ABSTRACT
VirK family [Pfam06903] consists of 14 bacterial VirK proteins of around 145 residues in length. The function of this family is unknown. Herein, using single-wavelength anomalous diffraction, we determined the crystal structure of lpg1832, a VirK family protein from Legionella pneumophila, at 2.0 Å resolution. This is the first structural determination of a VirK domain-containing protein. Lpg1832 is a type II secretion system-dependent extracellular protein that folds into a novel barrel-shaped structure. It is found to adopt a quaternary assembly comprising a homotetramer. The three-dimensional structure of lpg1832 provides the first structural information pertaining to the VirK family and allows us to possibly identify its functionally important regions.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray/methods , Legionella pneumophila/metabolism , Models, MolecularABSTRACT
OBJECTIVE: To evaluate the hematopoietic reconstitution of implanted NOD/SCID mice, after intra-bone marrow cavity injection (iBM) of human umbilical cord blood (CB) mononuclear cells (MNCs). METHODS: 24 female NOD/SCID mice were divided into different MNCs dosage iBM groups (3 x 10(6), 1 x 10(7), 3 x 10(7) cells), tail vein intravenous injection (iTV) group (3 x 10(7) cells) and control group (iBM of medium only). CB MNCs sorted by Ficoll-Hypaque were transplanted into left tibia bone marrow cavity of 6- to 8-week-old NOD/SCID mice, which were anesthetized and sublethally irradiated (270 cGy (137)Cs-gamma irradiation). The distribution of injected CB MNCs in noninjected right tibia of the same implanted mice was observed 24 hours after iBM. The establishment of hematopoiesis and the survival of mice were observed. BM cell surface CD marker expressions, dye Dil-CM tracing and human beta-actin from implanted mice were assessed 8 weeks after iBM or iTV. RESULTS: Dil-CM marker could be detected on BM cells from noninjected right tibia 24 hours after iBM. Fourteen engrafted mice survived at the end of our study. Among them two, four and five were of iBM-1, iBM-2 and iBM-3 groups respectively, and one of control group and two of iTV group. White blood cell reconstitution was better in iBM mice than in iTV and control mice. There were human markers including CD45, Dil-CM and beta-actin DNA in the marrow cells from the human CB MNC engrafted mice. CONCLUSION: The preliminary results showed that hematopoiesis reconstitution by iBM was significantly better than iTV.
Subject(s)
Cord Blood Stem Cell Transplantation/methods , Fetal Blood/cytology , Animals , Cells, Cultured , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Random Allocation , Transplantation, HeterologousABSTRACT
BACKGROUND AND OBJECTIVES: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder with abnormal megakaryocyte/platelet production. Recent studies have found that Bcl-xL, as a member of the bcl-2 family of proteins that inhibit apoptosis, is essential in megakaryocytic differentiation. In this study the expression of Bcl-xL was evaluated during megakaryocytic differentiation in ET patients. DESIGN AND METHODS: To study the role of Bcl-xL in megakaryocyte differentiation, we evaluated the effect of small interfering RNA (siRNA) on the expression of Bcl-xL. CD34+ cells from patients with ET, chronic myeloid leukemia (CML), polycythemia vera (PV) and normal individuals were cultured in serum-free medium supplemented with thrombopoietin (TPO). Immunocytochemical staining and flow cytometric analysis were used to evaluate the Bcl-xL expression during megakaryocytic differentiation of CD34+ cells. RESULTS: When exposured to si-Bcl-xL, the percentage of K562 cells induced into megakaryocytes in 72 hours was lower than the corresponding percentage of control cells. CD41a+ cells from the three groups of patients and the control group were cultured. At day 10, the percentage of Bcl-xL- cells in CD41a+ cells from ET patients was 61.0+/-28.1%, which was significantly higher than that from patients with CML (2.5+/-20.9%), PV (33.6+/-10.0%) or control subjects (15.1+/-13.0%).] INTERPRETATION AND CONCLUSIONS: These results demonstrate that Bcl-xL is down-regulated early during in vitro differentiation of megakaryocytes from ET patients; this might reflect an early entry of megakaryocytes into a degenerating mature stage.
