[Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure].

OBJECTIVE: To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA). METHODS: Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco's Modified Eagle's medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope. RESULTS: After incubating protamine sulfate, negative potential was transformed into positive one ( (-1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h ( P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less. CONCLUSION: Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

The additive effects of combined murine nuclear migration protein with murine thrombopoietin in vitro and in vivo on normal and myelosuppressed mice.

The human homolog of a fungal nuclear migration protein (hNUDC) has recently been shown in in vitro studies to overlap in function with the cytokine factor thrombopoietin (TPO) in its ability to induce human megakaryocyte development. In the present study, we sought to confirm the hypothesis that combination of TPO and NUDC may result in additive or synergistic effects on megakaryocyte maturation in bone marrow. For this purpose, murine bone marrow cells were stimulated with murine NUDC (mNUDC), either alone or in conjunction with murine thrombopoietin (mTPO), in serum-free medium. Studies showed that mNUDC + mTPO treatments resulted in the greatest number of colony-forming unit of megakaryocytes. Concomitantly, mNUDC + mTPO enhanced expression of CD61 and elicited the largest number of megakaryocytes, with higher ploidy and larger size. In addition, in vivo experiments revealed an elevation of platelet levels in normal and thrombocytopenic mice that had been administered mTPO + mNUDC. Moreover, mRNA levels of mNUDC and murine thrombopoietin receptor were much higher than the levels of mTPO mRNA at two different phases of normal murine megakaryocyte maturation. Furthermore, levels of ERK1/2 and Akt phosphorylation are higher with mNUDC in combination with mTPO. This study demonstrates that the combination of mTPO and mNUDC provides additive induction of megakaryocyte maturation in vitro and platelet production in vivo.

[hNUDC promotes proliferation and differentiation of megakaryocytopoiesis on human CD341+ cells].

AIM: aTo study the effect of human nuclear distribution C èhNUDCé on human megakaryocyte proliferation and differentiation from cord blood CD34(+) cells in vitro. METHODS: aHuman CD34(+) cells were isolated using the Dynal CD34 Progenitor Cell Selection System from umbilical cord blood. The CD34(+) cells were then cultured in serum free methylcellulose semi-solid media, the morphologic aspects and number of small, medium and large CFU-MK colonies were observed and scored on the day12 by microscopy analysis. The CD34(+) cells were cultured in serum free liquid media, cells were removed on day 10 and formation of CD41(+) in human megakaryocyte and its DNA polyploidization of nuclear were analyzed on a FACsort flowcytometer. RESULTS: ahNUDC supported the formation of small and medium CFU-MK colony in serum free semi-solid media. Furthermore, hNUDC induced a remarkable increase in expression of the megakaryocyte cell surface marker CD41(+) and stimulated the CD41(+) DNA polyploidization more effectively than TPO. CONCLUSION: hNUDC may play an important role in megakaryocyte proliferation and differentiation.

[Preparation of monoclonal antibody against human hNudC and its expression in human megakaryocytes].

AIM: To prepare and characterize the monoclonal antibody (mAB) against human nuclear distribution C (hNudC). METHODS: The coding region of hNudC was amplified from human embryo liver tissue and the recombinant prokaryotic expression vector pET28b was constructed. hNudC protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified recombinant hNudC protein was used to immunize BALB/c mice for preparing mAb. The hybridoma cells produced from mAB were screened by ELISA and the specificity of mAb was analyzed by immunohistochemical staining and Western blot. RESULTS: Two hybridoma cells (2C16 and 2D8) secreting mAb against hNudC were developed. The isotypes of the two mABs were IgGI and IgM, respectively. ELISA detection showed that the titer of mABs, 2C16 and 2D8 was 1:64, 1:32 in cultured supernatant and 1:1 x 10(5), 1:5 x 10(4) in ascites, respectively. They could specifically bind to recombinant hNudC. The results of immunohistochemical staining and Western blot indicated that mAb could specifically recognize hNudC in human Dami, Meg-01 cell lines and human marrow CD41+ megakaryocytes generated from umbilical core blood. CONCLUSION: Monoclonal antibodies against hNudC with high titers and specificity have been successfully prepared, which will be useful for assessing the function, native distribution and aberrant expression of hNudC protein.

Functional characterization of hNUDC as a novel accumulator that specifically acts on in vitro megakaryocytopoiesis and in vivo platelet production.

Human NUDC (hNUDC) has been previously described as a human homolog of a fungal nuclear migration protein. It is a multifunctional interactive protein that forms an association with the microtubule motor complex in a variety of cells. Our recent studies demonstrated that hNUDC could bind specifically to the thrombopoietin receptor (Mpl) and suggest a potential role for hNUDC in megakaryocytopoiesis and thrombopoiesis. The present study is designed to define its biological activity. We demonstrate that the recombinant hNUDC significantly increases megakaryocyte maturation in serum-free liquid-cultured human CD34(+) cells and stimulates colony formation in serum-free semi-solid cultures. Flow cytometry analyses also confirm the stimulatory effect of hNUDC on megakaryocyte polyploidization and in vitro platelet production. In vivo experiments further demonstrate that the administration of hNUDC substantially enhance the number of circulating platelets in normal mice.

A microtubule associated protein (hNUDC) binds to the extracellular domain of thrombopoietin receptor (Mpl).

Human NUDC (hNUDC) was initially characterized as a nuclear migration protein based on the similarity of its C-terminus to that of fungal NUDC from Aspergillus nidulans. However, hNUDC is a 331 amino acid protein whereas fungal NUDC is 198 amino acids in length. The extra N-terminal portion of hNUDC has no known function or homology to other proteins. In this study, we report the binding of hNUDC to the extracellular domain of the thrombopoietin receptor (Mpl) as detected by the yeast two-hybrid system, GST pull-down, and co-immunoprecipitation. Our deletion analysis demonstrated that amino acids between positions 100 and 238 as the critical domain mediating the hNUDC and Mpl interactions as detected by the two-hybrid system and GST pull-down assay. Immunofluorescence staining of human megakaryocyte cells indicated that hNUDC and Mpl colocalized at all stages of megakaryocyte development. Substantial colocalization of hNUDC with microtubules was also detected around nuclei and elongated microtubular structures, especially in proplatelet extensions.

Expression of the soluble extracellular domain of human thrombopoietin receptor using a maltose-binding protein-affinity fusion system.

The thrombopoietin (TPO) receptor (Mpl) belongs to the family of ligand-dependent cytokine receptors and plays a functional role in regulating platelet production. The signaling capacity largely depends on the binding of TPO to the extracellular domains of the TPO receptor (Mpl-EC). Because the expression level of Mpl in human tissue is very low, studies on the functional and spatial characteristics of its ligand-binding sites have been limited. In the present study, we report the expression and purification of Mpl-EC as a fusion with the maltose-binding protein (MBP), designated MBP-Mpl-EC. MBP-Mpl-EC was expressed in the cytoplasm of Escherichia coli as a soluble fusion protein. Specific binding of TPO to purified MBP-Mpl-EC was demonstrated by a dot-blot assay and surface plasmon resonance. We conclude that bacterial expression of MBP-Mpl-EC yields large amounts of protein with correct folding and that it can be used for further structure and function analyses.