Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; metabolism ; Bombyx ; enzymology ; CD13 Antigens ; metabolism ; Cadherins ; metabolism ; Endotoxins ; metabolism ; Hemolysin Proteins ; metabolism ; Larva

OBJECTIVE: To investigate the effect of osthole on epidermal growth factor receptor tyrosine kinase (EGFR-TPK), matrix-metalloproteinase-2 (MMP-2), aminopeptidase N (APN) in bladder cancer cell and the underlying mechanism.
 METHODS: The T24 cell lines were cultured. The inhibitory effects of osthole on EGFR-TPK, APN and MMP-2 were evaluated by spectrophotometric and MTT assay. The caspase-3 activity and the expression COX-2 and VEGF in T24 were examined. The activity of NF-κB was determined by electrophoretic mobility shift assay.
 RESULTS: The half inhibition concentrations (IC50) of osthole on EGFR-TPK, APN and MMP-2 were (45.33±3.98), (28.21±3.23) and (8.11±0.54) µmol/L, respectively. The growth inhibitory rates for T24 cells were increased in a dose-dependent manner (P<0.05). The caspase-3 activities were significantly increased in T24 cells in the osthole group compared with control group, while the expression of angiogenesis related-protein COX-2, VEGF, and NF-κB in T24 cells were decreased.
 CONCLUSION Through the inhibitory effect on EGFR-TPK, APN and MMP-2, osthole can decrease COX-2, VEGF and NF-κB expression while increase the activity of caspase-3, eventually blocking the growth and invasion of bladder cancer cell.
CD13 Antigens ; metabolism ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Coumarins ; pharmacology ; Cyclooxygenase 2 ; metabolism ; ErbB Receptors ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; NF-kappa B ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology ; Vascular Endothelial Growth Factor A ; metabolism

This study is to investigate the effect of downregulation histone deacetylases 1 (HDAC1) gene by the technology of RNA interference on the differentiation of HL-60 cells line. The optimal segment targeting HDAC1 gene was designed and transfected into HL-60 cells by Lipofectamine 2000. The HDAC1 mRNA and protein level were detected by RT-PCR and Western blotting. The morphologic change of HL-60 cells was detected by an optical microscope with Wright-Giemsa. Cell differentiation was tested by NBT reduction assay. Expression of CD13, CD33 and CD14 was measured by flow cytometry. The results indicated that HDAC1 mRNA and protein were markedly suppressed by the siRNA targeting HDAC1 in a concentration-dependent manner. HDAC1 siRNA promoted cell differentiation. HL-60 cells became more mature in morphology after transfected to HDAC1 siRNA at a concentration of 30-60 nmol x L(-1) for 24 h. NBT reduction ability of HDAC1 siRNA with 30 nmol x L(-1) was 0.31 +/- 0.09, compared with negative control (0.20 +/- 0.02) (t = -3.1, P < 0.01), and with 60 nmol x L(-1) was 0.25 +/- 0.02 in comparison with negative control (0.21 +/- 0.04) (t = -2.12, P < 0.05). But it has no change in HDAC1 siRNA > or = 120 nmol x L(-1). After transfection with 60 nmol x L(-1) HDAC1 siRNA to HL-60 cells, the expression of CD13 was (96.50 +/- 0.70)% in compared to siRNA-NC (3.39 +/- 0.68) % (t = 164.9, P < 0.000 5), CD33 was (66.73 +/- 0.50) % in compared to siRNA-NC (96.80 +/- 1.70) % (t = 43.4, P < 0.000 5). CD14 was (0.53 +/- 0.00) % by comparison with siRNA-NC (0.49 +/- 0.02) % (t = -0.97, P > 0.1). HDAC1 siRNA promoted cell differentiation in indicated concentration. HDAC1 might be one of the targets of gene therapy for leukemia.
CD13 Antigens ; metabolism ; Cell Differentiation ; Down-Regulation ; HL-60 Cells ; Histone Deacetylase 1 ; genetics ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Sialic Acid Binding Ig-like Lectin 3 ; metabolism ; Transfection

OBJECTIVETo establish and characterize a lung adenocarcinoma cell line from a female patient in Xuanwei, Yunnan province.

METHODSSurgical specimen of the lung adenocarcinoma was obtained and cultured immediately in RPMI 1640 medium with 10% fetal bovine serum and 10(5) U/L penicillin and 100 mg/L streptomycin. When stable proliferation of the cells was achieved after over 40 passages in culture, the biological features of the cell line were investigated by cell morphology, karyotyping, protein marker expression [cytokeratins (CKs), epithelial membrane antigen (EMA) and CD proteins], growth kinetics, cell cycle phase distribution, mitotic index, colony formation in soft agar, cell invasion and tumorigenicity in Balb/c nude mice.

RESULTSThe established cell line was stably cultured for over 80 passages during a one-year period as an anchorage-dependent monolayer of short spindle, polygonal to epithelioid cells under phase contrast microscope. Microglandular cavities and disordered microfilaments were observed under transmission electron microscope. The growth curve presented in an "S" shape with the cell population doubled every 46.7 hours. The mitotic index was 1.5% and the colony formation rate was 8.3%. The cell cycle distribution included 76.9% in G(0)/G(1), 15.1% in S and 8.0% in G(2)/M. The cell line displayed a hypotriploid karyotype with a mode of 66 chromosomes and a median of 64 chromosomes. The cells expressed CK7, CK8, CK (Pan) and EMA by immunohistochemistry. A high level of cell surface expression of CD13 and CD59 was evident by flow cytometry. The cells were able to penetrate Matrigel in vitro but failed to form a stable xenograft in nude mice.

CONCLUSIONA new human lung adenocarcinoma cell line, designated as XLA-07, is successfully established from a Xuanwei lung cancer patient.

Adenocarcinoma ; metabolism ; pathology ; Animals ; CD13 Antigens ; metabolism ; CD59 Antigens ; metabolism ; Cell Culture Techniques ; Cell Cycle ; Cell Line, Tumor ; ultrastructure ; Cell Proliferation ; Female ; Humans ; Karyotyping ; Keratins ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mucin-1 ; metabolism ; Neoplasm Transplantation ; Polyploidy ; Tumor Stem Cell Assay

This study is to investigate the effects of ubenimex on tumor cell invasion and apoptosis, dose relationship and mechanism. Immunofluorescence staining was performed to detect the expression of CD13 in HT-1080 cells. MTT assay was used to analyze the effect of ubenimex on cell proliferation. Annexin V-EGFP/PI was used to detect apoptotic cells by flow cytometry. Cell cycle was analyzed using flow cytometry. Ala-pNA was used as substrate to evaluate the effect of ubenimex on the aminopeptidase activity. Transwell assay was used to analyze the effect of ubenimex on cell invasion and migration ability. Western blotting was used to detect the expression level of CD13. MMP activity was analyzed using gelatin zymography. The results showed that ubenimex at high concentration inhibited the proliferation of HT-1080 cells (IC50: 3.8 mg x mL(-1)), and induced cell apoptosis. Cell cycle was blocked at G1 phase. Ubenimex at low concentration inhibited the aminopeptidase activity of HT-1080 cells (IC50: 8.3 microg x mL(-1)) and inhibited cell invasion, but it had no effects on the cell migration and proliferation. Ubenimex had no effects on CD13 expression and MMP activity. In conclusion, ubenimex at low concentration can inhibit the invasion ability of tumor cells by directly inhibiting the aminopeptidase activity; ubenimex at high concentration can inhibit the proliferation of tumor cells and induce cell apoptosis by a CD13-independent pathway.
Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; CD13 Antigens ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Fibrosarcoma ; metabolism ; pathology ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness

The objective of this study was to investigate the immunophenotype of T-lineage acute lymphoid leukemia (T-ALL) and to find valuable immunologic markers in T-ALL diagnosis and therapy. Four-color multiparametric flow cytometry(FCM) with CD45/SSC gating was used for immunophenotyping of 95 patients with newly diagnosed T-ALL. The results demonstrated that T-ALL occurred more frequently in males younger than 30 years of age and was usually accompanied by a high WBC count and tumor mass at diagnosis. Univariate analysis showed an influence on achievement of CR1 for age (< 30 years) but not for WBC count and tumor mass. According to WHO (2008) classification of tumors of haematopoietic and lymphoid tissues, 87 patients with confirmed subtype included 27 cases of Pro-T-ALL (31.0%), 31 cases of Pre-T-ALL (35.6%), 23 cases of cortical-T-ALL (26.4%), 6 cases of medullary-T-ALL (6.9%). CD34 expression in Pro-T-ALL was significantly higher than that of Pre-T-ALL (p = 0.001). After the first chemotherapy, the complete remission rate in Pro-T-ALL was statistically lower than that of Pre-T-ALL. Besides, the complete remission rate of immature T-ALL (including Pro-T-ALL and Pre-T-ALL) was also significantly lower than that in mature T-ALL (including cortical-T-ALL and medullary-T-ALL). Myeloid antigen (CD13, CD33) expression was associated with T-ALL subtype and treatment effect. While 66.7% of CD13(+) patients belonged to Pre-T-ALL, most (60.0%) of CD33(+) patients were classified into Pro-T-ALL; CD13 expression had no effect on CR1 rate whereas CD33(+) patients had worse treatment effect compared with CD33(-) groups (p = 0.001). Notably, the expression of CD117 reached up to 26.7% and the positive cases were primarily distributed in pro-T-TAll and pre-T-ALL. It is found that CD117 expression in CD34(-) group was homogeneous and CD117 expression level was less than 10% in 73.2% patients, but CD117 expression level in CD34(+) group was not homogenous, in which group the CD117 expression levels < 10%, 10% - 20% and > 20% were 44.2%, 17.3% and 38.5% respectively. As compared with CD34(-) group, the proportion of patients with CD117 expression levels < 10%, > 20% in CD34(+) group was higher, and there was significant difference between these 2 group. It is concluded that immunophenotype has great value in T-ALL diagnosis, classification as well as treatment. Flow cytometry provides access to find valuable immunologic markers for T-ALL biological research.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD13 Antigens ; metabolism ; Child ; Child, Preschool ; Female ; Humans ; Immunophenotyping ; Infant ; Male ; Middle Aged ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology ; therapy ; Proto-Oncogene Proteins c-kit ; metabolism ; Sialic Acid Binding Ig-like Lectin 3 ; metabolism ; Young Adult

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.
Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; CD13 Antigens ; antagonists & inhibitors ; metabolism ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Phosphorylation ; Tretinoin ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo screen and identify the peptides that specifically bind to CD13 on monocytes.

METHODSThe phages capable of specific binding to CD13 were screened in the phage-displayed 12-peptide library. The affinity of the selected phages with CD13 was verified with enzyme-linked immunosorbent assay (ELISA). The sequences of the peptides bound to the phages were deduced according to the phage DNA sequences, and the functional peptides aligned using the BLASTP on the Website NCBI were synthesized. To analyze the biological function of the screened peptides, the location of the peptides bound to THP-1 cells was detected using immunofluorescence assay. The blocking effect of WM15 on the peptide binding to THP-1 cells was assessed by immunofluorescence assay.

RESULTSThe phages that specifically bound to CD13 were effectively enriched to approach saturation after 4 rounds of panning. The recovery rate in the fourth round was 30 times that in the first round. Twenty selected phages were verified by ELISA, and the signals of 10 phages were higher than the control. The sequences of the peptides P9 and P7 showed 83% and 100% identity with those of human cytomegalovirus (HCMV) UL38 and UL105, respectively. The peptides bound to the cell membrane of THP-1 cells as shown by immunofluorescence assay. The binding of the peptides P9 and P7 to THP-1 cells was blocked by CD13-specific monoclonal antibody WM15 at different levels.

CONCLUSIONTwo peptides (P7 and P9) that can specifically bind to CD13 have been screened successfully, and these two peptides show specific binding to CD13 on the membrane of THP-1 cells.

Amino Acid Sequence ; Binding, Competitive ; CD13 Antigens ; analysis ; metabolism ; Cell Line ; Humans ; Molecular Sequence Data ; Peptide Library ; Peptides ; metabolism ; Protein Binding

To clone and express the gene encoding chicken aminopeptidase N (chAPN), and analysis the biological function of chAPN expressed in Escherichia coli (E. coli). The chAPN gene was amplified by RT-PCR from the kidney cells of chicken embryo and then cloned into the prokaryotic expression vector pCOLD-TF. Recombinant expression plasmid of pCOLD-TF-chAPN was constructed and then transformed into the competent E. coli BL21(DE3) cells for expression under different conditions such as induction time and inductor concentrations. Purified soluble recombinant chAPN was obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blotting assay. Its biological function was detected by its reaction with Leu-PNA and Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the expression product of chAPN gene in E. coli was soluble. It was able to bind infectious bronchitis virus (IBV) dose-dependently. In conclusion, chAPN gene has been successfully cloned and expressed in E. coli, which will establish a basis for further research the enzymatic activity and antiviral function.
Animals ; CD13 Antigens ; biosynthesis ; genetics ; metabolism ; Chickens ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets that leads to great economic losses in East Asia. It was reported that aminopeptidase N (APN) was the receptor for Transmissible gastroenteritis virus (TGEV), Human coronavirus 229E (HCoV-229E) and Feline coronavirus (FeCoV) which all belonged to group I coronavirus including PEDV. It was also confirmed previously that porcine aminopeptidase N (pAPN) could bind to PEDV, and anti-pAPN antibodies could inhibit the combination. To investigate whether pAPN was a receptor for PEDV, we transfected MDCK cells with porcine aminopeptidase (pAPN) cDNA and this enabled non-susceptible cells to support PEDV replication and serial viral propagation. Moreover, the infection was blocked by antibodies against pAPN, implying the critical role of pAPN during virus entry. In addition, immunofluorescence assays for detection of pAPN and PEDV antigens, together with neutralization assays using antibodies against pAPN, further confirmed the correlation between pAPN expression and viral replication in pAPN-transfected MDCK cells. These results indicated that pAPN is a functional receptor for PEDV.
Animals ; Antibodies ; pharmacology ; CD13 Antigens ; antagonists & inhibitors ; genetics ; metabolism ; Cell Line ; Coronavirus Infections ; enzymology ; metabolism ; Dogs ; Porcine epidemic diarrhea virus ; enzymology ; Swine