Analysis of the spatial expression pattern of seven Kip related proteins (KRPs) in the shoot apex of Arabidopsis thaliana

BACKGROUND AND AIMS: Kip-related-proteins (KRPs), negative regulators of cell division, have recently been discovered in plants but their in planta function is as yet unclear. In this study the spatial expression of all seven KRP genes in shoot apices of Arabidopsis thaliana were compared. METHODS: In situ hybridization analyses were performed on longitudinal sections of shoot apices from 2-month-old Arabidopsis plants. KEY RESULTS: The study provides evidence for different expression pattern groups. KRP1 and KRP2 expression is restricted to the endoreduplicating tissues. In contrast, KRP4 and KRP5 expression is mainly restricted to mitotically dividing cells. KRP3, KRP6 and KRP7 can be found in both mitotically dividing and endoreduplicating cells. CONCLUSION: The results suggest differential roles for the distinct KRPs. KRP1 and KRP2 might specifically be involved in the establishment of polyploidy. In contrast, KRP4 and KRP5 might be involved in regulating the progression through the mitotic cell cycle. KRP3, KRP6 and KRP7 might have a function in both types of cell cycle.     

Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation

Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.     

Tyrosine and serine phosphorylation regulate the conformation and subsequent threonine phosphorylation of the L1 cytoplasmic domain

Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both CKII- and PKC-blockade suppressed this modification, only CKII was capable of phosphorylating T1172 of a recombinant L1 cytoplasmic domain, suggesting the requirement for additional events to facilitate availability of T1172 to PKC. In this study, we demonstrate that the region around T1172 exists in distinct conformations based on both T1172 phosphorylation and the integrity of surrounding residues. We further demonstrate the role of membrane-proximal and membrane-distal residues in regulating cytoplasmic domain conformation, and that modification of 3 of the 4 tyrosines in the L1 cytoplasmic domain promote conformational changes that facilitate other events. In particular, phenylalanine-substitution of tyrosine-1151 or tyrosine-1229 promote opening up of the cytoplasmic domain in a manner that facilitates phosphorylation of the other 3 tyrosines, as well as phosphorylation of T1172 by PKCα. Importantly, we show that phosphorylation of serine-1181 is required for T1172 phosphorylation by CKII. These data define a specific role for secondary structure in regulating the availability of T1172 that facilitates phosphorylation by PKC.     

编码流感病毒血凝素基因和CD40L的核酸疫苗抗小鼠流感研究

为了检测表达CD40L的质粒能台作为核酸疫苗佐剂提高A／PR8／34流感病毒血凝素(HA)DNA疫苗的免疫应答,构建了表达鼠CD40L的质粒,行将它与A型流感病毒的HADNA疫苗用电击的方法共同免疫BALB／C小鼠(各30μg),免疫两次,间隔三周,第二次免疫后1周,用致死量流感病毒A／PR8／34攻击。发现与单独免疫30μgHA相比,血清中抗HA的IgG抗体节明显提高,且以IgG2a抗体提高为主,小鼠体重减轻非常少且体重恢复加快。实验结果显示,CD40L能作为流感病毒核酸疫苗佐剂,提高小鼠抗流感病毒攻击的能力。     

Identification of a novel gene family,paralogs of inhibitor of apoptosis proteins present in plants,fungi, and animals

Only few orthologs of animal apoptosis regulators have been found in plants. Recently, the ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has been shown to affect plant programmed cell death. Here, we identified two novel proteins homologous to Arabidopsis thaliana IAP-like protein (AtILP) 1 and 2 by applying an improved motif searching method. Furthermore, homologs of AtILP1 were found to occur as a novel gene family in other organisms such as fungi and animals including Homo sapiens (HsILP1). Like baculovirus IAP repeats (BIRs) in IAPs, ILPs contain two highly conserved BIR-like domains (BLDs) with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Homology modeling revealed that the three-dimensional structure of BLD in HsILP1 is similar to that of BIR. Transient expression of HsILP1 resulted in inhibition of etoposide-induced apoptosis in HEK293 and HeLaS3 cells. These findings suggest that ILPs are conserved in a wide range of eukaryotes including plants, and that their functions are closely related to those of IAPs.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.