OBJECTIVETo culture rat prostate glandular epithelial cells and study their barrier functions in vitro.

METHODSRat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test.

RESULTSCompact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs.

CONCLUSIONThe structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Animals ; Cell Membrane Permeability ; Cells, Cultured ; Claudin-1 ; metabolism ; Electric Impedance ; Epithelial Cells ; pathology ; physiology ; ultrastructure ; In Vitro Techniques ; Male ; Microscopy, Electron, Transmission ; Phenolsulfonphthalein ; pharmacokinetics ; Prostate ; metabolism ; pathology ; Rats ; Tight Junctions

OBJECTIVETo establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs.

METHODSTo obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E. coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain.

RESULTSWith the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K.

CONCLUSIONSThe compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.

Biological Assay ; Blotting, Western ; Cathepsin K ; Cathepsins ; antagonists & inhibitors ; genetics ; isolation & purification ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; Drug Evaluation, Preclinical ; Escherichia coli ; enzymology ; genetics ; Gene Expression ; Humans ; Osteoporosis ; drug therapy ; Papain ; antagonists & inhibitors ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.
Alcohol Oxidoreductases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; Benzoquinones ; metabolism ; Blotting, Southern ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Genetic Vectors ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; genetics ; physiology ; Polymerase Chain Reaction ; Streptomyces ; genetics ; metabolism

OBJECTIVETo investigate the clinicopathologic features of epididymal cyst in von Hippel-Lindau (VHL) syndrome.

METHODSWe reviewed the clinical data of 3 epididymal cyst patients treated by surgery, and detected the expressions of HIF-1alpha, VEGF, alpha-SMA and CD34 in the epididymal tissue samples by the immunohistochemistry SP method.

RESULTSAll the 3 patients underwent surgical removal of the epididymal cyst. Immunohistochemistry of the epididymal tissues showed HIF-1alpha, VEGF, alpha-SMA and CD34 to be positive. All the 3 cases were confirmed to be VHL syndrome, 1 right after surgery, and the other 2 within 8 years postoperatively.

CONCLUSIONEpididymal cyst is a usual benign disease, which may occur independently of or be complicated by VHL syndrome. If immunohistochemistry of epididymal tissues shows HIF-1alpha, VEGF, alpha-SMA and CD34 to be positive, VHL syndrome should be considered, and further clinical examinations and post-operation follow-up are necessitated.

Actins ; metabolism ; Adult ; Epididymis ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Middle Aged ; Spermatocele ; etiology ; metabolism ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; von Hippel-Lindau Disease ; complications ; metabolism ; pathology

BACKGROUNDTreponema pallidum (T. pallidum) subsp. pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis. Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies, but the results were controversial. In this research, the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed.

METHODST. pallidum subsp. pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted. The Tp0965 gene was amplified by polymerase chain reaction (PCR). Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid (Ni-NTA) purification system. The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay. The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay.

RESULTSRecombinant protein Tp0965 was expressed successfully in vitro. Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965 to all 28 uninfected sera. A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization.

CONCLUSIONSThe recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages. The results also demonstrate a potential application for the serodiagnosis of syphilis.

Animals ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Membrane Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; Rabbits ; Syphilis ; immunology ; microbiology ; Treponema pallidum ; immunology ; metabolism

Background Treponema pallidum (T.pallidum) subsp.pallidum is the causative agent of syphilis.Analysis of recombinant antigens of T.pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis.Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies,but the results were controversial.In this research,the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed.Methods T.pallidum subsp.pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted.The Tp0965 gene was amplified by polymerase chain reaction (PCR).Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid (Ni-NTA) purification system.The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay.The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay.Results Recombinant protein Tp0965 was expressed successfully in vitro.Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages.Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965,and lack of reactivity of Tp0965 to all 28 uninfected sera.A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization.Conclusions The recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages.The results also demonstrate a potential application for the serodiagnosis of syphilis.

Adult ; Aged ; Female ; Humans ; Joint Instability ; complications ; diagnosis ; physiopathology ; surgery ; Lumbar Vertebrae ; pathology ; physiopathology ; Male ; Middle Aged ; Spinal Canal ; pathology ; physiopathology ; Spinal Stenosis ; complications ; diagnosis ; physiopathology ; surgery

OBJECTIVETo search for the perfect therapy for the complications of breast augmentation by injecting polyacrylamide hydrogel.

METHODS15 patients whose complications were severe after hydrogel injection were included in this study. Open suction and irrigation of cavity were performed in all patients and all received immediately dual-plane augmentation mammaplasty with silicon gel prostheses.

RESULTS12 patient were followed up for 3 months to 1 year (mean 6.8 months) and no malposition or deformation occurred. 10 patients (20 breasts) had satisfactory results. The edges of the implant shell could be felt in 2 patients (3 breasts). 1 patient (1 breast) with breast firmness ranked Baker II .

CONCLUSIONSThe dual-plane breast augmentation is a valuable technique to treat the complications of polyacrylamide hydrogel injection.

Acrylic Resins ; adverse effects ; Adult ; Breast Implants ; adverse effects ; Female ; Humans ; Mammaplasty ; adverse effects ; methods ; Middle Aged ; Postoperative Complications ; surgery ; Young Adult

Objective To study the pollution and source apportionment differences of different periods PM2.5 in the residential community of suburb in Tianjin City during heating and non-heating periods. Methods From 2015 to 2016, daytime and nighttime PM2.5 samples were collected at a community in the suburb of Tianjin. The mass concentration of PM2.5 samples and major chemical components in PM2.5, including metal elements, polycyclic aromatic hydrocarbons (PAHs) and inorganic water-soluble ions were monitored. Positive matrix factorization (PMF) model was used to apportion potential sources of metal elements, PAHs and inorganic water-soluble ions in daytime and nighttime PM2.5. Results In the heating period, the concentrations of some metal elements of suburban residential community were higher in the daytime than in the nighttime. In the non-heating period, the concentrations of some PAHs and inorganic water-soluble ions of suburban residential community were higher in the nighttime than in the daytime. Meanwhile, the concentrations of some metal elements were greater in the daytime than in the nighttime. When in heating period, the main source of PM2.5 in the suburban residential community was coal combustion during daytime and its source contribution rate was 50.1% while secondary aerosol and fuel combustion emissions of gasoline and diesel vehicles were main sources during nighttime and their source contribution rates were 41.0% and 35.9%. The principal source of daytime PM2.5 in the suburban residential community was indoor activity emissions during non-heating period, and secondary aerosol was main source during nighttime and their source contribution rates were 29.8% and 31.1%. Conclusions The pollution status of PM2.5 in residential communities of suburban is serious, and the source apportionment of day and night PM2.5 samples has different in different heating periods.

Purpose: The experience with laparoscopic nephrectomy or nephroureterectomy was reported.Methods: Since Feb. 2000, 17 patients (Including 3 cases of pevic or upper-segment transitional cell tumour, 7cases of renal cell carcinoma, 3 cases of renal atrophy-induced hypertensin, 2 cases of non-functioning calculuskidney and 2 cases of renal tuberculosis) underwent peritoneoscopic or retroperitoneoscopic nephrectomy,radicalnephrectomy or nephroureterectomy with the latter in combination with cystoscope. Results:All operations weresuccessful except the first case. Operation time for nephrectomy was 90 to 180 min with an average of 117 min.All patients did not receive blood transfusion and had no obvious complication. Conclusions: Laparoscopicnephrectomy had the characteristics of minimal morbidity,minimal postoperative discomfort and a short hospitalstay. It may be used well in the future.