A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.     

Protein synthesis: its control in erythropoiesis

Erythropoiesis in the fetal mouse provides a model to study several important aspects of the regulation of cell differentiation and differentiated protein synthesis. Changes in the patterns of hemoglobins formed during fetal and postfetal development are shown to be associated with the substitution of the liver erythroid cell line. In the course of differentiation of yolk sac erythroid cells there are at least two classes of proteins distinguishable with respect to dependence on continued RNA formatoin. The bulk of nuclear proteins, "nondifferentiated" proteins, appear to be dependent on relatively short-lived messenger RNA while synthesis of differentiated proteins, the hemoglobins, proceeds on relatively stable molecules of messenger RNA. Hemoglobin formation occurs in those cells which are actively synthesizing DNA and dividing. On the average, two to three cell divisions may occur after the formation and stabilization of the messenger RNA for globin. Yolk sac erythropoiesis, at least from day 10 of gestation, is unresponsive to erythropoietin. By comparison, in fetal liver erythropoiesis, the hormone, erythropoietin, acts selectively on the most immature erythroid cell precursor to induce differentiation, cell replication, and hemoglobin formation. The erythropoietin responsive cell in the liver is apparently differentiated from the progenitor, pluripotential stem cell and committed to erythroblast formation and hemoglobin synthesis on exposure to the hormone. The initial effects of erythropoietin on macromolecular synthesis are to stimulate RNA synthesis, which temporally is followed by cell replication and the increase in hemoglobin formation. During liver erythropoiesis, there appears to be a transition from hemoglobin synthesis dependent on RNA formation to hemoglobin synthesis directed by relatively stable messenger RNA.     

Autonomous developmental control of human embryonic globin gene switching in transgenic mice

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.     

Graft versus host inhibition: fetal liver and thymus cells to minimize secondary disease

Long-lived radiation chimeras were produced in mice diflering at the major histocompatibility locus. Survival occurred in lethally irradiated recipients inoculated with allogeneic fetal liver and allogeneic fetal thymus cells in combination. The survival rate was equal or superior to that of mice with transplanted syngeneic fetal, neonatal, or adult hematopoietic cells.     

Globin composition and synthesis of hemoglobins in developing fetal mice erythroid cells

Fetal mouse erythropoiesis proceeds initially in yolk-sac blood islands (8 to 12 days) and, subsequently, in liver (12 to at least 16 days). Yolksac cells synthesize three hemoglobins, Hb E(I), Hb E(II) and Hb E(III). Hb E(I) has x- and y-globin chains; Hb E(II) has alpha and y; HB E(III), alpha and z. No detectable beta-globin is formed in these cells. Liver erythroid cells form only adult hemoglobin, composed of alpha- and beta-chains.     

The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function

The study of human hematopoietic cells and the human immune system is hampered by the lack of a suitable experimental model. Experimental data are presented showing that human fetal liver hematopoietic cells, human fetal thymus, and human fetal lymph node support the differentiation of mature human T cells and B cells after engraftment into mice with genetically determined severe combined immunodeficiency. The resultant SCID-hu mice are found to have a transient wave of human CD4+ and CD8+ T cells and human IgG (immunoglobulin G) in the peripheral circulation. The functional status of the human immune system within this mouse model is not yet known.     

磺胺喹噁啉对小鼠的急性毒性试验

研究了磺胺喹噁啉对小鼠的急性毒性作用。采用改良寇氏法测定小鼠口服磺胺喹噁啉的LD50。将小鼠随机分为5组,分别灌服不同剂量的磺胺喹噁啉和生理盐水。观察小鼠灌服不同剂量磺胺喹噁啉后的临床症状以及小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬活性,测定脏器指数。结果表明,磺胺喹噁啉对小鼠口服的LD50为4889.90 mg/kg,95%可信限为5471.71~4308.09 mg/kg。磺胺喹噁啉中毒后,小鼠巨噬细胞吞噬鸡红细胞的能力降低,心脏指数和脾脏指数降低,肝脏指数和肾脏指数升高。主要病理变化为心脏色泽暗淡,心肌纤维肿胀;肝脏和肾脏明显肿大,肾小管上皮细胞肿胀变性,部分肾小管上皮细胞破裂;脾脏肿大,脾小体萎缩,淋巴细胞减少。研究结果说明,小鼠磺胺喹噁啉中毒后,非特异性免疫功能活性降低,心脏、肝脏、脾脏和肾脏等主要器官都受到一定程度的损害。     

Neonatal hepatitis induced by alpha 1-antitrypsin: a transgenic mouse model

Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of the human alpha 1-antitrypsin (alpha 1-Pi) gene. All of the alpha 1-Pi transgenic mice expressed the human protein in the liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The human M-allele protein was secreted normally into the serum. However, the human Z-allele protein accumulated in several cell types, but particularly in hepatocytes, and was found in serum in tenfold lower concentrations than the M-allele protein. Mice in one lineage carrying the mutant Z allele expressed high levels of human alpha 1-Pi RNA and displayed significant runting (50% of normal weight) in the neonatal period. This lineage was found to have alpha 1-Pi-induced liver pathology in the neonatal period, concomitant with the accumulation of human Z protein in diastase-resistant cytoplasmic globules that could be revealed in the Periodic acid-Schiff reaction (PAS). The phenotype of mice in the strain expressing high levels of the Z allele is remarkably similar to human neonatal hepatitis, and this strain may prove to be a useful animal model for studying this disease.     

小鼠胚胎发育不同阶段肝脏差异蛋白质组分析

利用固相pH梯度双向凝胶电泳技术分离胎龄12,13,14,16,18d的胎鼠及成鼠肝脏总蛋白,考染显色．PDQuest2DE软件分析获得6个不同发育时期的蛋白质表达谱。从总体看,14,16,18d胎鼠与成鼠肝脏的蛋白质表达谱较为一致,而12d和13d的肝脏蛋白质表达谱则与它们有较明显的差异,其蛋白质表达量显著降低,许多蛋白甚至缺失。以18d的胎肝图谱为参考,14,16d与其匹配率分别为57％,61％,成鼠与其匹配率为69％。与18d胎肝图谱相比,14d表达上调3倍以上的点有27个,16d表达上调3倍以上的点有13个,成鼠表达上调3倍以上的点有15个。并应用基质辅助激光解吸电离飞行时间质谱对14个差异蛋白质进行分析鉴定,经数据库查询,其中6个蛋白点获得了有意义的结果,分别为细胞周期依赖激酶调节亚基1、凡科尼蛋白、酪氨酸蛋白激酶BLK、通用转录因子3C多肽4、TBC1家族因子13、巨噬细胞受体MARCO。研究结果表明,在小鼠胚胎发育过程中,肝脏合成蛋白的种类和数量均迅速增加,并且细胞的增殖和生长、免疫功能的发育和完善以及蛋白质与核酸的合成和运输在其胎肝发育过程中起着非常重要的作用。     

MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity.

The role of major histocompatibility complex (MHC) class I expression in natural killer (NK) cell target recognition is controversial. Normal T cell blasts from MHC class I-deficient mutant mice were found to serve as target cells for NK cells in vitro, which suggests that MHC class I molecules are directly involved in NK cell recognition. Spleen cells from the mutant mice were deficient in their ability to lyse MHC class I-deficient target cells or NK-susceptible tumor targets, and mutant mice could not reject allogeneic bone marrow. Thus, class I molecules may participate in the positive selection or tolerance induction of NK cells.     

Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP

JNPL3 transgenic mice expressing a mutant tau protein, which develop neurofibrillary tangles and progressive motor disturbance, were crossed with Tg2576 transgenic mice expressing mutant beta-amyloid precursor protein (APP), thus modulating the APP-Abeta (beta-amyloid peptide) environment. The resulting double mutant (tau/APP) progeny and the Tg2576 parental strain developed Abeta deposits at the same age; however, relative to JNPL3 mice, the double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and olfactory cortex. These results indicate that either APP or Abeta influences the formation of neurofibrillary tangles. The interaction between Abeta and tau pathologies in these mice supports the hypothesis that a similar interaction occurs in Alzheimer's disease.     

Activation of developmentally mutated human globin genes by cell fusion

Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.     

Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice

Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the engulfment of apoptotic cells by macrophages. We report that tingible body macrophages in the germinal centers of the spleen and lymph nodes strongly express MFG-E8. Many apoptotic lymphocytes were found on the MFG-E8-/- tingible body macrophages, but they were not efficiently engulfed. The MFG-E8-/- mice developed splenomegaly, with the formation of numerous germinal centers, and suffered from glomerulonephritis as a result of autoantibody production. These data demonstrate that MFG-E8 has a critical role in removing apoptotic B cells in the germinal centers and that its failure can lead to autoimmune diseases.     

Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis

The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.     

Liver mitochondria from manganese-deficient and pallid mice: function and ultrastructure

Oxidative phosphorylation was studied in isolated liver mitochondria from manganese-deficient mice and in those from a mutant strain, pallid. In mitochondria from manganese-deficient mice, ratios of adenosine triphosphate formed to oxygen consumed were normal, but oxygen uptake was reduced. Electron microscopy of these mitochondria revealed ultrastructural abnormalities including elongation and reorientation of cristae. No biochemical or structural abnormalities were found in mitochondria from pallid mice.     

Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes

Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents their precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C (Nppc) messenger RNA (mRNA), whereas cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC increased cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.     

Lysosomal stability during phagocytosis of Aspergillus flavus spores by alveolar macrophages of cortisone-treated mice

Control mice and those treated with cortisone were exposed to aerosols of viable spores of Aspergillus flavus. Fifteen to 20 minutes later, animals were killed, and alveolar macrophages were obtained by tracheobronchial lavage. Electron-microscopic examination of these cells revealed that, whereas the lysosomes of control macrophages showed extensive attraction and fusion with the phagocytic membranes surrounding spores, the lysosomes of macrophages from animals treated with cortisone revealed little, if any, interaction. This diminished lysosomal response in forming phagocytic vacuoles may be important in the subsequent development of hyphal bronchopneumonia which frequently, occurs in cortisonetreated mice exposed to spores of A. flavus.     

孕鼠DEHP暴露影响胎儿早期卵母细胞H3K27me3表达的研究

本文以胎鼠卵母细胞为研究对象,分析了妊娠母鼠孕期暴露邻苯二甲酸二(2-乙基)己酯(DEHP)对胎鼠卵母细胞早期发育过程中组蛋白甲基化修饰程度的影响,结果发现:妊娠母鼠在12.5 dpc到16.5 dpc 期间暴露40 μg/kg DEHP,第一次减数分裂前期的胎鼠雌性生殖细胞的H3K27me3表达受到了显著影响,导致H3K27me3强阳性细胞比例显著减少.该研究结果说明DEHP可通过孕鼠影响胎鼠卵母细胞早期发育过程中的组蛋白甲基化修饰.