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

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

This study was aimed to investigate the effect of exogenous Wnt5a on directional differentiation of K562 cells. Wnt5a and GFP condition mediums were prepared by recombinant adenoviral vector AdWnt5a and AdGFP transfecting CHO cells. K562 cells were treated with Wnt5a and the GFP condition mediums for 1 - 7 days as Wnt5a treated group and control group respectively. The morphological changes of K562 cells were observed by light microscope and electron microscope; the differentiation phenotypes of K562 cells were identified by the cytochemical staining of POX, PAS, alpha-NAE and immunocytochemistry of CD13, CD14, CD68, and the effect of Wnt5a on cell cycle distribution of K562 cells was detected by flow cytometry. The results showed that the morphology and ultrastructure of K562 cells treated by Wnt5a displayed differentiation mature feature; both POX and PAS staining showed higher positive ratio in Wnt5a treated group than that in control group; the alpha-NAE staining also was positive, but positive intensity in Wnt5a treated group could be inhibited up to 70% by NaF. The expressions of monocytic differentiation antigens of CD14, CD68 in Wnt5a treated group were higher than those in control group, but the expression differences of granulocytic differentiation antigen CD13 between Wnt5a treated group and control group were not significant. The cell cycle in treated group was blocked at G2 phase as compared with control group. It is concluded that exogenous Wnt5a can induce K562 cells to differentiate towards mature and K562 cells treated with Wnt5a displays features of differentiation towards monocytic lineage.
Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD13 Antigens ; metabolism ; Cell Cycle ; drug effects ; Cell Transformation, Neoplastic ; drug effects ; Culture Media ; Humans ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; Phenotype ; Proto-Oncogene Proteins ; metabolism ; pharmacology ; Wnt Proteins ; metabolism ; pharmacology ; Wnt-5a Protein

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

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

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

Porcine epidemic diarrhea virus (PEDV) causes an acute enteritis in pigs of all ages, often fatality for neonates. PEDV occupies an intermediate position between two well characterized members of the coronavirus group I, human coronavirus (HCoV-229E)and transmissible gastroenteritis virus (TGEV) which uses aminopeptidase N (APN), a 150 kDa protein, as their receptors. However, the receptor of the PEDV has not been identified yet. A virus overlay protein binding assay (VOPBA) was used to identify PEDV binding protein in permissive cells. The binding ability of PEDV to porcine APN (pAPN) and the effects of pAPN on infectivity of PEDV in Vero cells were also investigated. VOPBA identified a 150 kDa protein, as a putative PEDV receptor in enterocytes and swine testicle (ST) cells. Further the PEDV binding to pAPN was blocked by anti-pAPN and pAPN enhanced PEDV infectivity in Vero cells. In conclusion, these results suggested that pAPN may act as a receptor of PEDV.
Animals ; Antigens, CD13/*metabolism ; Cercopithecus aethiops ; Coronavirus/*metabolism ; Coronavirus Infections/*veterinary/virology ; Digestive System Diseases/metabolism/*veterinary/virology ; Enterocytes/enzymology/metabolism/virology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Male ; Protein Binding ; Receptors, Virus/*metabolism ; Swine ; Swine Diseases/metabolism/*virology ; Vero Cells

OBJECTIVETo investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression.

METHODSThe results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases.

RESULTSEleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers.

CONCLUSIONSFlow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD13 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD5 Antigens ; metabolism ; CD79 Antigens ; metabolism ; Diagnosis, Differential ; Flow Cytometry ; Humans ; Leukemia, B-Cell ; diagnosis ; immunology ; Leukemia, T-Cell ; diagnosis ; immunology ; Lymphoma, Mantle-Cell ; diagnosis ; immunology ; Peroxidase ; metabolism ; Retrospective Studies ; Sialic Acid Binding Ig-like Lectin 3

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

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