Cultivation of the liver forms of Plasmodium vivax in human hepatocytes

The blood schizogonic cycle of human malaria parasites has thus far been the most exhaustively studied phase of parasite development. However, before entering red blood cells (RBCs), the parasite undergoes its first multiplication not in blood, but in hepatic cells. These hepatic stages were the last to be discovered and only a few studies have been performed in humans and other primates. Despite recent advances, in vivo studies have limitations and other approaches such as cultures of these liver forms may be necessary to investigate their chemosensitivity and their biochemical or immunological properties. Recently, sporozoites of species of rodent malaria have been made to infect cultured cell lines or primary hepatocyte cultures. We report here that the complete cycle of the human malaria parasite Plasmodium vivax can be obtained in primary cultures of human hepatocytes up to release of merozoites able to penetrate RBCs.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry

The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.     

A proteomic view of the Plasmodium falciparum life cycle

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.     

Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum

Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of approximately 155,000 (refs 1-7). RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites. It is thought to be released through the apical pore from the rhoptry at the time of merozoite invasion. Because antibodies directed against this antigen strongly inhibit parasite growth in vitro, RESA may be useful in developing a vaccine against this parasite Here we describe an immunization trial using Aotus monkeys and Escherichia coli-derived fused polypeptides corresponding to various regions of the RESA molecule. Some monkeys in all test groups, but not in the control group, were protected against overwhelming infection. Strikingly, protection correlated with antibody responses to either of two different repetitive sequences in RESA.     

A rhoptry-protein-associated mechanism of clonal phenotypic variation in rodent malaria

The recognition and invasion of host cells are mediated by components of the apical complex of the ookinete, sporozoite and merozoite stages of Plasmodium parasites. The paired rhoptries (organelles involved in host-cell recognition) in the apical complex contain many proteins of as-yet unknown function. In the rodent malaria agent P. yoelii yoelii, a multigene family codes for merozoite rhoptry proteins of relative molecular mass 235,000 (p235 proteins); these proteins are thought to determine the subset of erythrocytes that the parasites invade. Further support for this idea came from the identification of a region in p235 with weak but significant homology to reticulocyte-binding protein-2 of P. vivax and the demonstration that at least one p235 member binds to the erythrocyte surface membrane. Here, using single, micromanipulated P.y.yoelii parasites, we describe a new mechanism of gene expression by which the merozoites originating from a single schizont each express a distinct member of this multigene family. We propose that this new type of clonal phenotypic variation provides the parasite with a survival strategy in the mammalian host; this strategy contributes to the observed chronicity of malarial infections. This phenomenon is genetically and functionally distinct from classical antigenic variation, which is mediated by the var multigene family of P. falciparum.     

Competence for genetic transformation in pneumococcus depends on synthesis of a small set of proteins.

In bacterial genetic transformation the uptake of DNA and its integration into the resident chromosome is dependent on a special cellular state, termed competence. In those species where appearance of competence has been studied, specific (but often poorly defined) growth conditions lead to a simultaneous development of competence in a substantial fraction of the cells in a culture. In Bacillus subtilis, and in Haemophilus species, competence appears in the stationary phase of growth or in certain other growth-limiting conditions. Streptococcus pneumoniae (pneumococcus) is perhaps unusual in that virtually all cells of a culture become competent, for a short period at a specific cell density during logarithmic growth, without perturbing the growth rate. The synchronous appearance of competence in pneumococcal cultures results from an autocatalytic effect of a small protein released by the cells that induces competence. The response to competence factor has been shown to require protein synthesis. We report here additional information on the nature of competence in pneumococcus: pulse-labelling studies show that for the brief period of competence protein synthesis is restricted to a few specific polypeptides.     

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed. We now describe a modified culture system in which the 'early' BFU-E develop into 'late' BFU-E in response to added erythropoietin. Further maturation of these cells into CFU-E and non-nucleated erythrocytes can be achieved by mechanical agitation of the long-term cultures or by transferring the cells into dishes which do not allow cell attachment to occur.     

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

Mechanism of sequential induction of cell-type specific mRNAs in Dictyostelium differentiation

Upon starvation, the cellular slime mould Dictyostelium discoideum initiates a 24-h programme of differentiation. Within 6 h, cells move towards aggregation centres in response to pulsatile synthesis and secretion of cyclic AMP. At about 12 h, aggregates of 10(5) cells are formed, held together by newly made surface adhesion molecules. The cells then differentiate into the two principal types found in the terminal stage of development, spores and stalks. Here we show that the chemotaxis and aggregation stages of this developmental programme can be described as a series of sequential events in which these extracellular signals--starvation, cyclic AMP and cell-cell contact--induce specific, sequential changes in the pattern of gene expression.     

Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones

One model of synaptic transmission suggests that transmitters modify postsynaptic permeability through the intermediary of cyclic AMP. Thus, serotonin (5-hydroxytryptamine) evokes in molluscan neurones a decrease in a voltage-dependent K+ conductance which in turn generates a slow inward current when studied in steady voltage-clamp conditions. The serotonin-induced increase of the plateau phase of the spike of an Aplysia sensory neurone can be mimicked by both intracellularly injected cyclic AMP and extracellularly applied phosphodiesterase inhibitors, suggesting that cyclic AMP mediates the effect. We have tested whether a similar mechanism could account for the serotonin slow inward current in identified snail neurones and have found that the intracellular injection of cyclic AMP, but not of cyclic GMP or 5'-AMP, evokes a slow inward current showing similar voltage dependence, inversion potential and ionic properties to the serotonin slow inward current. Phosphodiesterase inhibitors at low concentrations (1-20 microM) potentiate the serotonin slow inward current and at higher concentrations evoke by themselves an inward current, partially or totally occluding the serotonin and cyclic AMP currents. Finally, we have found that in homogenates of pooled identified snail neurones serotonin stimulates the adenylate cyclase, increasing its activity by 50-100%.     

Tumour promoter uncouples beta-adrenergic receptor from adenyl cyclase in mouse epidermis

Alterations in beta-adrenergic receptor number and function and in the hormonal responsiveness of adenylate cyclase have been observed in transformed cells, and tumours. Phorbol myristate acetate (PMA), a potent tumour promoter in mouse skin, induces a dramatic loss of epidermal responsiveness to catecholamines in vivo, although basal levels of cyclic AMP are not affected. In other work we have shown that PMA treatment does not alter the number or affinity of epidermal beta-receptors, although accumulation of cyclic AMP in response to isoprenaline injection is sharply inhibited. Evidence is presented here that PMA exerts this effect by uncoupling epidermal beta-receptors from adenylate cyclase.     

波浪荷载作用后软黏土静强度弱化及软基加固研究

利用土工静力-动力液压三轴-扭转多功能剪切仪,针对取自于长江口的海洋原状饱和淤泥质软黏土,在不固结不排水条件下,分别进行了动三轴、45°线耦合以及圆耦合等多种复杂循环剪切试验及循环荷载作用后静三轴试验;通过室内试验模拟堆载预压加固软基,并针对加固后土体进行了上述系列试验.结果表明,对应相同循环剪切次数,双向耦合大于单向...     

Reversible cyclic AMP-dependent change in distribution of myosin thick filaments in Dictyostelium

Myosin is thought to act as a major mechanochemical transducer in non-muscle cell motility, but the in situ organization of the molecules has not yet been determined. Here we report the localization of myosin 'rods', analogous to the thick filaments of muscle, by ameliorated immunofluorescence and demonstrate the dynamic translocation of these rods in response to exogenously added cyclic AMP, which is a chemoattractant for Dictyostelium amoebae. On addition of cyclic AMP, we observed instantaneous shedding of the endoplasmic myosin followed by an increase in cortical rods, the original distribution being recovered in a few minutes. We conclude that myosin filaments mediate Dictyostelium cell movement, probably by an assembly/disassembly cycle of the molecules in response to a chemotactic stimulus.     

冷锻模具材料高速基体钢应变控制室温疲劳性能

对冷锻模具材料高速基体钢进行了室温下的单轴静态拉伸试验和应变控制单轴对称拉压低周疲劳试验,获得了静态拉伸力学性能和低周疲劳性能参数.通过对试验数据进行拟合,得到了高速基体钢材料在室温下静态单调拉伸应力-应变关系、循环应力-应变关系以及循环应变-寿命曲线.试验结果表明,该模具材料在本试验控制的循环应变幅范围内发生了循环软化,疲劳断裂之前未观察到明显的应力饱和现象.以控制应变幅为1.5%的试样为例,对材料的低周疲劳行为特性进行了分析.材料的循环应力响应分为明显软化,软化效应减弱和瞬时断裂3个阶段.