Pregnancy: a cloned horse born to its dam twin

Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.     

Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor.

A critical point during mammalian pregnancy is the implantation of the blastocyst when the embryo attaches to the wall of the uterus. The autonomously developing preimplantation embryo then becomes dependent on the maternal environment for its continued development. Little is known about the regulation of implantation, except that a complex interaction between peptide and steroid hormones synchronizes the preparation of the uterus for implantation with the development of the embryo. Whether the implantation event is under maternal or embryonic control is also unclear (reviewed in refs 1, 2). We have previously shown that a cytokine, leukaemia inhibitory factor (LIF), is expressed in the uterine endometrial glands specifically on the fourth day of pregnancy. This burst of expression is under maternal control and always precedes implantation of the blastocyst. Here we report that transient expression of LIF in mice is essential for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and do not develop. The blastocysts, however, are viable and, when transferred to wild-type pseudopregnant recipients, they can implant and develop to term.     

A transient placental source of serotonin for the fetal forebrain

Serotonin (5-hydroxytryptamine or 5-HT) is thought to regulate neurodevelopmental processes through maternal-fetal interactions that have long-term mental health implications. It is thought that beyond fetal 5-HT neurons there are significant maternal contributions to fetal 5-HT during pregnancy but this has not been tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used Pet1(-/-) (also called Fev) mice in which most dorsal raphe neurons lack 5-HT. We detected previously unknown differences in accumulation of 5-HT between the forebrain and hindbrain during early and late fetal stages, through an exogenous source of 5-HT which is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source. We uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor in both mice and humans. This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and indicates that maternal-placental-fetal interactions could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.     

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.     

Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy

In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear. Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure. Unexpectedly, corin expression was detected in the pregnant uterus. Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal-fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.     

A new approach to protein fold recognition.

The prediction of protein tertiary structure from sequence using molecular energy calculations has not yet been successful; an alternative strategy of recognizing known motifs or folds in sequences looks more promising. We present here a new approach to fold recognition, whereby sequences are fitted directly onto the backbone coordinates of known protein structures. Our method for protein fold recognition involves automatic modelling of protein structures using a given sequence, and is based on the frameworks of known protein folds. The plausibility of each model, and hence the degree of compatibility between the sequence and the proposed structure, is evaluated by means of a set of empirical potentials derived from proteins of known structure. The novel aspect of our approach is that the matching of sequences to backbone coordinates is performed in full three-dimensional space, incorporating specific pair interactions explicitly.     

Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes

A prominent hypothesis in embryology is that localized maternal factors are important in specifying cell fate. There are, however, only a few examples of maternal molecules that have been shown to be localized and very little is known about how such factors are physically localized within an egg (for review see ref. 1). Previously, cDNA clones were obtained for a class of localized maternal mRNAs from Xenopus laevis. These mRNAs are unusual in that they are concentrated at either the animal or vegetal pole of unfertilized eggs. In the present study the synthesis and intracellular distribution of one of them, Vg1, has been examined during oogenesis. The results show that Vg1 mRNA is localized as a crescent at the vegetal pole of mature oocytes. Surprisingly, this mRNA is uniformly distributed in the cytoplasm of immature oocytes. These findings suggest that a single cell, the frog oocyte, has some mechanism for translocating specific RNAs like Vg1. The process that moves Vg1 mRNA is evidently a cytoplasmic localization machinery which is not directly coupled to the synthesis of Vg1 RNA.     

Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia

Pre-eclampsia is a principal cause of maternal morbidity and mortality, affecting 5-10% of first pregnancies worldwide. Manifestations include increased blood pressure, proteinuria, coagulopathy and peripheral and cerebral oedema. Although the aetiology and pathogenesis remain to be elucidated, the placenta is undoubtedly involved, as termination of pregnancy eradicates the disease. Here we have cloned a complementary DNA from human placental messenger RNA encoding a precursor protein of 121 amino acids which gives rise to a mature peptide identical to the neuropeptide neurokinin B (NKB) of other mammalian species. In female rats, concentrations of NKB several-fold above that of an animal 20 days into pregnancy caused substantial pressor activity. In human pregnancy, the expression of NKB was confined to the outer syncytiotrophoblast of the placenta, significant concentrations of NKB could be detected in plasma as early as week 9, and plasma concentrations of NKB were grossly elevated in pregnancy-induced hypertension and pre-eclampsia. We conclude that elevated levels of NKB in early pregnancy may be an indicator of hypertension and pre-eclampsia, and that treatment with certain neurokinin receptor antagonists may be useful in alleviating the symptoms.     

A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.     

The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours

In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.     

The structural basis for activation of plant immunity by bacterial effector protein AvrPto

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.     

Interaction of peptide antigens and class II major histocompatibility complex antigens

T lymphocytes require a foreign antigen to be presented on a cell surface in association with a self-transplantation antigen before they can recognize it effectively. This phenomenon is known as major histocompatibility complex (MHC) restriction. It is not clear how an incalculably large number of foreign proteins form unique complexes with a very limited number of MHC molecules. We studied the recognition properties of T cells specific for a peptide derived from bacteriophage lambda cI protein. Analogues of this peptide, as well as peptides derived from other unrelated antigens which can be presented in the context of the same MHC molecule, can competitively inhibit activation of these T cells by the cI peptide. Furthermore, these unrelated antigens can stimulate cI-specific T cells if certain specific amino-acid residues are replaced. Here we suggest a model in which all antigens give rise to peptides that can bind to the same site on the MHC molecule. T-cell recognition of this site (which is presumed to be polymorphic) with or without antigen bound can explain self-selection in the thymus and MHC restriction.     

BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.     

Stimulation of connective tissue cell growth by substance P and substance K

Connective tissue cells proliferate actively when cultured in the presence of serum. Platelet-derived growth factor (PDGF), a basic protein of relative molecular mass approximately 30,000, has been identified as the major serum mitogen for these cells; its main physiological/pathophysiological role may be to initiate wound healing in connection with tissue injury. However, growth of cultured cells is also influenced by several other factors, including epidermal growth factor, fibroblast growth factor, insulin and somatomedins. Furthermore, Rozengurt and Sinnett-Smith recently showed that bombesin, a neuroendocrine peptide isolated from frog skin, stimulates DNA synthesis and cell division in cultures of a specific subtype of 3T3 cells. Substance P and substance K (also known as neurokinin A or neuromedin L) are mammalian peptides belonging to the tachykinin family. Substance P has been studied extensively; it is distributed widely throughout the central and peripheral nervous system, including primary sensory neurones, and can be released in the periphery from axon collaterals of stimulated pain fibres and contribute to the inflammatory response. Substance K is a member of the tachykinin family isolated from mammalian spinal cord; Nawa et al. determined the primary structure of two types of substance P precursors, one of which contained a sequence homologous to substance K, as well as the sequence of substance P. We report here that substance P and substance K stimulate DNA synthesis in cultured arterial smooth muscle cells and human skin fibroblasts, and that this stimulation is inhibited by the substance P-antagonist spantide.     

CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.     

实质重于形式原则对所得税的影响

《企业会计制度》中新增加的实质重于形式原则，要求企业应当按照交易或事项的经济实质进行会计核算，而不应当仅仅按照它们的法律形式作为会计核算的依据。实质重于形式原则的运用使得会计制度与税法在融资租入固定资产的确认与计价、借款费用资本化的确认、收入的确认和资产减值准备的计提等方面的处理存在差异，当企业向税务机关缴纳所得税时，必须将企业的会计所得调整为纳税所得。     

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75-110 of influenza virus haemagglutinin) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding.     

基于T检验与支持向量机的蛋白质质谱数据分析

对蛋白质质谱数据进行模式识别成为癌症诊断的一种新方法,但质谱数据存在高维小样本问题,因而数据分析面临着巨大挑战.在对原始数据进行基线校正与标准化并用分箱法进行降维预处理的基础上,提出用T检验方法选取特征,对蛋白质质谱数据进行分析研究.实验对卵巢质谱数据集进行分类,用10-fold交叉验证法选择训练和测试样本,以支持向量...