Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.

The applicator therapy is a unique method to treat infant diarrhea in traditional Chinese medicines and widely applied in clinical practice. Currently, many researchers have proved the rationality of the therapy based on the traditional Chinese medicine mechanism and on the data from clinical practice, but its action mechanism is uncertain at present. In this study, with the assistance of pediatric practitioners, the automated ribosomal intergenic-spacer analysis (ARISA) was adopted to study the effect of the adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants, in which Dingguier umbilical paste served as the adjuvant therapy in oral traditional Chinese medicines and fecal samples of infants with different diarrhea symptoms were collected and used as the study materials. The results showed that the adjuvant therapy had a significant effect on the shift of intestinal flora, which was associated with the decrease in the similarity difference to the normal control group and the increase in the number of operational taxonomic units (OTUs) shared with the normal control group. Additionally, adjuvant therapy with Dingguier umbilical paste also showed long action duration and increased OTUs number. These results indicated that Dingguier umbilical paste has the effect in restoring the micro-ecosystem of unbalanced intestinal bacteria. Intestinal flora may be one of major targets for the applicator therapy for the infant diarrhea, but not for the single oral traditional Chinese medicine for infant diarrhea.
Adjuvants, Pharmaceutic ; therapeutic use ; Diarrhea ; drug therapy ; microbiology ; Feces ; microbiology ; Female ; Humans ; Infant ; Intestines ; drug effects ; microbiology ; Male ; Medicine, Chinese Traditional ; methods ; Ointments ; Treatment Outcome ; Umbilicus

OBJECTIVETo observe the expression and effect of human calcitonin gene-related peptide (hCGRP) gene mediated by recombinant adeno-associated virus (rAAV) in primary cultured corporal cavernosum smooth muscle cells of the rat and explore the possibility of using CGRP gene for gene therapy in erectile dysfunction.

METHODSThe primary cultured corporal cavernosum smooth muscle cells of the rat were randomly divided into 4 groups and infected with recombinant virus VssHGCMV-hCGRP, VssHGCMV, VssC-MV-GFP and the untreated, respectively. CGRP-like immunoreactivity was measured by protein dot blot assay in the 24 h-culture medium, and intracellular cAMP and cGMP levels in the cultured cells were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP.

RESULTSThe exogenous gene was transferred into primary corporal cavernosum smooth muscle cells by VssHGCMV-hCGRP infection and efficiently expressed. Compared with the control group, intracellular cAMP level in the cell infected by VssHGCMV-hCGRP was significantly increased (48.7 +/- 1.1 nmol/L vs 7.8 +/- 1.4 nmol/L, P < 0.01), whereas cGMP level remained unchanged in two groups, and CGRP-like immunoreactivity was also detected in the culture medium infected by VssHGCMV- hCGRP.

CONCLUSIONThe system of secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in corporal cavernosum smooth muscle cells and affect cAMP level in the corporal cavernosum smooth muscle cells of the rat.

Animals ; Calcitonin Gene-Related Peptide ; biosynthesis ; genetics ; Cells, Cultured ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Dependovirus ; genetics ; Male ; Muscle, Smooth ; cytology ; metabolism ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombination, Genetic ; Transfection

OBJECTIVETo determine whether recombinant adeno-associated virus-mediated overexpression of hCGRP in the corpus cavernosum can affect the continuous production of hCGRP in the penile tissue and enhance erectile responses in STZ-induced diabetic rats.

METHODSDiabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg streptozotocin in male SD rats. VssHGCMV-hCGRP, VssCMV-GFP and rAAV solution were injected into the corporal cavernosum of STZ-induced diabetic rats, respectively. The corporal tissue was obtained from groups of 8 rats on day 5 post-injection, and the expressions of CGRP and GFP in cavernosal tissue were detected using immunohistochemistry and laser scanning confocal microscopy, respectively. Cavemosal tissue cAMP and cGMP levels were measured using radioimmunoassay. On day 5 post-injection, intracavernous pressure induced by electrostimulation of penile dorsal nerves was measured and recorded with a biological signal processing system in each group rat.

RESULTSrAAV transduction efficiency of GFP reporter gene was measured by laser scanning confocal microscopy and was observed in the penile tissue, especially in the corporal cavernosum and the vessel 5 days after transfection with VssCMV-GFP. Immunohistochemistry showed that the CGRP increased in the corporal cavernosum. In addition, both cAMP and cGMP levels in the corpora cavernosa transfected with VssHGCMV-hCGRP were significantly increased, compared with controls [(48.4 +/- 6.5) nmol/L and (21.2 +/- 13.6) nmol/L vs (16.7 +/- 2.5) nmol/L and (0.42 +/- 0.12) nmol/L, respectively]. More importantly, 5 days after administration of VssHGCMV-hCGRP,a significant increase was observed in the erectile response to penile dorsal nerve stimulation in the diabetic rat [(60.5 +/- 4.5) mm Hg vs (22. 3 +/- 1.3) mm Hg].

CONCLUSIONSThis results demonstrate that rAAV-mediated transfer of the CGRP gene can increase production of endogenous CGRP, cAMP and cGMP in corpora cavernosa of STZ-induced diabetic rats. Moreover, overexpression of CGRP enhances ICP and the erectile response to penile dorsal nerve stimulation in the diabetic rat.

Adenoviridae ; genetics ; Animals ; Calcitonin Gene-Related Peptide ; biosynthesis ; genetics ; Diabetes Mellitus, Experimental ; physiopathology ; Green Fluorescent Proteins ; biosynthesis ; Humans ; Male ; Penile Erection ; physiology ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo constitute a model of B immunoblastic lymphomas in the Hu-PBL-SCID mice.

METHODSThe SCID mice were reconstituted by intraperitoneal injection (i.p.) of 5 x 10(7) human lymphocytes from Epstein-Barr virus (EBV) seronegative individuals. After one week, the SCID mice were inoculated with EBV by i.p. injection, and subjected to the investigation of whether there was any tumor in the abdomen of such SCID mice four weeks later. The characteristics of the found tumor was observed by the methods of Hematoxylin-eosin (HE) stain, immunohistochemical staining and polymerase chain reaction (PCR).

RESULTSCompared with the control groups, all the EBV-infected Hu-PBL-SCID mice had abdominal solid tumors [(32 +/- 12.5) mm3] developed, often located in the liver. HE staining and immunohistochemical staining showed the tumors were human B cell lymphomas. EBV DNA could be detected in the tumors by the PCR.

CONCLUSIONSThe model of B immunoblastic lymphomas in the Hu-PBL-SCID mice is successfully constituted, and may well be useful to the human tumor immunological study.

Animals ; Disease Models, Animal ; Herpesvirus 4, Human ; physiology ; Humans ; Lymphoma, Large-Cell, Immunoblastic ; Mice ; Mice, SCID

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.
Animals ; Antiviral Agents ; metabolism ; toxicity ; Bacillus subtilis ; metabolism ; Chick Embryo ; cytology ; Fibroblasts ; cytology ; drug effects ; Infectious bursal disease virus ; drug effects ; Newcastle disease virus ; drug effects

OBJECTIVETo evaluate the frequency of microdeletions in the long arm of Y chromosome of idiopathic infertile males with azoospermia and oligospermia in Shaanxi province in China and to investigate the relevance of sperm count to Y microdeletion frequencies.

METHODSAccording to the sequence of sequence-tagged sits (STS) AZFa, AZFb, AZFc and SRY, 4 of the azoospermic factor regions on Y chromosome long-term supplied by GenBank, 5 sets of primers were synthesized. The Y microdeletions in AZF regions were screened by polymerase chain reaction (PCR) in 64 idiopathic cases of azoospermia and oligospermia and 20 men of known fertility.

RESULTSNo microdeletion was detected in the 20 normospermic subjects. Deletion of the AZFc/DAZ was detected in 11 individuals and one patient had both AZFb and AZFc deletion; no deletion of AZFa and SRY region was found. The frequency of Y microdeletions in the subgroups with different sperm count showed the highest value among azoospermic men (3 cases, 21.4%). The percentage progressively decreased with the deletion frequency (20.0%, 17.9% and 8.3%) in the subgroups with sperm counts of < 1 x 10(6)/ml, < (1-5) x 10(6)/ml and < (1 to approximately 10) x 10(6)/ml, respectively.

CONCLUSIONY chromosome microdeletions are specifically associated with severe spermatogenic failure. The rate of deletion involving AZF region of the Y-chromosome is higher in infertile men with azoospermia and oligospermia. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

Adult ; China ; Chromosome Deletion ; Chromosomes, Human, Y ; Humans ; Male ; Oligospermia ; genetics ; Polymerase Chain Reaction ; Sequence Tagged Sites

Objective To investigate the related mechanism of Slug inhibiting the proliferation of cervical cancer cell through CDH3/β-catenin/C-myc. Methods SiHa cells with stable Slug expression were screened. The expression of CDH3 in Slug-overexpressed SiHa cell was detected by RNA-sequence, Real-time PCR, Western blot and immunocytochemistry. The expression of CDH3 in SiHa and HeLa cells were detected by Western blot and immunocytochemistry. The protein level of CDH3 was up-regulated in HeLa cells or rescued in SiHa-Slug cells by transient transfection of CDH3 expression vector. The protein levels of β-catenin and C-myc were detected by Western blot, the cell growth was detected by cell counting and CCK-8 assays. Luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) were performed to detect the effect of Slug on regulating the promoter region of CDH3. Results SiHa cell line with stable Slug expression was successfully constructed. Slug overexpression inhibited CDH3 expression in SiHa cells. CDH3 promoted cell proliferation and up-regulated the protein level of β-catenin and C-myc in HeLa and SiHa-Slug cells. Slug could recognize and bind to the E-boxes in the CDH3 promoter region and inhibited the transcription of CDH3 in SiHa cells. Conclusion Slug could inhibit the expression of β-catenin and C-myc by inhibiting CDH3 transcription in SiHa cells, and then attenuate the growth of SiHa cells.

Objective To observe the therapeutic efficacy of hyperbaric oxygen (HBO) for focal cerebral infraction influenced by the treatment time after permanent middle cerebral artery occlusion (MCAO) in rabbits. Methods Seventy-five male rabbits were randomly divided into simple MCAO group (n=25), MCAO+HBO group (100% O2, 250 kPa, 1 h/d, from 1 d after MCAO, n=25) and MCAO+DHBO group (100% O2, 250 kPa, 1 h/d, from 7 d after MCAO, n=25). Behaviors and volumes of infarction were observed, and microdialysis was applied to monitor the concentrations of glucose, lactate, pymvate and glutamate around the infarct foci at 1, 3, 10 and 30 d after permanent MCAO. Results Behaviors'score was lower in MCAO+HBO group than the others (P<0.05). The infarct volume from day 3 to day 30 was significantly smaller in MCAO+HBO group than in the other 2 groups (P<0.05). The lactate and pyruvate ratio was increased after MCAO in three groups, but they were lower in the MCAO+HBO group than in the others at day 1 and day 3 (P<0.05). The glutamate concentration was increased after MCAO, peaked at 3 d, but at day 1 and day 3 the glutamate concentration was lower in the MCAO+HBO group (P<0.05). Conclusions HBO treatment could protect the brain from infarction through improving the energy metabolism and decreasing the excitatory amino acids disorders around the infarct foci after MCAO in rabbits. In order to improve the therapeutic efficacy of HBO, it should be performed as possible.

OBJECTIVEA comparative proteomic approach was used to identify and analyze proteins relevant to metastasis of hepatocellular carcinoma (HCC).

METHODSProteins extracted from 12 liver tumor tissue specimens (6 with metastases and 6 without) were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns between the two groups were done using computerized image analysis. Selected proteins exhibiting statistically significant alternations were identified by mass spectrometry. Immunohistochemistry, Western blotting and RT-PCR were performed to examine the expressions of the candidate proteins.

RESULTS16 proteins including HSP27, S100A11, CK18 were identified using mass spectrometry, which were related to cell mobility, signal transduction, and energy metabolism respectively. Of these, HSP27 was found to be uniquely over-expressed in 2-DE maps of all metastatic HCCs when compared to the non-metastatic HCC tissues. Immunohistochemistry and Western blotting of HCC tissues confirmed this difference while RT-PCR did not.

CONCLUSIONThere are different proteins working together that affect the metastasis of HCCs. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets to the metastatic phenotype of HCC. The role of HSP27 in HCC metastasis warrants further investigation.

Carcinoma, Hepatocellular ; chemistry ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Neoplastic ; HSP27 Heat-Shock Proteins ; Heat-Shock Proteins ; analysis ; Humans ; Liver Neoplasms ; chemistry ; pathology ; Mass Spectrometry ; Neoplasm Proteins ; analysis ; Proteome ; analysis ; S100 Proteins ; analysis