Objective:To optimize the renaturation procedure of denatured LexA,prepare the repressor LexA from Pseudomonas aeruginosa(PA),which have the satisfactory biologic activity.Methods:The LexA was renatured by the GSH/GSSG dilution method,and the renatured protein were purified by Ni2+ chelate affinity chromatography and gel filtration chromatography,following desalination by Sephadex G25 gel column.The renaturation result were detected by the native polyacrylamide gel electrophoresis and RPHPLC.The immunological activity of all LexA proteins,including the denatured,renatured protein and the renatured protein that was treated with the DTT,were determined by Western blot.Results:The renatured LexA appears both monomer and multimer,which is confirmed by the native polyacrylamide gel electrophoresis analysis and RPHPLC.Gel retardation experiments shows that the renatured LexA have satisfactory biologic activity.

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Carbon-Nitrogen Ligases ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Flow Cytometry ; Gene Expression ; HLA-A Antigens ; chemistry ; genetics ; metabolism ; HLA-A24 Antigen ; Humans ; Oligopeptides ; genetics ; metabolism ; Phosphoproteins ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Repressor Proteins ; metabolism ; Substrate Specificity ; T-Lymphocytes, Cytotoxic ; cytology ; metabolism ; Viral Matrix Proteins ; chemistry ; genetics ; metabolism

OBJECTIVEThis research intends to amplify the entire coding region sequences of SLC25A13 mRNA which encodes citrin, and to investigate sequence features of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidence for prenatal diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) at mRNA level.

METHODSOne amniocyte sample was collected from a pregnant woman who underwent prenatal diagnosis of citrin deficiency and whose fetus has proven a carrier of 851del4 mutation by genomic DNA analysis. Another amniocyte sample, as a control, was from a fetus without family history of citrin deficiency. Total RNA was extracted from cultured amniocytes, cDNA was synthesized, and then nested-PCR was performed to amplify the entire coding region sequences of SLC25A13. The PCR products were cloned and analyzed by sequencing.

RESULTSThe entire coding region of SLC25A13 gene was successful amplified from two cultured human amniocytes. The splice variant of SLC25A13, SLCA (normal mRNA), was identified in the two samples. SLCB (CAG insertion between exon 9-10) was identified in the control. SLCC (exon 5-11 skipping), but not transcriptional product from the allele with 851del4 mutation, was identified in the 851del4 mutation carrier.

CONCLUSIONSThis study demonstrated that the entire coding region of SLC25A13 cDNA can be successfully amplified from two cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA predominated in the transcripts in normal control and 851del4 mutation carrier, suggesting that the two fetuses were not at risk for NICCD. These SLC25A13 transcription features provided laboratory evidence for prenatal diagnosis of NICCD.

Amniotic Fluid ; cytology ; metabolism ; Calcium-Binding Proteins ; deficiency ; Cholestasis, Intrahepatic ; diagnosis ; Cloning, Molecular ; Female ; Humans ; Mitochondrial Membrane Transport Proteins ; genetics ; Organic Anion Transporters ; deficiency ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods ; RNA, Messenger ; analysis ; Sequence Analysis, DNA ; Transcription, Genetic

OBJECTIVETo construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice.

METHODSFifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry.

RESULTSComparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups.

CONCLUSIONSOur results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.

Animals ; Carcinoma, Hepatocellular ; blood ; pathology ; therapy ; Cell Line, Tumor ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Immunotherapy ; Interferon-gamma ; blood ; Interleukin-2 ; blood ; Interleukins ; genetics ; Killer Cells, Natural ; immunology ; Liver Neoplasms ; blood ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Plasmids ; therapeutic use ; Random Allocation ; Recombinant Proteins ; therapeutic use ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Burden

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.
Animals ; Antibodies ; immunology ; isolation & purification ; Antigens, CD ; genetics ; immunology ; metabolism ; B7-H1 Antigen ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Humans ; Immune Sera ; immunology ; Immunoblotting ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; Solubility

Objective:To investigate the predictive value of MRI balanced steady-state free precession(b SSFP)synergistic voxel incoherent motion diffusion weighted imaging(IVIM-DWI)and Gd-DTPA enhanced scanning for the status of extramural vascular invasion(EMVI)in rectal cancer before surgery.Methods:A total of 105 rectal cancer patients from the People's Hospital of Lujiang County,Anhui Province,were retrospectively selected and included.All patients were confirmed by postoperative pathology and underwent preoperative b SSFP sequences,IVIM-DWI functional imaging,and Gd-DTPA-enhanced multiparameter MRI scans.Three seven-point schemes based on individual b SSFP sequences,IVIM-DWI functional imaging,and Gd-DTPA en-hancement,two-by-two synergy,and multi-sequence combined diagnosis were utilized in con-junction with conventional MRI sequences for preoperative prediction of EMVI status.The diag-nostic efficacy of T2WI and b SSFP sequences was compared with that of postoperative patho-logic results.ROC curves were plotted to obtain the corresponding area under the ROC curve(AUC),specificity,and sensitivity.Results:The AUC for predicting the preoperative vascular status outside the rectal wall was 0.572(95％CI:0.408～0.737)for the conventional T2 lipid sup-pression sequence,with a specificity of 0.811 and a sensitivity of 0.667.The AUC for the b SSPF sequence was 0.817(95％CI:0.680～0.954),with a specificity of 0.900 and a sensitivity of 0.733.All of the statistical parameters were higher than the diagnostic efficacy of conventional T2 lipid suppression sequences.The multi-sequence MRI co-diagnosis had an AUC of 0.961(95％CI:0.886～1.000),with a specificity of 0.988 and a sensitivity of 0.875(P＜0.05).Conclusion:Mag-netic resonance b SSFP sequence synergized with IVIM-DWI and Gd-DTPA-enhanced multipa-rameter scanning has high clinical application value for the preoperative prediction of EMVI inva-sion in rectal cancer.

Maca (Lepidium meyenii) is an herbaceous plant that grows in high plateaus and has been used as both food and folk medicine for centuries because of its benefits to human health. In the present study, ITS (internal transcribed spacer) sequences of forty-three maca samples, collected from different regions or vendors, were amplified and analyzed. The ITS sequences of nineteen potential adulterants of maca were also collected and analyzed. The results indicated that the ITS sequence of maca was consistent in all samples and unique when compared with its adulterants. Therefore, this DNA-barcoding approach based on the ITS sequence can be used for the molecular identification of maca and its adulterants.
DNA Barcoding, Taxonomic ; methods ; DNA, Intergenic ; analysis ; DNA, Plant ; analysis ; Drug Contamination ; prevention & control ; Humans ; Lepidium ; genetics ; Phytotherapy