Objective To examine the expression of Toll-like receptor(TLR)4 protein and the transcription of TLR4 mRNA in the peripheral blood mononuclear cells(PBMC)from patients with hepatitis B virus(HBV)infection and explore the relationship between TLR4 and chronic HBV infec- tion.Methods The expression level and transcription level of TLR4 were determined by flow cytometre and reverse transcriptase polymerase chain reaction respectively in PBMC from 37 chronic hepatitis patients,28 liver cirrhosis patients,31 severe hepatitis patients and 27 healthy controls. Meanwhile,liver function,as well as blood routine test,prothrombin test activity(PTA)and HBV DNA was measured.Results The expression level and transcription level of TLR4 in patients were higher than those in healthy controls(P

Background Fundus photography is a traditional method for detecting local retinal nerve fiber layer (RNFL) defects,but the evaluation of its result depends on the observer's experience.The spectral domain optical coherence tomography (OCT) exhibit the defects of RNFL very clearly.Objective This study was to evaluate the diagnosis value and correlation between topographic profiles of localized RNFL defects determined by spectral domain and time domain OCT with fundus photography.Methods Forty-one normal eyes of 41 subjects and 55 eyes of 55 glaucomatous patients with localized,wedge-shaped RNFL defects identified by two glaucoma specialists in fundus photographs were enrolled in the clinical study.The angular location and width of RNFL defects determined on the images of fundus photography,Cirrus HD-OCT and Stratus OCT were analyzed respectively using Pearson's correlation coefficient and linear regression analysis.This study followed the Helsinki declaration and was approved by Ethic Committee of Shenzhen Eye Hospital.Written informed consent was obtained from each individual before the clinical examination.Results Seventy-five RNFL defects were identified in 55 eyes by two glaucoma specialists unanimously with the defect position at superior-temporal and inferior-temporal quadrants.The sensitivity of Cirrus HD-OCT and Stratus OCT to determining RNFL defects were 88.0% and 69.3% respectively and their specificities were 92.7% and 97.6% respectively.The angular locations of RNFL defects by Cirrus HD-OCT and Stratus OCT were highly correlated with those by fundus photography(r=0.993,r=0.992,P＜0.001);while the angular widths of RNFL defects by Cirrus HD-OCT and Stratus OCT were moderately correlated with those by fundus photography(r=0.420,r=0.432,P=0.019,P=0.002).No significant differences were found in the defect width of RNFL between Cirrus HD-OCT or Stratus OCT and fundus photography(Cirrus HD-OCT:P=0.114;Stratus OCT:P=0.074),and significant difference was found in that between Cirrus HD-OCT and Stratus OCT(P=0.002).Conclusion Spectral domain OCT and time domain OCT can localize RNFL defects with high sensitivity and specificity.The measure value of Cirrus HD-OCT and Stratus OCT for RNFL defects shows a good diagnostic agreement with fundus photography.

Genomic DNA of two fungi Thamnidium elegans and Umbelopsis isabellina were extracted with an amended Cetyltrimethyl Ammonium Bromide (CTAB) method. This modified method uses repeated freezing in liquid nitrogen and thawing with combination of shocking with glass beads to replace of the tra- ditional method. Quality and concentration of DNA extracted by the modified methodwere tested. Compared with the traditional method, higher yield and purity of genomic DNA were obtained with less amount ofmy- celium. The result indicted that this is a simple and highly efficient method, which is suitable to treat many samples at one time and for basic molecular experiments, such as restriction endonuclease reaction and PCR.

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.
Cloning, Molecular ; Fatty Acids ; analysis ; Gas Chromatography-Mass Spectrometry ; Linoleoyl-CoA Desaturase ; genetics ; Mortierella ; enzymology ; genetics ; Pichia ; genetics ; Plasmids ; gamma-Linolenic Acid ; analysis

This study was aimed to investigate the expression and regulation mechanism of DNA-dependent protein kinase catalylic subunit (DNA-PKcs) in chronic myeloid leukemia (CML) and its role in blast crisis of CML. Expression of DNA-PKcs mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and DNA-PKcs protein by Western blot in 62 CML patients and K562, as compared to those of 23 normal individual controls. In 26 CML patients received allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients treated with imatinib, the expression of bcr-abl mRNA and DNA-PKcs protein was detected by RT-PCR and Western blot, respectively. After treatment with imatinib in mononuclear cell (MNC) of CML patients and K562 in vitro, expression of DNA-PKcs mRNA was detected by RT-PCR and DNA-PKcs protein level, tyrosine phosphorylation of bcr-abl fusion protein were detected by Western blot. The results showed that the expression of DNA-PKcs protein was significantly lower in CML and K562 than those in normal control (P<0.05). In 26 CML patients received allo-PBSCT and 4 CML patients treated with imatinib, the expression of DNA-PKcs protein was enhanced while the expression of bcr-abl mRNA decreased. After treatment of MNC of CML and K562 with imatinib in vitro, the expression of DNA-PKcs protein was enhanced while tyrosine phosphorylation of bcr-abl fusion protein decreased. It is concluded that the expression of DNA-PKcs protein is down-regulate by bcr-abl fusion gene, and the bcr-abl fusion gene down-regulate the expression of DNA-PKcs protein by post-transcriptional mechanism; the decrease of DNA-PKcs protein expression may be one of mechanisms underlying the acute transformation of CML.
Adult ; Aged ; Benzamides ; Bone Marrow Cells ; metabolism ; DNA-Activated Protein Kinase ; biosynthesis ; genetics ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; therapy ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Piperazines ; therapeutic use ; Pyrimidines ; therapeutic use ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the expression and regulation mechanism of mismatch repair (MMR) genes in chronic myeloid leukemia (CML).

METHODSExpression of MMR genes hMSH2, hMSH3, hMSH6, hMLH1 and hPMS2 mRNAs in 62 CML patients and K562 cell line were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of bcr-abl mRNA and MMR genes mRNA were detected by RT-PCR in 26 CML patients with allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients on imatinib treatment. Expression of bcr-abl mRNA was detected by RT-PCR and tyrosine phosphorylation of BCR-ABL fusion protein by Western blot.

RESULTSExpression of hMSH2, hMSH3 and hMLH1 mRNA was significantly lower in CML and K562 cells than in normal control (P < 0.05). In 26 CML with allo-PBSCT and 4 CML patients on imatinib treatment, expressions of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while expression of bcr-abl mRNA decreased. In CML MNC after imatinib treatment and in K562 cells, expression of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while tyrosine phosphorylation of BCR-ABL fusion protein decreased.

CONCLUSIONExpressions of hMSH2, hMSH3 and hMLH1 mRNA were down-regulated by bcr-abl fusion gene.

Adult ; Aged ; Antineoplastic Agents ; pharmacology ; Benzamides ; DNA Mismatch Repair ; Female ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Humans ; Imatinib Mesylate ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Adolescent ; Adult ; Carrier State ; metabolism ; pathology ; Collagen Type III ; blood ; Collagen Type IV ; blood ; Female ; Hepatitis B, Chronic ; complications ; metabolism ; pathology ; Humans ; Interleukin-17 ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; etiology ; metabolism ; pathology ; Male ; Middle Aged ; Severity of Illness Index ; Young Adult

AIMTo investigate the ultrastructural changes of penile corpus cavernosum and tunica albuginea in rats treated with castration or finasteride.

METHODSEighteen male Sprague-Dawley rats of nine weeks old were randomly divided into three groups with 6 rats each. Group A served as the control, Group B was castrated and Group C, treated with finasteride. Four weeks later, rats were anesthetized and blood samples obtained for the determination of serum testosterone (T) and dihydrotestosterone (DHT) levels; penile tissues were taken for scanning electron microscopy.

RESULTSThe T, free T and DHT levels in Group B and the DHT level in Group C were significantly lower than those in Group A (P<0.05). The tunica albuginea was significantly thinner in Group B than that in Group A (P<0.05), but there was no significant difference between Group C and Group A (P>0.05). Elastic fibers in the tunica albuginea of Group A were very rich and arranged regularly and undulatedly, but in Group B, most of the elastic fibers were replaced by collagenous fibers. In Group C, the tunica albuginea was mainly composed of thick and irregular-arranged collagenous fibers. In Group A, there were abundant smooth muscle fibers in the trabeculae of corpus cavernosum, but they were much less in Group C and scarce or even disappeared in Group B. In Groups B and C, the diminished/disappeared smooth muscle fibers were replaced by irregularly arranged collagenous fibers.

CONCLUSIONIn rats, androgen is essential for maintaining the normal structure of penile tunica albuginea and corpus cavernosum.

Animals ; Dihydrotestosterone ; blood ; Enzyme Inhibitors ; pharmacology ; Finasteride ; pharmacology ; Male ; Microscopy, Electron, Scanning ; Orchiectomy ; Penis ; pathology ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood ; deficiency

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.
Cloning, Molecular ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Engineering ; methods ; Phylogeny ; gamma-Linolenic Acid ; biosynthesis

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology