Identification and roles of proteins for seed development in mungbean (Vigna radiata L.) seed proteomes.

Proteomic analysis of developing mungbean (Vigna radiata L.) seeds has not yet been investigated in detail. Fifty-seven proteins were separated by 2-DE, identified by nanoelectrospray mass spectrometry from the present protein databases, and categorized according to their functions. Many of the identified enzymes were involved in central carbon metabolism; thus, a pathway illustrating starch synthesis/breakdown, sugar conversion for glycolysis, and tricarboxylic acid (TCA) cycle was proposed. Quantitative comparison of the protein expression revealed that during developmental process (11-21 days after flowering, DAF), proteins involved in glycolysis, TCA cycle, and alcoholic fermentation showed a trend to be down-regulated, whereas storage proteins were generally up-regulated. The downward tendency of central carbon metabolic proteins suggests a reduction in ATP and oxygen consumption associated with accumulation of storage compounds. UDP-glucose-1-pyrophosphorylase, an upstream enzyme in the starch ADP-Glc pathway, was found as a stably expressed protein throughout the growth stage, demonstrating its importance in mungbean starch biosynthesis. The temporal expression of metabolic enzymes suggests the coordination of an acclimation mechanism and cellular processes associated with accumulation of storage compounds in seed development.

UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons.

UNC-31 or its mammalian homologue, Ca(2+)-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca(2+)-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca(2+) level (pre-flash Ca(2+) was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca(2+) evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans.

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a "fluorescent timer" through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this "timer" changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this "timer", we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this "timer". We anticipate that more new "fluorescent timers" with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.

Synaptotagmin IV regulates dense core vesicle (DCV) release in LbetaT2 cells.

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca(2+)-triggered exocytosis of dense core vesicles (DCVs) in LbetaT2 cells. Syt I silencing, however, had no effect on vesicular release.