[Show abstract][Hide abstract] ABSTRACT: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis.
We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence.
Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose.
Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.
[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum (ER) is a complex and multifunctional organelle. It is the intracellular compartment of protein folding, a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. The ER is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity and responds by activating signal transduction pathways, known as unfolded protein response (UPR). Three major classes of transducer are known, inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), which sense with their endoluminal domain the state of protein folding, although the exact mechanism(s) involved is not entirely clear. Depending on whether the homeostatic response of the UPR is successful in restoring an equilibrium between ER load and protein folding or not, the two possible outcomes of the UPR so far considered have been life or death. Indeed, recent efforts have been devoted to understand the life/death switch mechanisms. However, recent data suggest that what appears to be a pure binary decision may in fact be more complex, and survival may be achieved at the expenses of luxury cell functions, such as expression of differentiation genes.
Histology and histopathology 01/2012; 27(1):1-12. · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plants respond to environmental stimuli, such as heat shock, by re-programming cellular activity through differential gene expression, mainly controlled at the transcription level. The current study refers to two sunflower small heat shock protein (sHSP) genes arranged in tandem in head-to-head orientation and linked by a 3809 bp region. These genes exhibit only slight structural differences in the coding portion. They code for cytosolic class I sHSPs and are named HaHSP17.6a and HaHSP17.6b according to the molecular weight of the putative proteins. The genomic organization of these genes is consistent with the idea that many HSP genes originate from duplication events; in this case, probably an inversion and duplication occurred. The HaHSP17.6a and HaHSP17.6b genes are characterized by different expression levels under various heat stress conditions; moreover, their expression is differently induced by various elicitors. The differential regulation observed for HaHSP17.6a and HaHSP17.6b genes differs from previous observations on duplicated sHSP genes in plants.
[Show abstract][Hide abstract] ABSTRACT: A full-length cDNA encoding a novel small heat shock protein (HaHSP17.9) was isolated from a cDNA library of sunflower (Helianthus annuus cv. Gloriasol). The deduced amino acid sequence exhibited high degree homology to the class I cytosolic sHSPs from other plant species, and contained all the conserved regions characteristic of this class of proteins. Northern analyses showed that the transcript homologous to HaHSP17.9 accumulates during heat shock in suspension cultured cells and in the different parts of sunflower seedlings.
DNA Sequence 01/2002; 12(5-6):397-400. DOI:10.3109/10425170109084464 · 0.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(A)+RNA isolated from durum and common wheat seeds exposed to different thermal regimes during ripening, was translated in vitro using a rabbit reticulocyte system. The modification of protein synthesis was studied with particular regard to the heat shock proteins produced under high temperature conditions. One-dimensional polyacrylamide gel electrophoresis analysis showed products ranging in size from 14 to 100 kDa, some of which were present only when mRNA samples from high temperature-treated plants were translated. The mRNAs were also analysed by Northern hybridization with specific probes for heat shock proteins. The results clearly show that wheat plants respond to thermal stress by triggering the typical mechanisms of the heat shock response including activation of the heat shock genes, in developing grains as well as other plant parts.
[Show abstract][Hide abstract] ABSTRACT: High temperatures during grain filling are considered one of the factors that can modify dough properties and quality in wheat. In this study we analysed four Italian wheat cultivars grown under different temperature conditions to study the influence of high temperature on storage-protein-gene expression. Plants were grown both in the field and in growth cabinets, and were subjected to different thermal regimes. PolyA+ mRNAs were extracted from control and stressed plants at different stages of kernel development. Northern blot hybridisations were performed using probes for storage and heat shock proteins to monitor the expression of the relative genes under different temperature conditions. Northern analyses, performed using storage protein probes, indicated that temperature variation does not influence the synthesis of any of the storage protein mRNAs. On the contrary, the hybridisation signals obtained using heat shock probes were more intense in the stressed samples, indicating that the expression of heat shock genes is modulated by the temperature variation.