Beginning to understand autophagy, an intracellular self-degradation system in plants

Autophagy is an evolutionarily conserved intracellular process for the vacuolar degradation of cytoplasmic components. There is no doubt that autophagy is very important to plant life, especially because plants are immobile and must survive in environmental extremes. Early studies of autophagy provided our first insights into the structural characteristics of the process in plants, but for a long time the molecular mechanisms and the physiological roles of autophagy were not understood. Genetic analyses of autophagy in the yeast Saccharomyces cerevisiae have greatly expanded our knowledge of the molecular aspects of autophagy in plants as well as in animals. Until recently our knowledge of plant autophagy was in its infancy compared with autophagy research in yeast and animals, but recent efforts by plant researchers have made many advances in our understanding of plant autophagy. Here I will introduce an overview of autophagy in plants, present current findings and discuss the physiological roles of self-degradation.     

Autophagy in development and stress responses of plants

The uptake and degradation of cytoplasmic material by vacuolar autophagy in plants has been studied extensively by electron microscopy and shown to be involved in developmental processes such as vacuole formation, deposition of seed storage proteins and senescence, and in the response of plants to nutrient starvation and to pathogens. The isolation of genes required for autophagy in yeast has allowed the identification of many of the corresponding Arabidopsis genes based on sequence similarity. Knockout mutations in some of these Arabidopsis genes have revealed physiological roles for autophagy in nutrient recycling during nitrogen deficiency and in senescence. Recently, markers for monitoring autophagy in whole plants have been developed, opening the way for future studies to decipher the mechanisms and pathways of autophagy, and the function of these pathways in plant development and stress responses.     

Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes

Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.     

Isolation and Characterization of Helicobacter Species from the Stomach of the House Musk Shrew (Suncus murinus) with Chronic Gastritis

A Gram-negative, motile bacterium with bipolar sheathed flagella (one at each end) was isolated from the stomach of house musk shrews (Suncus murinus) with chronic gastritis. The isolates grew at 37°C under microaerophilic conditions, but not under aerobic conditions; rapidly hydrolyzed urea; were catalase, oxidase, alkaline phosphatase, and arginine aminopeptidase positive; reduced nitrate to nitrite; and were resistant to cephalothin and nalidixic acid, but sensitive to tetracycline, erythromycin, and chloramphenicol. This bacterium was found on gastric epithelial cells by electron microscopy. In addition, a coccoid form of the bacteria was found in vacuoles formed in the epithelial cells of some of the house musk shrews tested. These results, including 16S rRNA gene sequence analysis, strongly suggested that this bacterium should be classified as a novel Helicobacter species. It is proposed that this bacterium should be called “Helicobacter suncus.” Received: 22 December 1997 / Accepted: 26 January 1998     

Polycystin-1L2 is a novel G-protein-binding protein

Mutations in genes encoding polycystin-1 (PC1) and polycystin-2 cause autosomal dominant polycystic kidney disease. The polycystin protein family is composed of Ca2+-permeable pore-forming subunits and receptor-like integral membrane proteins. Here we describe a novel member of the polycystin-1-like subfamily, polycystin-1L2 (PC1L2), encoded by PKD1L2, which has various alternative splicing forms with two translation initiation sites. PC1L2 short form starts in exon 12 of the long form. The longest open reading frame of PKD1L2 short form, determined from human testis cDNA, encodes a 1775-amino-acid protein and 32 exons, whereas the long form is predicted to encode a 2460-residue protein. Both forms have a small receptor for egg jelly domain, a G-protein-coupled receptor proteolytic site, an LH2/PLAT, and 11 putative transmembrane domains, as well as a number of rhodopsin-like G-protein-coupled receptor signatures. RT-PCR analysis shows that the short form, but not the long form, of human PKD1L2 is expressed in the developing and adult heart and kidney. Furthermore, by GST pull-down assay we observed that PC1L2 and polycystin-1L1 are able to bind to specific G-protein subunits. We also show that PC1 C-terminal cytosolic domain binds to Galpha12, Galphas, and Galphai1, while it weakly interacts with Galphai2. Our results indicate that both PC1-like molecules may act as G-protein-coupled receptors.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.