Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Animals ; Mice ; Humans ; Adenoviruses, Human/genetics* ; Escherichia coli/genetics* ; HEK293 Cells ; Isopropyl Thiogalactoside ; Blotting, Western ; Immunoglobulin G ; Antibodies, Monoclonal ; Antibody Specificity ; Mice, Inbred BALB C

The present study was designed to determine the major chemical constituents of the leaves of Rhododendron dauricum L. Compounds were isolated and purified by various chromatographic methods, and their structures were elucidated by physicochemical properties and spectral data. The present study identified two new C-methyl flavanones, 5, 7, 3', 5'-tetrahydroxy-6, 8-di-C-methyl flavanone (1) and 5, 4'-dihydroxy-8-C-methylflavanone-7-O-β-D-glucopyranoside (2), and one new flavonoid glycoside, quercetin-3-O-β-D-(6"-O-cinnamoyl)-galactoside (3), along with seven known compounds, including syzalterin (4), poriolin (5), farrerol-7-O-β-D-glucopyranoside (6), myrciacetin (7), quercetin-3-O-β-D-(6-p-hydroxy-benzoyl)-galactoside (8), quercetin-3-O-β-D-(6-p-coumaroyl)-galactoside (9), and 5, 7, 3', 5'-tetrahydroxyl flavanone (10). Compounds 1-3 were determined to be new flavonoids; compounds 4-6 were isolated from this species for the first time; and compounds 7-10 were reported for the first time from this genus.
Flavanones ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Galactosides ; chemistry ; isolation & purification ; Glucosides ; chemistry ; isolation & purification ; Molecular Structure ; Plant Extracts ; chemistry ; Plant Leaves ; chemistry ; Quercetin ; analogs & derivatives ; chemistry ; isolation & purification ; Rhododendron ; chemistry

OBJECTIVETo investigate whether the antioxidation and the regulation on the Extracellular Regulated Protein Kinases (ERK) signaling pathway are involved in the protective effects of blueberry on central nervous system.

METHODS30 Senescence-accelerated mice prone 8 (SAMP8) mice were divided into three groups and treated with normal diet, blueberry extracts (200 mg/kg•bw/day) and cyaniding-3-O-galactoside (Cy-3-GAL) (50 mg/kg•bw/day) from blueberry for 8 weeks. 10 SAMR1 mice were set as control group. The capacity of spatial memory was assessed by Passive avoidance task and Morris water maze. Histological analyses on hippocampus were completed. Malondialdehyde (MDA) levels, Superoxide Dismutase (SOD) activity and the expression of ERK were detected.

RESULTSBoth Cy-3-GAL and blueberry extracts were shown effective functions to relieve cellular injury, improve hippocampal neurons survival and inhibit the pyramidal cell layer damage. Cy-3-GAL and blueberry extracts also increased SOD activity and reduced MDA content in brain tissues and plasma, and increased hippocampal phosphorylated ERK (p-ERK) expression in SAMP8 mice. Further more, the passive avoidance task test showed that both the latency time and the number of errors were improved by Cy-3-GAL treatment, and the Morris Water Maze test showed significant decreases of latency were detected by Cy-3-GAL and blueberry extracts treatment on day 4.

CONCLUSIONBlueberry extracts may reverse the declines of cognitive and behavioral function in the ageing process through several pathways, including enhancing the capacity of antioxidation, altering stress signaling. Cy-3-GAL may be an important active ingredient for these biological effects.

Aging ; drug effects ; Animals ; Anthocyanins ; pharmacology ; Avoidance Learning ; Blueberry Plants ; chemistry ; Dietary Supplements ; Galactosides ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Malondialdehyde ; metabolism ; Maze Learning ; Memory ; drug effects ; Mice ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Plant Extracts ; pharmacology ; Superoxide Dismutase ; metabolism

Constructing robust gene circuits is a fundamental work for synthetic biology. Bacteria with suicide gene circuit based on quorum-sensing will kill themselves in a controllable pattern upon certain cell density. In the media of different IPTG inducer concentration, we observed the growth and suicidal behavior of the Escherichia coli. Top10F' with such gene circuit, screened the mutants and determined their mutated loci. The results show that, with higher IPTG concentration, the more wild type bacteria were killed; as well the mutants emerged earlier and spread over the population more quickly. The sequence of plasmids in those mutants revealed that a transposon inserted into the luxR gene and therefore disrupted Quorum-Sensing of these individuals. Furthermore, the insertion sequence of the plasmid can solely result in the mutants escaping from suicide.
Culture Media ; chemistry ; DNA Transposable Elements ; genetics ; Escherichia coli ; genetics ; growth & development ; Gene Expression Regulation, Bacterial ; Genes, Synthetic ; genetics ; Genes, Transgenic, Suicide ; Isopropyl Thiogalactoside ; chemistry ; Mutation ; Quorum Sensing ; genetics ; Repressor Proteins ; genetics ; Trans-Activators ; genetics

OBJECTIVETo investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model.

METHODSThe susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal). Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopic bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed.

RESULTSGene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 cells with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal cells infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was (39 ± 3) % and (49 ± 5) % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle (P < 0.05). AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAlacZ, gemcitabine, AxdAdB-3, and AxdAdB-3 + gemcitabine were 400.6, 126.4, 82. 0, 40.4 mg, respectively. Either AxdAdB-3 (P < 0.0001) and gemcitabine (P < 0.0001) suppressed the tumor growth in nude mice, and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P < 0.0001) or gemcitabine (P < 0.0001) alone.

CONCLUSIONSIntravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse, and further relevant clinical studies are guaranteed.

Adenoviridae ; genetics ; Administration, Intravesical ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; therapeutic use ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacology ; therapeutic use ; Galactosides ; Indoles ; Mice ; Mice, Nude ; Models, Animal ; Urinary Bladder Neoplasms ; drug therapy

OBJECTIVE: To optimize the induction condition of human NOR1 gene expression in E.coli. and purify NOR1 recombinant proteins. METHODS: A full-length cDNA of human NOR1 was inserted into the corresponding region of pET28b expression vector to yield recombinant prokaryotic expression vector pET28b-NOR1. The prokaryotic expression vector pET28b-NOR1 was introduced into the bacterial host E.coli Rosettablue(DE3). Recombinant NOR1 protein was induced at different conditions. Induction condition was optimized to obtain high yield of recombinant protein. At last, the recombinant NOR1 protein was purified by Ni-IDE chromatography resin. RESULTS: Recombinant NOR1 protein was induced by IPTG in a dose-dependent manner. Increase of kanamycin concentration and induction temperature resulted in high yield of recombinant protein. The most recombinant protein was found in inclusion bodies. The recombinant His-NOR1 protein was purified with Ni-IDE chromatography resin under denature condition. CONCLUSION IPTG, kanamycin concentration and temperature can affect the expression of recombinant NOR1 protein in pET28b system. High yield of recombinant NOR1 protein is achieved by inducing 1 mmol/L IPTG and 200 μg/mL kanamycin at 37 degree. Recombinant His-NOR1 protein with high purity is purified.
Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Membrane Transport Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Tumor Suppressor Proteins ; biosynthesis ; genetics

PURPOSE: Peritoneal dialysis associated peritonitis (PD peritonitis) is an important complication in maintaining. There have been only a few reports on the clinical outcome of initial no-growth peritonitis (INGP). METHODS: We reviewed 332 episodes of PD peritonitis between January 2002 and August 2009. INGP was defined as PD peritonitis with no growth of etiologic microorganism within 3 days of peritonitis. INGP was compared with initial positive growth peritonitis (IPGP) in view of clinical manifestations and outcomes. RESULTS: We divided PD peritonitis episodes into two groups: INGP (n=90) and IPGP (n=242). Peritonitis-related mortality was 5.6% in INGP, while 0.8% in IPGP (p=0.017). Further relapse was noted in INGP (10.0%) than in IPGP (vs. 4.1%; p=0.041). Salvage antibiotics were used more frequently in INGP (21.1%) than in IPGP (vs. 11.6%; p=0.027). Odds ratio of INGP to IPGP for peritonitis-related mortality was 7.14 (95% CI 1.36-37.51; p=0.017). Growth of mycobacteria or fungi increased the risk of peritonitis-related mortality with an odds ratio of 18.11 (95% CI 2.99-109.89; p=0.013). In multivariate analysis, growth of mycobacteria or fungi was the only independent risk factor for peritonitis-related mortality with an odds ratio of 10.63 (95% CI 1.27-88.75; p=0.029). CONCLUSION: INGP revealed poorer outcome than IPGP. Higher growth rate of mycobacteria or fungi in INGP than in IPGP accounted for the poor outcome. Thus one should make vigorous efforts to detect surreptitious organism when there is no growth by 3 days, especially for the possibility of either mycobacteria or fungi.
Anti-Bacterial Agents ; Fatal Outcome ; Fungi ; Isopropyl Thiogalactoside ; Multivariate Analysis ; Mycobacterium ; Odds Ratio ; Peritoneal Dialysis ; Peritonitis ; Recurrence ; Risk Factors

To investigate the chemical constituents of Psidium Guajava L, the EtOH/H2O extract of the fresh leaves was subjected to various chromatography. Five constituents with galloyl moiety were isolated and elucidated as 1-O-(1, 2-propanediol)-6-O-galloyl-beta-D-glucopyranoside (1), gallic acid (2), ellagic acid (3), ellagic acid-4-O-beta-D-glucopyranoside (4) and quercetin-3-O-(6"-galloyl) beta-D-galactopyranoside (5) by spectroscopic methods, including 2D NMR and HR-ESI-MS spectrometry as well as by comparison with published data. Compounds 4 and 5 were obtained from P. guajava for the first time, and compound 1 is a new polyhydroxyl compound.
Drugs, Chinese Herbal ; chemistry ; Ellagic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; Galactosides ; chemistry ; isolation & purification ; Gallic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; Glucosides ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Psidium ; chemistry ; Quercetin ; analogs & derivatives ; chemistry ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization

To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.
Animals ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Insect Proteins ; biosynthesis ; genetics ; Inteins ; genetics ; Isopropyl Thiogalactoside ; pharmacology ; Molecular Sequence Data ; Recombinant Proteins ; analysis ; biosynthesis ; genetics