Heterocytolysis by macrophages activated by bacillus Calmette-Guérin: lysosome exocytosis into tumor cells

The cytotoxic activity of activated macrophages against tumorigenic target cells appears to be mediated by lysosomal enzymes of activated macrophage origin. Lysosomes of activated macrophages are secreted directly into the cytoplasm of susceptible target cells, which subsequently undergo heterolysis. This reaction can be inhibited by agents which prevent the exocytosis of macrophage lysosomes (hydrocortisone) or which interfere with the action of lysosomal enzymes (trypan blue).     

Protein kinase G from pathogenic mycobacteria promotes survival within macrophages

Pathogenic mycobacteria resist lysosomal delivery after uptake into macrophages, allowing them to survive intracellularly. We found that the eukaryotic-like serine/threonine protein kinase G from pathogenic mycobacteria was secreted within macrophage phagosomes, inhibiting phagosome-lysosome fusion and mediating intracellular survival of mycobacteria. Inactivation of protein kinase G by gene disruption or chemical inhibition resulted in lysosomal localization and mycobacterial cell death in infected macrophages. Besides identifying a target for the control of mycobacterial infections, these findings suggest that pathogenic mycobacteria have evolved eukaryotic-like signal transduction mechanisms capable of modulating host cell trafficking pathways.     

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation

Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.     

LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis

Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here, we identified the Caenorhabditis elegans lysosomal lysine/arginine transporter LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlargement by cysteamine, a drug that alleviates cystinosis by converting cystine to a lysine analog. LAAT-1 also maintained availability of cytosolic lysine/arginine during embryogenesis. Thus, LAAT-1 is the lysosomal lysine/arginine transporter, which suggests a molecular explanation for how cysteamine alleviates a lysosomal storage disease.     

A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism

Mucolipidosis II is a severe lysosomal storage disorder caused by defects in the α and β subunits of the hexameric N-acetylglucosamine-1-phosphotransferase complex essential for the formation of the mannose 6-phosphate targeting signal on lysosomal enzymes. Cleavage of the membrane-bound α/β-subunit precursor by an unknown protease is required for catalytic activity. Here we found that the α/β-subunit precursor is cleaved by the site-1 protease (S1P) that activates sterol regulatory element-binding proteins in response to cholesterol deprivation. S1P-deficient cells failed to activate the α/β-subunit precursor and exhibited a mucolipidosis II-like phenotype. Thus, S1P functions in the biogenesis of lysosomes, and lipid-independent phenotypes of S1P deficiency may be caused by lysosomal dysfunction.     

Transmembrane molecular pump activity of Niemann-Pick C1 protein

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.     

Contact-mediated bone resorption by human monocytes in vitro

Human circulating monocytes in tissue culture are capable of resorbing devitalized adult and fetal bone. An important component of this process is the adhesion of the cells to the mineralized substrate and the localized removal of matrix from beneath the attached cells. The process appears to involve both release of lysosomal enzymes onto the substrate and intracellular accumulation (transport) of resorbed matrix.     

Identification of HE1 as the second gene of Niemann-Pick C disease

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.     

A clonogenic bone marrow progenitor specific for macrophages and dendritic cells

Macrophages and dendritic cells (DCs) are crucial for immune and inflammatory responses and belong to a network of cells that has been termed the mononuclear phagocyte system (MPS). However, the origin and lineage of these cells remain poorly understood. Here, we describe the isolation and clonal analysis of a mouse bone marrow progenitor that is specific for monocytes, several macrophage subsets, and resident spleen DCs in vivo. It was also possible to recapitulate this differentiation in vitro by using treatment with the cytokines macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Thus, macrophages and DCs appear to renew from a common progenitor, providing a cellular and molecular basis for the concept of the MPS.     

(S)-9-(2,3-Dihydroxypropyl)adenine: An Aliphatic Nucleoside Analog with Broad-Spectrum Antiviral Activity

(S)-9-(2,3-Dihydroxypropyl)adenine, a novel nucleoside analog, the sugar moiety of which is replaced by an aliphatic chain, inhibits the replication in vitro of several DNA and RNA viruses, including vaccinia, herpes simplex (types 1 and 2), measles, and vesicular stomatitis. It is also effective in vivo in reducing the mortality rate of mice inoculated intranasally with vesicular stomatitis virus.     

Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate

Antigen-presenting cells (APCs) internalize antigens and present antigen-derived peptides to T cells. Although APCs have been thought to exhibit a well-developed capacity for lysosomal proteolysis, here we found that they can exhibit two distinct strategies upon antigen encounter. Whereas macrophages contained high levels of lysosomal proteases and rapidly degraded internalized proteins, dendritic cells (DCs) and B lymphocytes were protease-poor, resulting in a limited capacity for lysosomal degradation. Consistent with these findings, DCs in vivo degraded internalized antigens slowly and thus retained antigen in lymphoid organs for extended periods. Limited lysosomal proteolysis also favored antigen presentation. These results help explain why DCs are able to efficiently accumulate, process, and disseminate antigens and microbes systemically for purposes of tolerance and immunity.     

Lysosomal glycosphingolipid recognition by NKT cells

NKT cells represent a distinct lineage of T cells that coexpress a conserved alphabeta T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking beta-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.     

仙人掌多糖对THP–1巨噬细胞源性泡沫细胞胆固醇蓄积及其相关基因表达的影响

采用PMA和ox–LDL构建THP–1巨噬细胞源性泡沫细胞模型,观察不同浓度(5、10、15 nmol/L)仙人掌多糖(ODP–Ia)对泡沫细胞内脂质、胆固醇蓄积以及介导胆固醇流出的ABCA1、ABCG1、SR–BI基因表达的影响。结果显示:THP–1巨噬细胞泡沫化后胞内蓄积了大量脂质,高剂量(15 nmol/L)ODP–Ia能够显著减少胞内脂质、胆固醇蓄积,并增强ABCA1、ABCG1、SR–BI m RNA及蛋白表达(与单独ox–LDL处理组比较,P0.05),但效果均次于阳性对照依折麦布组(P0.05),低、中剂量(5、10 nmol/L)ODP–Ia作用效果均不显著。本研究结果表明,ODP–Ia可通过增强ABCA1、ABCG1、SR–BI基因表达来减少胞内胆固醇蓄积。     

RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.     

Inhibition of purine nucleoside phosphorylase by 8-aminoguanosine: selective toxicity for T lymphoblasts

The guanosine analog 8-aminoguanosine is an effective inhibitor of the purine degradative enzyme purine nucleoside phosphorylase, both in vitro and in intact lymphoid cells. In a human lymphoblast tissue culture system, 8-aminoguanosine, in combination with low concentrations of 2'-deoxyguanosine, causes toxicity toward T cells but not B cells. The selective T cell toxicity correlates with increased accumulation of deoxyguanosine triphosphate in the treated T lymphoblasts.     

Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes

The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of 22Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.     

A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development

Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their target messenger RNAs. Here, I show that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene, APETALA2, regulates APETALA2 expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of APETALA2 in Arabidopsis flower development.     

城市土地混合利用测度研究——以深圳市为例

采用了熵指数和相异指数两种土地混合利用测度,以深圳市第二次土地调查的数据为处理对象,研究深圳市土地混合利用现状。结果表明,深圳市土地类型中林地和工矿仓储用地的熵指数最高,特殊用地最低;住宅用地和非住宅用地之间的占地面积和分布程度的差异都很大。由此可知,深圳市的城区土地是以工业用地为主,工业用地面积远大于其他用地,进而推得深圳市的经济发展也以第二产业为主。     

Bond order and charge localization in nucleoside phosphorothioates

In the recent literature on nucleoside phosphorothioate anions the structural formulas show a double bond between phosphorus and sulfur and a single bond between phosphorus and oxygen with a negative charge localized on oxygen. However, a review of physical data on these compounds shows the reverse to be the case; that is, in phosphorothioate anions the P-S bond is a single bond with a negative charge localized on sulfur, while the P-O bond order for exocyclic and nonbridging oxygens is greater than 1, approaching 2 in O-alkyl phosphorothioate monoanions and O,O-dialkyl phosphorothioates. The P-O bond orders in phosphorothioate dianions and trianions approach 1 1/2 and 1 1/3, respectively, owing to delocalization of negative charge among two or three oxygens. These conclusions are based on bond lengths obtained from x-ray crystallographic data and electron diffraction, the magnitudes of the effects of 18O on the 31P-nuclear magnetic resonance chemical shifts of phosphorus in nucleoside [18O]phosphorothioates, the pH-dependence of 17O-NMR chemical shifts in [17O]phosphate and [17O]thiophosphate, the vibrational spectra of thiophosphate di- and trianions, and the pKa (dissociation constant) values for phosphoric and thiophosphoric acids.     

Lymphokine production by cultured human T cells transformed by human T-cell leukemia-lymphoma virus-I

Cell-free conditioned media from human T cells transformed by human T-cell leukemia-lymphoma virus (HTLV-I) were tested for the production of soluble biologically active factors, including several known lymphokines. The cell lines used were established from patients with T-cell leukemia-lymphoma and from human umbilical cord blood and bone marrow leukocytes transformed by HTLV-I in vitro. All of the cell lines liberated constitutively one or more of the 12 biological activities assayed. These included macrophage migration inhibitory factor (MIF), leukocyte migration inhibitory factor (LIF), leukocyte migration enhancing factor (MEF), macrophage activating factor (MAF), differentiation inducing factor (DIF), colony stimulating factor (CSF), eosinophil growth and maturation activity (eos. GMA), fibroblast activating factor (FAF), gamma-interferon and, in rare instances, T-cell growth factor (TCGF). Some cell lines produced interleukin 3 (IL-3), platelet-derived growth factor (PDGF), or B-cell growth factors (BCGF). Such cells should prove useful for the production of lymphokines and as sources of specific messenger RNA's for their genetic cloning.