Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.

In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.

Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling.

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase.

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.

Detection of tumorigenic cells in Friend virus-infected mice: an in vivo methodological investigation.

Tumor formation by subcutaneous transplants of spleens from erythroleukemic mice infected with Friend virus complex inducing polycythemia (FLV-P) is successful only during the late phase of the disease. To determine whether this observation is due to the absence of tumorigenic cells in the early phase of such leukemia or to the inability of standard procedures to detect these cells, the sensitivities of different routes of inoculation in sublethally irradiated or unirradiated syngeneic recipients were examined. The omentum of the sublethally irradiated mouse was found to be a suitable site for the homing and proliferation of recently isolated tumorigenic cells from FLV-P-infected mice, since it proved 1,000 times more sensitive than the usual subcutaneous sites in unirradiated mice. When this sensitive graft in the omentum was applied to detection of tumorigenic cells in the spleens of FLV-P-infected mice, the mean detection time was 20 days after virus infection, compared to 36 days with the usual subcutaneous graft method.

The 'WS motif' common to v-mpl and members of the cytokine receptor superfamily is dispensable for myeloproliferative leukemia virus pathogenicity.

Several motifs are conserved in the extracellular domain of the cloned chains of the recently described cytokine receptor superfamily. One of them, usually close to the transmembrane region, is the 'WS motif'. Its function remains unknown, but it has been recently shown that the integrity of this motif is essential for interleukin 2 receptor beta-chain and erythropoietin receptor activity [Miyazaki, T., Maruyama, M., Yamada, G., Hatakeyama, M. & Taniguchi, T. (1991). EMBO J., 10, 3191-3197; Watowich, S.S., Yoshimura, A., Longmore, G.D., Hilton, D.J., Hoshimura, Y. & Lodish, H.R. (1992). Proc. Natl. Acad. Sci. USA, 89, 2140-2144]. This WS motif is present in the v-mpl oncogene, which has been transduced in the myeloproliferative leukemia virus (MPLV). v-mpl encodes a truncated transmembrane protein that belongs to this growth factor receptor family. We demonstrate that determinants of MPLV pathogenesis are encoded by the env-mpl fusion gene and that the complete deletion of the WS motif does not abolish MPLV oncogenic properties.

Expression and activation of B-Raf kinase isoforms in human and murine leukemia cell lines.

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms previously shown to be expressed predominantly in neural tissues. We report here that B-Raf proteins of 95 and 72 kDa are also expressed in various human and murine hematopoietic cell lines. Their relative level of expression is variable depending on the cell line examined. The highest level of expression of p95B-raf was found in UT-7 cells, a human pluripotent cell line established from a patient with a megakaryoblastic leukemia. These cells are able to differentiate toward erythroid or myeloid lineage phenotypes in presence of erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. We show that treatment of UT-7 cells with EPO, GM-CSF or stem cell factor (SCF) rapidly induces phosphorylation of p95B-raf as indicated by a shift of electrophoretic mobility. This increase in phosphorylation is correlated with a three-fold increase of B-Raf kinase activity. B-Raf activation also increases in a dose-dependent manner in response to EPO and GM-CSF. We also show that both p95B-raf and p72B-raf can be activated by IL-3 in murine BAF-3 pro-B cells and by anti-CD3 in human Jurkat cells, respectively. These observations provide the first evidence that the B-Raf kinase is involved in signal transduction pathways regulating proliferation and differentiation of hematopoietic cells of both myeloid and lymphoid lineages.

Crystallization and preliminary X-ray diffraction study of the nickel-binding protein NikA of Escherichia coli.

NikA, a periplasmic nickel-binding protein involved in nickel-repellent chemotaxis has been crystallized. The crystals are hexagonal with space group P6(2) (or its enantiomorph) with a = 160.3 A and c = 138.4 A and they diffract to at least 3.0 A resolution in the laboratory. NikA presents sequence homology with several periplasmic solute-binding proteins from Gram-negative bacteria.

Rearrangements of the Pim-1, c-myc, and p53 genes in Friend helper virus-induced mouse erythroleukemias.

The Friend helper leukemia virus (F-MuLV) induces in mice leukemias of the erythroid, lymphoid, and myeloblastic lineages. Erythroleukemic cell DNAs were examined for genetic alterations at loci described as common proviral integration regions in MuLV-induced myeloid or lymphoid leukemias or in Friend complex-induced erythroleukemias. No alteration of the Fim-1, Fim-2, Fim-3, pvt-1, and Spi-1 loci were detected in 17 erythroleukemias, p53 gene rearrangement was observed in 6 (30%) erythroleukemias and was always associated with a loss of the germ line allele. Interestingly, genetic alterations were also detected at two loci, c-myc and Pim-1, previously described as common provirus integration regions in T lymphoid leukemias. Rearrangements of these two genes were often associated with p53 gene alteration within the same tumor.

The effects of filtration on protein nucleation in different growth media.

Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease dependent on the membrane cut-off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results. The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time-dependent change in the size or the number of crystals per drop. Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time-dependent effect.

[Mapping of the cardiac electric activity. Applications to experimental surgery]. / Cartographie de l'activité électrique cardiaque. Applications à la chirurgie expérimentale.

The results reported by French and other groups of workers have shown that peroperative epicardial mapping is a valuable technique for locating the accessory pathway in the Wolff-Parkinson-White syndrome and also in the surgical treatment of certain forms of chronic ventricular tachycardia. The aim of this study was to develop a completely automatic method of mapping potentials recorded at many points during the same systole. A system of acquisition and automatic treatment of 256 cardiac potentials called SATAPEC was constructed at the University of Caen. The system comprises specialized recorders, a multiplexing system on 256 leads with analogical-digital conversion and a PDP 11-23 mini computer. The results presented are based on the location of the His bundle and the right bundle branch in the dog from recordings of the H potential. The recording device with 240 (20 x 12) measuring points is a silk net on which 50 micrometers diameter silver wires are attached. The wires are insulated except at their tips and are separated one from another by a distance of 2 mm. After infundibulotomy the recording device was placed over the interventricular or right interatrial septum. The 240 unipolar electrograms were recorded during a single systole. The programmes which have been developed automatically produce maps of the amplitude of the H potential. The computer detects the presence or absence of a H potential on each electrogram and then displays on a cathode ray tube points of variable size indicating the locations of the electrodes which have recorded a H potential.(ABSTRACT TRUNCATED AT 250 WORDS)

MPLV: a retrovirus complex inducing an acute myeloproliferative leukemic disorder in adult mice.

A novel murine retrovirus complex was derived from the in vivo passage of a molecularly cloned Friend ecotropic helper virus. The virus isolate, myeloproliferative leukemia virus (MPLV), causes an acute (2-3 weeks) and generalized myeloproliferative disorder in adult mice. All strains of mice examined, including the C57BL/6J strain, developed the acute syndrome. This syndrome is characterized by a rapid hepatosplenomegaly, no thymus or lymph node involvement, granulocytosis, thrombocytosis, and erythroblastosis leading to polycythemia. The most prominent feature at the terminal phase of the disease is a granulocytic hyperplasia. The MPLV isolate replicates in vitro on NIH 3T3 fibroblasts but does not induce foci of transformed cells. Thus, MPLV exhibits unique biological properties that distinguish it either from the Friend virus complexes or from acutely transforming sarcomatogenic murine retrovirus which also induced a rapid splenomegaly.

Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors.

The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, we have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10(4) foci per microgram of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, we found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which we had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo (M. Souyri, C. F. Koehne, P. V. O'Donnel, T. H. Aldrich, M. E. Furth, and E. Fleissner, Virology 158:69-78). However, similarly poor oncogenicity was also observed when the v-H-ras coding sequence was inserted in pSV(X) vector, which indicated that the vector sequences play a crucial role in the pathogenicity of a given oncogene. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.

Factor-independent erythropoietic progenitor cells in leukemia induced by the myeloproliferative leukemia virus.

The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform fibroblasts, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of growth factor requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (16%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few factor-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.

PBR: a heavy-atom refinement and phasing procedure to reduce phase bias when heavy-atom derivatives contain common sites.

A procedure, called PBR (phase-bias reduction), has been developed to properly refine heavy-atom derivatives and to generate less biased heavy-atom phases when these derivatives contain common heavy-atom sites. Two independent events are obtained by splitting the refinement and phasing calculations into two stages, the first in which one of the derivatives having common sites is used together with the native amplitudes and the second in which both derivatives with common sites are used simultaneously, with one of them being used as the native data set. Improved centroid phases and the corresponding figures of merit are obtained by phase combination. This procedure has been used in the structure determination of the iron-cluster-containing protein -pyruvate-ferredoxin oxidoreductase. When the common heavy-atom sites are properly treated by the PBR procedure, the resulting calculated centroid phases are improved with respect to classical heavy-atom refinement centroid phases where all derivatives are refined together. This leads to improved electron-density distributions, since anomalous difference Fourier maps calculated with the PBR-refined centroid phases and corresponding figures of merit show more clearly the positions of the iron sites.

Crystallographic studies of the interaction between the ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena: looking for the elusive ferredoxin molecule.

Ferredoxin-NADP(+) reductase (FNR) and its physiological electron donor ferredoxin (Fd) from the cyanobacterium Anabaena PCC7119 have been co-crystallized. The unit-cell parameters are a = b = 63.72, c = 158.02 A and the space group is P2(1)2(1)2(1). The crystal structure has been solved with 2.4 A resolution synchrotron data by molecular replacement, anomalous dispersion and R(min) search methods. For the computations, the crystal was treated as a merohedral twin. The asymmetric unit contains two FNR molecules and one ferredoxin molecule. The packing of the FNR molecules displays a nearly tetragonal symmetry (space group P4(3)2(1)2), whereas the ferredoxin arrangement is orthorhombic. This study provides the first crystallographic model of a dissociable complex between FNR and Fd.

Interleukin-3, erythropoietin, and prolactin activate a STAT5-like factor in lymphoid cells.

Interleukin-3 (IL-3)-, erythropoietin (EPO)-, and prolactin (PRL)-induced signal transduction via the JAK/STAT pathway was studied in the IL-3-dependent BAF-3 lymphoid cell line. Transfected cells expressing either the long form of the PRL receptor or the EPO receptor were used. We demonstrated that IL-3, EPO, and PRL activated a transcription factor related to the mammary transcription factor STAT5 but not to STAT1, -2, -3, or -4 as opposed to interferon gamma (IFN gamma) which activated STAT1 in the same cells. Similarly, PRL and EPO activated a STAT5-like factor (STAT5-L) in the rat Nb2 and the human UT7 cells expressing endogenous PRL and EPO receptors, respectively. The hematopoietic STAT5-L activated by IL-3, EPO, or PRL was identified as a 97-kDa tyrosine-phosphorylated protein. These results confer to STAT5 a much broader role than previously suggested.

Characterization of mpl cytoplasmic domain sequences required for myeloproliferative leukemia virus pathogenicity.

v-mpl is a truncated form of a receptor-like chain which belongs to the cytokine receptor superfamily. This sequence has been transduced in the myeloproliferative leukemia virus as an env-mpl fusion gene responsible for an acute myeloproliferative disorder in mice. We constructed a series of viral mutants in the mpl sequence. Analysis of their oncogenic potential in vivo indicated that a critical 69-amino-acid-long cytoplasmic domain of v-Mpl is required for myoproliferative leukemia virus pathogenicity. We also developed an in vitro assay and showed that expression of the env-mpl gene confers growth factor independence to murine as well as to human hematopoietic growth factor-dependent cell lines. These findings strongly suggest that v-Mpl delivers a constitutive proliferative signal through a limited region of its cytoplasmic domain.

Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization.

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this truncated cytokine receptor and found that the rearrangement of the env sequences in the env-mpl fusion gene was not required for oncogenicity. A pathogenic variant, DEL3MPLV, was generated, which differs from MPLV by the deletions of 22 amino acids of the Env signal peptide, all of the mature Env sequences, and 18 N-terminal amino acids of the v-Mpl extracellular domain. The resulting del3-mpl oncogene product conserves in its extracellular region the first 12 amino acids of the Env signal sequence including a cysteine residue, and 25 amino acids of the v-Mpl. We show here that a mutation converting this cysteine to a glycine completely abolishes del3-mpl oncogenicity and that the del3-mpl oncogene product is constitutively activated by disulfide-linked homodimerization.