The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants. INTRODUCTIONSince their discovery in the early to mid-1980s, the enzymes responsible for D-3 phosphorylation of phosphoinositides, that is, the phosphatidylinositol (PtdIns) 3-kinases, have been the subject of intense study (reviewed in Carpenter and Cantley, 1996). The PtdIns 3-kinases are now known to be involved in a plethora of cellular processes, ranging from mitogenesis, membrane trafficking and ruffling to glucose uptake, oxidative burst responses, chemotaxis, and apoptosis.Several distinct PtdIns 3-kinase isoforms are known to exist in eukaryotic cells. The currently identified PtdIns 3-kinases can be divided broadly into four families based on sequence homology and their preferred inositol lipid substrate or substrates. However, only one family has been found in all the eukaryotic cells studied so far, namely, the PtdIns-specific Vps34p-related 3-kinases. The other families presumably are more organism specific and have evolved later from a common primordial PtdIns 3-kinase family. Despite the high degree of sequence homology between members of this family and the other PtdIns 3-kinases, the Vps34p-related kinases all have the distinguishing biochemical feature that they phosphorylate only PtdIns, both in vivo and in vitro, and are unable to utilize PtdIns 4-monophosphate (PtdIns(4)P) or PtdIns 4,5-bisphosphate as substrates. The VPS34 gene product, Vps34p, was first identified in a screen of yeast mutants defective in vesicle sorting (Stack and Emr, 1994). Subsequently, a human homolog was identified, and Vps34p-related PtdIns 3-kinas...
The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.
Cross-sectional comparisons indicated that minimal-moderate AD patients produced more semantic paraphasias, phonological paraphasias, and empty and indefinite phrases, whilst producing fewer pictorial themes, repairing fewer errors, and producing shorter and less complex sentences than controls. The two groups could not be distinguished on visual paraphasias. Longitudinal follow-up, however, suggested that visual processing deteriorates over time, where the prevalence of visual errors increased over 12 months. Discussion The findings suggest that the deterioration of writing skills observed in the spontaneous writings of AD patients shows a pattern of impairment dominated by semantic errors with a secondary impairment in phonological processing, which is later joined by a disruption of visuospatial and graphomotor processing.
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