The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.

• MeSH
• Citrobacter rodentium immunology   MeSH
• Enterobacteriaceae Infections genetics   immunology   MeSH
• Homeostasis drug effects   genetics   immunology   MeSH
• Lymphocytes drug effects   immunology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Immunity, Innate * drug effects   genetics   MeSH
• Gene Expression Regulation drug effects   MeSH
• Signal Transduction drug effects   genetics   MeSH
• Intestines drug effects   immunology   microbiology   MeSH
• Kruppel-Like Transcription Factors genetics   physiology   MeSH
• Tretinoin metabolism   pharmacology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T-cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T-cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.

• MeSH
• Homeostasis MeSH
• Immunity MeSH
• Interleukin-17 metabolism   MeSH
• Cells, Cultured MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Gene Expression Regulation MeSH
• Repressor Proteins metabolism   MeSH
• Mucous Membrane physiology   MeSH
• Intestines physiology   MeSH
• T-Lymphocytes physiology   MeSH
• Kruppel-Like Transcription Factors genetics   metabolism   MeSH
• Tretinoin metabolism   MeSH
• Inflammation immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Antioxidants metabolism   pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cell Respiration drug effects   MeSH
• Cell Line MeSH
• Energy Metabolism drug effects   MeSH
• Glutathione metabolism   MeSH
• Homeostasis MeSH
• Kinetics MeSH
• Humans MeSH
• Mitochondrial Membranes drug effects   metabolism   MeSH
• Molecular Structure MeSH
• Molecular Weight MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress drug effects   MeSH
• Polyethyleneimine chemistry   toxicity   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Oxygen Consumption drug effects   MeSH
• Transfection methods   MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.

• MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• Cytokinins biosynthesis   metabolism   MeSH
• Endoreduplication radiation effects   MeSH
• Homeostasis radiation effects   MeSH
• Hypocotyl cytology   growth & development   metabolism   radiation effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Germination drug effects   radiation effects   MeSH
• Abscisic Acid metabolism   MeSH
• Solanum lycopersicum cytology   growth & development   metabolism   radiation effects   MeSH
• Darkness * MeSH
• Plant Development drug effects   radiation effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We studied hsBAFF activity in in vitro mouse splenic B cells. hsBAFF effects on intracellular free Ca2+ concentration ([Ca2+]i) were assayed, using a laser scanning confocal microscope with fluorescent probe, Fluo-3/AM. We showed that treatment of B cells with 0.5-5 µg/ml hsBAFF resulted in significantly higher [Ca2+]i levels in a dose-dependent fashion at 12 and 24 h, respectively (p<0.05 or p<0.01 vs. control). Furthermore, we noticed that 2.5 µg/ml hsBAFF-treated cells were significantly resistant to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca2+-ATPase inhibitor (p<0.05 hsBAFF plus Tg group vs. Tg group). Thus hsBAFF may promote B cell survival by direct upregulation of [Ca2+]i physiological homeostasis contributing to prevention of [Ca2+]i dysfunction. Using immunocytochemistry and Western blot analysis, we found that the activation of ERK1/2 due to hsBAFF was triggered by a [Ca2+]i-dependent pathway, leading to elevation of B cell proliferation. This is supported by the findings that intracellular Ca2+ chelator BAPTA/AM attenuated phosphorylated ERK1/2 expression and cell proliferation in hsBAFF-stimulated B cells. hsBAFF-stimulated B cell proliferation was obviously reduced by mitogen extracellular kinase 1/2 (MEK1/2, upstream of ERK1/2) inhibitor U0126. Taken together, the main finding of this study is that hsBAFF elicits higher but homeostatic [Ca2+]i levels, which regulates ERK1/2 activity and cell proliferation in in vitro B cells

• MeSH
• B-Lymphocytes enzymology   MeSH
• Butadienes pharmacology   MeSH
• Time Factors MeSH
• Chelating Agents pharmacology   MeSH
• Egtazic Acid analogs & derivatives   pharmacology   MeSH
• B-Cell Activating Factor metabolism   MeSH
• Financing, Organized MeSH
• Phosphorylation MeSH
• Homeostasis MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinase 1 metabolism   MeSH
• Mitogen-Activated Protein Kinase 3 metabolism   MeSH
• Mice, Inbred ICR MeSH
• Mice MeSH
• Nitriles pharmacology   MeSH
• Cell Proliferation MeSH
• Sarcoplasmic Reticulum Calcium-Transporting ATPases antagonists & inhibitors   metabolism   MeSH
• Thapsigargin pharmacology   MeSH
• Calcium metabolism   MeSH
• Cell Survival MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

• MeSH
• beta-Glucosidase physiology   metabolism   MeSH
• Cytokinins metabolism   MeSH
• Financing, Organized MeSH
• Plants, Genetically Modified anatomy & histology   enzymology   drug effects   MeSH
• Homeostasis MeSH
• Kinetics MeSH
• Zea mays enzymology   genetics   MeSH
• Culture Media MeSH
• Abscisic Acid metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Nicotiana genetics   MeSH
• Zeatin pharmacology   metabolism   MeSH