cells were cultured for 9 days, the flasks were added into dexamethasone (10(-8) mol/L), beta-glycerophosphate sodium (10 mmol/L) and L-ascorbic acid (50 micrograms/ml). After 5 weeks, the cultured cells were collected and marked by 5-Bromo-2'-dexyouridine (BrdU). The grand sum of cells seeded on a piece of SDBM was about (4-6) x 10(6). The composites of cells and SDBM (tissue engineered chip, TEC) were implanted into muscles and bone defects of radius in rabbits. A standard procedure was applied to make a 10 mm long defect bilaterally in the radius of nine skeletally mature male New Zealand white rabbits. All of the 18 defects were randomly divided into three groups: group I, six defects were grafted by TEC; group II, six defects were grafted with SDBM alone; group III, six defects were empty.

RESULTSThe results of radiographic and histological evaluation showed that all of the defects were repaired in group I and group II at 6 weeks, none of the defects was repaired in group III. The results of BrdU staining showed that the staining was positive in group I, but negative in group II. Biomechanical test showed that the compressive ultimate strength (CUS) of new bone in TEC implanted group was comparable with normal radius (P = 0.623) and in SDBM implanted group was significant lower than normal radius (P = 0.038).

CONCLUSIONSThe TEC can form cartilage and bone tissue in muscles and repair segmental bone defects. SDBM is a kind of effective natural scaffold in tissue engineering of bone.

Animals ; Bone Demineralization Technique ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Bone Matrix ; Implants, Experimental ; Male ; Rabbits ; Radius Fractures ; surgery ; Random Allocation ; Stem Cells ; cytology ; Tissue Engineering

A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.
Colorimetry ; methods ; DNA Primers ; chemistry ; genetics ; Genotype ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 6 ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; Papillomavirus Infections ; virology

OBJECTIVETo explore the therapeutic effect of natural vitamin E (VitE) on oligospermia and asthenospermia in in- fertile men.

METHODSWe conducted a prospective multi-centered randomized controlled study on 64 infertile men with oligospermia (31 as controls treated with Tamoxifen 10 mg bid and 33 as experimental cases treated with Tamoxifen 10 mg bid + VitE 100 mg tid) and 42 cases of asthenospermia (20 as controls treated with Levocarnitine oral solution 1 bottle bid and 22 as experimental cases treated with Levocarnitine oral solution 1 bottle bid + VitE 100 mg tid). We compared the control and experimental groups in sperm concentration and percentage of progressively motile sperm before and 3 months after medication, as well as the rate of clinical pregnancy and adverse events.

RESULTSAmong the oligospermia patients, the average sperm concentrations in the control and experimental groups were 8.00 x 10(6)/ml and 10.66 x 10(6)/ml before medication (P > 0.05). After medication, the numbers of cases evaluated as with no, slight, moderate and marked improvement in sperm concentration were 10 and 9 (P > 0.05), 16 and 14 (P > 0.05), 5 and 4 (P > 0.05) and 0 and 0 (P >0.05); and the numbers of natural pregnancies were 0 and 6 in the control and experimental groups (P < 0.01). Among the asthenospermia patients, the average rates of progressively motile sperm were 17.00% and 18.10% in the control and experimental groups before medication (P > 0.05). After medication, the numbers of cases evaluated as with no, slight, moderate and marked improvement in the percentage of progressively motile sperm were 7 and 2 (P < 0.01), 4 and 8 (P < 0.01), 3 and 2 (P > 0.05) and 1 and 1 (P > 0.05), and the numbers of natural pregnancies were 5 and 9 in the two groups (P < 0.01), but no adverse events were observed.

CONCLUSIONAs a safe and effective adjuvant agent for the treatment of oligospermia and asthenospermia, vitamin E can improve sperm concentration, the percentage of progressively motile sperm, and finally the rate of natural pregnancy.

Adult ; Asthenozoospermia ; drug therapy ; Carnitine ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Oligospermia ; drug therapy ; Pregnancy ; Pregnancy Rate ; Prospective Studies ; Tamoxifen ; therapeutic use ; Treatment Outcome ; Vitamin E ; therapeutic use ; Young Adult

BACKGROUNDThe congenital Long QT syndrome (LQTS) is a hereditary cardiac channelopathy that is characterized by a prolonged QT interval, syncope, ventricular arrhythmias, and sudden death. The chromosome 7-linked type 2 congenital LQTS (LQT2) is caused by gene mutations in the human ether-a-go-go-related gene (HERG).

METHODSA Chinese family diagnosed with LQTS were screened for KCNQ1, HERG and SCN5A, using polymerase chain reaction (PCR), direct sequencing, and clong sequencing. We also investigated the mRNA expression of the HERG gene.

RESULTSWe identified a novel I414fs + 98X mutation in the HERG gene. The deletion mutation of 14-bp in the first transmembrane segment (S1) introduced premature termination codons (PTCs) at the end of exon 6. This mutation would result in a serious phenotype if the truncated proteins co-assembled with normal subunit to form the defective channels. But only the proband was symptomatic.

CONCLUSIONSWe found that the mRNA level of the HERG gene was significantly lower in I414fs + 98X carriers than in noncarriers. We found a novel I414fs + 98X mutation. The mRNA level supports that NMD mechanism might regulate the novel mutation.

Adult ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; genetics ; Female ; Frameshift Mutation ; Humans ; Long QT Syndrome ; genetics ; RNA, Messenger ; analysis

BACKGROUNDRemodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-beta1 (TGFbeta1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFbeta1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.

METHODSAdenoviral vector containing TGFbeta1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFbeta1, the expression of VEGF165 and TGFbeta1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFbeta1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.

RESULTSThe results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFbeta1 and VEGF165 genes. Co-expression of TGFbeta1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.

CONCLUSIONCo-expression of TGFbeta1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

Adenoviridae ; genetics ; Animals ; Anterior Cruciate Ligament ; cytology ; metabolism ; Cell Movement ; Cells, Cultured ; Collagen ; genetics ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; physiology ; Fibronectins ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; Wound Healing