Partial recovery of grape energy metabolism upon aeration following anaerobic stress

The metabolic changes of mature grape berries during periodsof anoxia and upon return to air were examined using two cultivars.Glutamate declined during anoxia, together with a correspondingaccumulation of -aminobutyrate (GABA). The reverse occurredduring a 24 h period of air. Total adenine nucleotide (AdN),ATP level, and adenylate energy charge (AEC) all declined duringperiods of anoxia but showed a reversible pattern upon subsequentaeration. However, aerobic recovery of metabolite levels wasnot observed when the duration of anoxia at 30°C exceeded6 d. Accumulated ethanol during anoxia (up to 0.22 M) triggered afurther increase in the rate of ethanol synthesis when stressedberries were returned to air. Ethanol may be the principal determinantgoverning the ability of grapes to withstand and recover fromanoxic stress. We propose that the imbalance between aerobicand fermentative pathways may be due to the ability of ethanolto impair mitochondrial membrane function and uncouple oxidativephosphorylation, the rate of anaerobic respiration being insufficientto meet energy requirements. Key words: Grape, energy metabolism, anaerobic stress, aeration, ethanol production     

Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.     

Mechanisms of pre‐apoptotic calreticulin exposure in immunogenic cell death

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre‐apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)‐sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2α, followed by partial activation of caspase‐8 (but not caspase‐3), caspase‐8‐mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE‐dependent exocytosis. Knock‐in mutation of eIF2α (to make it non‐phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase‐8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase‐8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.     

Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.     

Molecular characteristics of immunogenic cancer cell death

Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activation), elements of the endoplasmic reticulum stress response (such as eukaryotic initiation factor 2alpha phosphorylation), as well as elements of the apoptotic response (such as caspase activation). Depending on the signal-transduction pathway, tumor cells responding to chemotherapy or radiotherapy can express 'danger' and 'eat me' signals on the cell surface (such as NKG2D ligands, heat-shock proteins and calreticulin) or can secrete/release immunostimulatory factors (such as cytokines and high-mobility group box 1) to stimulate innate immune effectors. Likewise, the precise sequence of such events influences the 'decision' of the immune system to mount a cognate response or not. We therefore anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.     

Calreticulin exposure dictates the immunogenicity of cancer cell death

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.     

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.