Case Report: The First Report of NUP214-ABL1 Fusion Gene in Acute Myeloid Leukemia Patient Detected by Next-Generation Sequencing.

The NUP214-ABL1 fusion gene is a constitutively active tyrosine kinase that can be detected in 6% of T-cell acute lymphoblastic leukemia (T-ALL) patients, and it can also be found in B-cell acute lymphoblastic leukaemia (B-ALL). However the NUP214-ABL1 fusion in acute myeloid leukemia (AML) has not yet been reported. Up to now, the sensitivity of NUP214-ABL1-positive patients to tyrosine kinase inhibitor (TKI) is still controversial. Here we report the first case of an AML patient carrying NUP214-ABL1 fusion gene. The conventional AML chemotherapy regimen for the patient was successful. Identification of additional AML patients with NUP214-ABL1 fusion gene will provide treatment experience and prognostic evaluation.

[Analysis on bioactivity of HIV-1 integrase by ELISA method].

OBJECTIVE: To develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors. METHODS: After expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates. RESULT: (1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L. CONCLUSION: The non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.

[Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets].

OBJECTIVE: To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin ß3 on platelet signal transduction. METHODS: A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin ß3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested. RESULTS: myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation. CONCLUSION: The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin ß3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

[Effect of the Integrin ß3 Cytoplasmic NITY Motif on α II bß3-Mediated Cell Functions in CHO Cell Model].

UNLABELLED: OBJLECTIVE: To investigate the effect of integrin ß3 cytoplasmic NITY motif on αIIbß3-mediated cell functions. METHODS: Stable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type ß3 or mutant ß3ΔNITY (ß3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and ß3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions. RESULTS: CHO-αIIbß3, CHO-αIIbß3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbß3 had the ability of adhesion and spreading. Compared with CHO-αIIbß3 cells, CHO-αIIbß3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbß3. The ß3ΔNITY mutation substantially reduced kindlin-2 association. CONCLUSION: Deletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin ß3, thereby partially inhibit the integrin ß3 signaling.

[Transgenic mouse models of the truncated platelet integrin ß3 cytoplasmic tail established by stem cell transplantation].

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin ß3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin ß3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type ß3, ß3-Δ759 (R(760) GT(762) truncated ß3) and ß3-Δ754 (T(755) NITYRGT(762) truncated ß3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the ß3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface ß3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type ß3, ß3-Δ759 and ß3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the ß3 expression of transgenic mouse reached heterozygote (ß3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin ß3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

[Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana].

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.