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  • br Application of the screening system


    Application of the screening system for ITP-binding proteins Inosine is a nucleoside with hypoxanthine as a base. Inosine nucleotides are relatively abundant in human Direct Mouse Genotyping Kit because inosine monophosphate (IMP) is a general precursor molecule for the de novo synthesis of AMP and GMP or a product of enzymatic deamination of AMP. ITP and dITP are known to be generated by nitrosative deamination of ATP or dATP, respectively. The phosphorylation of IMP to IDP and ITP or the reduction of IDP to dIDP and its conversion to dITP by phosphorylation can also occur. ITP and dITP may be toxic for organisms ranging from bacteria to mammals [14], [15], [16]. To identify novel inosine nucleotides hydrolyzing enzymes and to understand the molecular basis of the toxicities of ITP and dITP, we have performed screens for ITP-binding proteins in mouse or human cell extracts.
    Functional analyses of ITP-binding proteins identified in the comprehensive screens
    Discussion and conclusions
    Conflict of interest statement
    Acknowledgments These works were supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan [20013034 to YN, 21117512 to DT]; the Japan Society for the Promotion of Science [19390114 to DT, 08J03650 to TI] and Kyushu University Global COE program [YN].
    Introduction Cold storage is one of the most widely used technologies to slow respiration and other metabolic processes in order to preserve postharvest life of horticultural products (Wang, 1994). However, citrus fruit are susceptible to chilling injury (CI) when exposed to temperatures of less than 2–5°C. Chilling injury is expressed as pitting, staining and necrotic areas in the peel that increase in number and size over time (Sanchez-Ballesta et al., 2003). Several postharvest treatments have been reported to reduce chilling injury symptoms in citrus including intermittent warming (Porat et al., 2003, Kluge et al., 2003) low temperature conditioning (Hofman et al., 2003) and high temperature treatments using air (Sala and Lafuente, 2000) and water (Fallik, 2004). Hot water treatments may also control postharvest rots of horticultural crops (Porat et al., 2000, Fallik, 2004). It is widely accepted that symptoms of CI are a consequence of oxidative stress in the tissues (Sala, 1998) occurring when active oxygen species (AOS) such as hydrogen peroxides, superoxides and hydroxyl radicals are in excess of the scavenging capacity of fresh tissue (Hodges et al., 2004). Involvement of antioxidant enzymes in regulation of AOS can be followed by measuring guaiacol peroxidase (POX; EC and catalase (CAT; EC activity during postharvest storage (Sala, 1998). Preconditioning treatments of fruit with hot water (Fallik, 2004, Schirra and D’hallewin, 1997) may induce chilling tolerance by modulating antioxidant systems that would prevent the accumulation of AOS (Martinez-Tellez and Lafuente, 1997, Sala and Lafuente, 2000). Lafuente et al., 2004 also noted that ethylene was involved in inducing chilling tolerance in ‘Fortune’ mandarins. Sustaining antioxidant levels of treated fruit during cold storage periods may also be important for preserving chilling tolerance (Rivera et al., 2004). Citrus fruit show limited changes in fruit quality attributes during postharvest periods, although the low metabolism activity through prolonged storage can result in internal metabolite changes. One change which we seek to maximise in NZ fruit is a decrease in the acidity in order to meet Japanese market requirements, this being possible using temperature conditioning (Burdon et al., 2007). Declining organic acid levels in fruit during cold storage may be indicative of changes in energy metabolism, pH stability, and defence compounds that prevent or repair damage caused by chilling temperature (Maldonado et al., 2004). The V-ATPase pumps protons from the cytoplasm into the Direct Mouse Genotyping Kit vacuole using the energy released by ATP hydrolysis, thereby creating an electrochemical H-gradient that is the driving force for citrate uptake into the vacuole (Müller et al., 1997). The hydrolysis of inorganic pyrophosphate (PPi) can also generate a proton motive force across the tonoplast (Rea and Sanders, 1987). V-PPase also plays a role in unloading protons during de-acidification of some citrus cultivars (Marsh et al., 2001). The activity of the V-ATPase and V-PPase is modulated to cope with environmental and metabolic changes (Dietz et al., 2001), and Darley et al. (1995) suggested that the high activity of V-PPase was linked with a specific role in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.