Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels

CLC-ec1 is an E. coli homologue of the CLC family of Cl- channels, which are widespread throughout eukaryotic organisms. The structure of this membrane protein is known, and its physiological role has been described, but our knowledge of its functional characteristics is severely limited by the absence of electrophysiological recordings. High-density reconstitution and incorporation of crystallization-quality CLC-ec1 in planar lipid bilayers failed to yield measurable CLC-ec1 currents due to porin contamination. A procedure developed to prepare the protein at a very high level of purity allowed us to measure macroscopic CLC-ec1 currents in lipid bilayers. The current is Cl- selective, and its pH dependence mimics that observed with a 36Cl- flux assay in reconstituted liposomes. The unitary conductance is estimated to be <0.2 pS. Surprisingly, the currents have a subnernstian reversal potential in a KCl gradient, indicating imperfect selectivity for anions over cations. Mutation of a conserved glutamate residue found in the selectivity filter eliminates the pH-dependence of both currents and 36Cl- flux and appears to trap CLC-ec1 in a constitutively active state. These effects correlate well with known characteristics of eukaryotic CLC channels. The E148A mutant displays nearly ideal Cl- selectivity.     

Suberoylanilide hydroxamic acid (SAHA) at subtoxic concentrations increases the adhesivity of human leukemic cells to fibronectin

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL‐MK1, K562, CML‐T1, Karpas‐299, HL‐60, and ML‐2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose‐dependent and cell type‐dependent cell death which displayed apoptotic features (caspase‐3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5–1 µM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma‐derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin β1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA‐induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate). J. Cell. Biochem. 109: 184–195, 2010. © 2009 Wiley‐Liss, Inc.     

转录因子与造血调控

血细胞生成是一个极其复杂的过程,转录因子在这个过程中起到了重要的调控作用。而转录因子的表达具有阶段和细胞系特异性。在造血干细胞的增殖和分化、髓系和淋巴系细胞等的成熟过程中,众多转录因子既相互作用又表现出各自的特异性。转录因子数目较多,该文仅就一些与造血细胞分化、成熟相关的转录因子近年来的研究进展作一介绍。     

巨核细胞生成调控相关细胞因子及其作用研究进展

造血干细胞分化生成巨核细胞是一个十分复杂的过程,包括造血干细胞动员及其向巨核系祖细胞分化,巨核系祖细胞增殖、分化生成未成熟巨核细胞,巨核细胞的成熟和血小板释放等过程。研究发现,造血干细胞动员及其向各系细胞分化的大部分过程都在一种称为"龛"的结构中进行,多种龛内信号分子参与了造血干细胞的动员和分化调控。该文对造血干细胞龛内参与造血干细胞动员和分化生成巨核细胞的几种重要细胞因子及其调控作用进行综述。     

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage-restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage-restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage-restricted progenitor cells.     

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion.     

AML1-ETO interacts with Sp1 and antagonizes Sp1 transactivity through RUNT domain

AML1-ETO fusion protein is observed in approximately 12% of acute myeloid leukemia. In the present research, we found that AML1-ETO is able to inhibit Sp1 transactivity. We also found that this inhibition of Sp1 transactivity by AML1-ETO is achieved by interaction between Sp1 and RUNT domain of AML1. AML1b is able to abrogate the inhibition of AML1-ETO. Since Sp1 is involved in hematopoietic cell differentiation, we proposed that AML1-ETO promotes leukemogenesis by blocking cell differentiation through inhibition of Sp1 transactivity.     

Importance of Lipids for Bacteriorhodopsin Structure, Photocycle, and Function

This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the regulation of the pK for the purple to blue transition caused by deionization, and the reformation of trimers from monomers after exposure of the purple membrane to Triton X-100. Most of the review is devoted to newer studies which indicate an important role for the neutral lipid squalene in the functional stability of the fast-decaying M-intermediate, for its decay through a pathway involving the O-intermediate, and for the regulation of the relative amounts of slow-decaying and fast-decaying forms of M. Participation of a peripheral acidic amino acid in the overall expression of fast-decaying M is also discussed. Initial studies suggest that the acidic amino acid may be Asp36 and/or Asp38.     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.