The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related

Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose.     

Isolation of an ozone-sensitive and jasmonate-semi-insensitive Arabidopsis mutant (oji1)

A novel ozone-sensitive mutant was isolated from Arabidopsis T-DNA tagging lines. This mutant revealed severe foliar injury and higher ethylene emission than the wild type under ozone exposure. The ozone-induced injury and ethylene emission were suppressed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis, both in this mutant and wild-type plants. Pretreatment with methyl-jasmonate (MeJA) at 10 micro M, however, suppressed the ozone-induced ethylene emission and foliar injury only in the wild-type plants. This mutant was less sensitive to jasmonate than the wild type, estimated by the MeJA-induced inhibition of root elongation and ozone-induced expression of AtVSP1, a jasmonate-inducible gene. Thus, this mutant was named oji1 (ozone-sensitive and jasmonate-insensitive 1). These results suggest that the ozone sensitivity of oji1 is caused by the increase in ozone-induced emission of ethylene as a result of low sensitivity to jasmonate, which plays defensive roles under stress conditions.     

Differential ozone sensitivity among <Emphasis Type="Italic">Arabidopsis</Emphasis> accessions and its relevance to ethylene synthesis

We compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to ozone (O(3)) exposure. Observation of visible injury as well as ion-leakage analysis demonstrated clear differences between the O(3)-tolerant accession Col and the O(3)-sensitive accession Ws. RNA-blot analysis showed that O(3)-induced increases in mRNA levels of several ethylene-inducible genes and a salicylic acid-inducible gene were substantially higher in Ws than in Col. The time-course of induction of various mRNA levels shows that the expression of ethylene-inducible genes was rapidly, and more strongly, induced by O(3) in Ws than in Col, suggesting that Ws exhibits higher ethylene-signaling. Both the level of mRNA for an O(3)-inducible 1-aminocyclopropane-1-carboxylate synthase and the level of ethylene generation after 3 h of O(3)-exposure were higher in Ws than in Col. O(3)-induced leaf damage was attenuated by pretreatment with ethylene biosynthesis- and signaling-inhibitors, indicating that ethylene signaling is required for O(3)-induced leaf injury in Ws. On the other hand, an ethylene-overproducing mutant of Col, eto1-1, displayed significantly increased O(3)-induced leaf injury compared to wild type plants. These results indicate that the difference in O(3) sensitivity is dependent on the difference in ethylene production rate between these two accessions. Finally, we investigated the relationship between the degree of leaf damage and the level of ethylene evolution in 20 different Arabidopsis accessions. Based on the result, the accessions were classified into four types. However, most of them showed significant correlation between the ethylene production level and the degree of leaf injury, suggesting that ethylene signaling is an important factor in the natural variety of O(3) sensitivity among Arabidopsis accessions.     

Profile analysis of 24-hours measurements of blood pressure

A method is proposed for classifying subjects according to their convex, flat, or concave change patterns of 24-hours blood pressure measurements. To obtain such a classification is useful for detecting subjects who show abnormal change patterns and giving them appropriate medical treatments. Therefore, an appropriate statistic is proposed for detecting a systematic change along the time axis, as well as a statistic with its inverse characteristic appropriate for evaluating the noise variation. The method is based on the ratio of those two types of statistics; it is verified to work well on real data, giving a classification of subjects into four types of subgroups: extreme dipper, dipper, nondipper, and inverted dipper. It also suggests that there might be an ultra-extreme dipper subgroup.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

Site-directed mutagenesis alters DnaK-dependent folding process

The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE). When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine. These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK. This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.     

Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.