The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins--a family of four related small guanosine triphosphatases (GTPases)--interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.     

Organization of ion channels in the myelinated nerve fiber

The functional organization of the mammalian myelinated nerve fiber is complex and elegant. In contrast to nonmyelinated axons, whose membranes have a relatively uniform structure, the mammalian myelinated axon exhibits a high degree of regional specialization that extends to the location of voltage-dependent ion channels within the axon membrane. Sodium and potassium channels are segregated into complementary membrane domains, with a distribution reflecting that of the overlying Schwann or glial cells. This complexity of organization has important implications for physiology and pathophysiology, particularly with respect to the development of myelinated fibers.     

mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H(+)-adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid-sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing.     

Perineurium originates from fibroblasts: demonstration in vitro with a retroviral marker

A cellular sheath, the perineurium, forms a protective barrier around fascicles of nerve fibers throughout the peripheral nervous system. In a study to determine the cellular origin of perineurium, a culture system was used in which perineurium forms after purified populations of sensory neurons, Schwann cells, and fibroblasts are recombined. Before recombination, the Schwann cells or the fibroblasts were labeled by infection with a defective recombinant retrovirus whose gene product, beta-galactosidase, is histochemically detectable in the progeny of infected cells. Perineurial cells were labeled when fibroblasts had been infected but not when Schwann cells had been infected. Thus, perineurium arises from fibroblasts in vitro and, by implication, in vivo as well.     

A Mycobacterium leprae-specific human T cell epitope cross-reactive with an HLA-DR2 peptide

Mycobacterium leprae induces T cell reactivity and protective immunity in the majority of exposed individuals, but the minority that develop leprosy exhibit various types of immunopathology. Thus, the definition of epitopes on M. leprae antigens that are recognized by T cells from different individuals might result in the development of an effective vaccine against leprosy. A sequence from the 65-kD protein of this organism was recognized by two HLA-DR2-restricted, M. leprae-specific helper T cell clones that were derived from a tuberculoid leprosy patient. Synthetic peptides were used to define this epitope as Leu-Gln-Ala-Ala-Pro-Ala-Leu-Asp-Lys-Leu. A similar peptide that was derived from the third hypervariable region of the HLA-DR2 chain, Glu-Gln-Ala-Arg-Ala-Ala-Val-Asp-Thr-Tyr, also activated the same clones. The unexpected cross-reactivity of this M. leprae-specific DR2-restricted T cell epitope with a DR2 peptide may have to be considered in the design of subunit vaccines against leprosy.     

Experimental leprosy in three species of monkeys

Eleven mangabey monkeys inoculated with Mycobacterium leprae developed lepromatous-type leprosy. Nine of the mangabeys were inoculated with M. leprae isolated from a mangabey with naturally acquired lepromatous leprosy. Immune function was depressed in some of these animals after dissemination of the disease. Two mangabeys developed lepromatous leprosy after inoculation with human M. leprae passaged in an armadillo. Three rhesus and three African green monkeys inoculated with mangabey-derived M. leprae also developed lepromatous leprosy. Mangabeys may be the first reported nonhuman primate model for the study of leprosy. Rhesus and African green monkeys may also prove to be reproducibly susceptible to the disease.     

Control of peripheral nerve myelination by the beta-secretase BACE1

Although BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is essential for the generation of amyloid-b peptide in Alzheimer's disease, its physiological function is unclear. We found that very high levels of BACE1 were expressed at time points when peripheral nerves become myelinated. Deficiency of BACE1 resulted in the accumulation of unprocessed neuregulin 1 (NRG1), an axonally expressed factor required for glial cell development and myelination. BACE1-/- mice displayed hypomyelination of peripheral nerves and aberrant axonal segregation of small-diameter afferent fibers, very similar to that seen in mice with mutations in type III NRG1 or Schwann cell-specific ErbB2 knockouts. Thus, BACE1 is required for myelination and correct bundling of axons by Schwann cells, probably through processing of type III NRG1.     

Axonal neuregulin-1 regulates myelin sheath thickness

In the nervous system of vertebrates, myelination is essential for rapid and accurate impulse conduction. Myelin thickness depends on axon fiber size. We use mutant and transgenic mouse lines to show that axonal Neuregulin-1 (Nrg1) signals information about axon size to Schwann cells. Reduced Nrg1 expression causes hypomyelination and reduced nerve conduction velocity. Neuronal overexpression of Nrg1 induces hypermyelination and demonstrates that Nrg1 type III is the responsible isoform. We suggest a model by which myelin-forming Schwann cells integrate axonal Nrg1 signals as a biochemical measure of axon size.     

Strophanthidin-sensitive transport of cesium and sodium in muscle cells

Uptake of cesium-134 ions into muscle cells is reduced to very low values by the presence of 10(-5)M strophanthidin in the Ringer solution. Cesium ions can induce extrusion of sodium from muscle cells in which the intracellular sodium content is elevated. The cesium-induced extra efflux of sodium-22 is inhibited by the external presence of 10(-5)M strophanthidin. The coupling between inward movement of cesium and outward movement of sodium appears to be chemical in nature. The evidence suggests that cesium ions are transported into muscle cells by a system of sites or carriers that requires a source of metabolic energy for ion turnover to occur.     

雷公藤内酯醇对AD细胞模型炎性因子的影响

[目的]以Aβ1-42寡聚体为工具药建立AD细胞模型,观察雷公藤内酯醇（T10）对AD的防治作用并初步探讨其作用机制。[方法]用不同浓度的T10（10-11、10-10、10-9、10-8mol/L）预孵育小胶质细胞12h,然后加入10μmol/LAβ1-42共孵育12h,ELISA法检测上清TNF-α、IL-1β的含量,收获条件培养基,加入海马神经元中,作用24h,MTT法检测神经元存活率。[结果]在体外T10可减少Aβ1-42诱导的小胶质细胞TNF-α、IL-1β的释放,提高海马神经元的存活率。[结论]T10可通过抑制小胶质细胞激活保护海马神经元。     

Development of a human adaptive immune system in cord blood cell-transplanted mice

Because ethical restrictions limit in vivo studies of the human hemato-lymphoid system, substitute human to small animal xenotransplantation models have been employed. Existing models, however, sustain only limited development and maintenance of human lymphoid cells and rarely produce immune responses. Here we show that intrahepatic injection of CD34+ human cord blood cells into conditioned newborn Rag2-/-gammac-/- mice leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of functional immune responses. This provides a valuable model to study development and function of the human adaptive immune system in vivo.     

Neurofibromas in NF1: Schwann cell origin and role of tumor environment

Neurofibromatosis type 1 (NF1) is one of the most prevalent dominantly inherited genetic diseases of the nervous system. NF1 encodes a tumor suppressor whose functional loss results in the development of benign neurofibromas that can progress to malignancy. Neurofibromas are complex tumors composed of axonal processes, Schwann cells, fibroblasts, perineurial cells, and mast cells. Through use of a conditional (cre/lox) allele, we show that loss of NF1 in the Schwann cell lineage is sufficient to generate tumors. In addition, complete NF1-mediated tumorigenicity requires both a loss of NF1 in cells destined to become neoplastic as well as heterozygosity in non-neoplastic cells. The requirement for a permissive haploinsufficient environment to allow tumorigenesis may have therapeutic implications for NF1 and other familial cancers.     

维拉帕米和三苯氧胺逆转卵巢癌细胞对阿霉素耐药性的实验研究

目的 了解维拉帕米和三苯氧胺体外逆转卵巢癌细胞阿霉素耐药性的效果及临床意义.方法 分别以阿霉素、维拉帕米和三苯氧胺作用于人卵巢癌亲本细胞SKOV3和阿霉素耐药细胞SKOV3/ADM后,采用MTI法检测药物对癌细胞的抑制作用,以细胞内阿霉素聚集量测定和流式细胞术检测凋亡.结果 维拉帕米和三苯氧胺能部分逆转耐药细胞对阿霉素的耐药性,且维拉帕米能显著增加耐药细胞内阿霉素的聚集量并促进其凋亡.结论 虽然维拉帕米和三苯氧胺能部分逆转耐药细胞对阿霉素的耐药性,但用于临床的意义不大.     

牛胎肺间充质干细胞体外培养及生物学特性研究

[目的]建立牛胎肺间充质干细胞（Mesenchymal stem cells,MSCs）体外培养体系并对其进行生物学特性研究。[方法]取3～5月龄牛胎儿肺脏,得到MSCs。通过控制胰酶消化时间,去除难消化的上皮样贴壁细胞,获得纯化的牛肺MSCs,研究其形态、增殖情况及体外诱导分化潜能。[结果]牛胎肺MSCs可在体外扩增培养,传至24代以上,表达CD29、CIM4和CDl66,不表达CDM、CIM5和BOL-DR,并具有向成骨细胞诱导分化的潜能。[结论]从牛胎肺中可以分离出MSCs,可以在体外培养,并可诱导分化为成骨细胞。     

Response of Schwann cells to action potentials in development

Sensory axons become functional late in development when Schwann cells (SC) stop proliferating and differentiate into distinct phenotypes. We report that impulse activity in premyelinated axons can inhibit proliferation and differentiation of SCs. This neuron-glial signaling is mediated by adenosine triphosphate acting through P2 receptors on SCs and intracellular signaling pathways involving Ca2+, Ca2+/calmodulin kinase, mitogen-activated protein kinase, cyclic adenosine 3',5'-monophosphate response element binding protein, and expression of c-fos and Krox-24. Adenosine triphosphate arrests maturation of SCs in an immature morphological stage and prevents expression of O4, myelin basic protein, and the formation of myelin. Through this mechanism, functional activity in the developing nervous system could delay terminal differentiation of SCs until exposure to appropriate axon-derived signals.     

The polarity protein Par-3 directly interacts with p75NTR to regulate myelination

Cell polarity is critical in various cellular processes ranging from cell migration to asymmetric cell division and axon and dendrite specification. Similarly, myelination by Schwann cells is polarized, but the mechanisms involved remain unclear. Here, we show that the polarity protein Par-3 localizes asymmetrically in Schwann cells at the axon-glial junction and that disruption of Par-3 localization, by overexpression and knockdown, inhibits myelination. Additionally, we show that Par-3 directly associates and recruits the p75 neurotrophin receptor to the axon-glial junction, forming a complex necessary for myelination. Together, these results point to a critical role in the establishment of cell polarity for myelination.     

颗粒细胞或卵丘细胞提高小鼠未成熟卵母细胞恢复减数分裂能力的研究

本研究通过12～13d小鼠颗粒细胞或21d小鼠卵丘细胞与14d小鼠直径55～65μm的卵母细胞共培养7d,发现单独培养、与颗粒细胞共培养、与卵丘细胞共培养的直径分别为54.2±1.6μm、65.2±2.3μm和63.4±3.4μm。而且,共培养提高了卵母细胞GSH的表达量(p<0.05)。在与颗粒细胞共培养7d后,11.1%(27/254)的卵母细胞能完成第一次减数分裂达到MII期。说明了小鼠颗粒细胞或卵丘细胞能够促进未成熟卵母细胞的发育及减数分裂的恢复,为研究哺育动物卵母细胞的发育调控提供了依据。     

Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.