Unfolding of poly-L-glutamic acid by microbubbling of supercritical carbon dioxide

The conformational changes in alpha-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, alpha-helix content decreased to 37%, while without the filter it was 68%. The alpha-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.     

Ganglioside GT1b in rat brain binds to p58, a brain-specific sodium-dependent inorganic phosphate cotransporter: expression cloning with a specific monoclonal antibody to ganglioside GT1b-binding protein

To evidence the notion that gangliosides involve neuronal cell interactions in the brain, we surveyed the presence of ganglioside-binding proteins in membrane lysates of adult rat cerebellum. Three proteins (p58, p90, and p160) were identified as GT1b-binding proteins by incubation of the blot of the membrane lysate with GT1b micelles. We generated a monoclonal antibody (mAb) specific to the polypeptide portion of the GT1b-binding proteins (YAK-2). The YAK-2 mAb specifically reacted with all three proteins on blots of proteins pretreated under nonreducing conditions for SDS-PAGE, but reacted mainly with p58 under reducing conditions, showing that p90 and p160 are oligomeric forms of p58. The binding activity of the YAK-2 mAb was completely inhibited by the presence of GT1b micelles, indicating the specificity of YAK-2 mAb for p58 and its oligomers. Immunohistochemical investigations revealed that both p58 and GT1b colocalize within the granular layer of adult rat cerebellum. Expression cloning of p58 cDNA was performed using YAK-2 mAb, and five putative clones were obtained. Among them, the nucleotide sequence of one cDNA completely matched that of rat brain-specific sodium-dependent inorganic phosphate cotransporter (rBNPI), a 61 kDa membrane protein. COS7 cells were transfected with a Flag-chimeric construct containing the rBNPI/p58 cDNA, and the membrane lysate was subjected to immunoprecipitation with anti-Flag antibody. One protein (64 kDa) was detected only with YAK-2 mAb, and the membrane lysate specifically bound to GT1b micelles. Taking together, we propose that rBNPI/p58 functions as a GT1b-binding protein in neuronal cells.     

Apoptosis in the aging process

Although many hypotheses have been proposed to explain the aging process, the exact mechanisms are not well defined. Recent accumulating evidence indicates that dysregulation of the apoptotic process may be involved in some aging processes; however, it is still debatable how exactly apoptosis is expressed during aging in vivo. In this review, we discuss recent findings related to apoptosis of individual organs during aging and their significance. We demonstrate that aging enhances apoptosis and susceptibility to apoptosis in several types of intact cells. In contrast, in certain genetically damaged, initiated, and preneoplastic cells, aging suppresses these age-associated apoptotic changes. In various cells, apoptosis enhances the elimination of damaged and dysfunctional cells presumably caused by oxidative stress, glycation, and DNA damage. In these cases, the incidence of apoptosis correlates with the level of accumulated injury. It is concluded that apoptosis plays an important role in the aging process and tumorigenesis in vivo probably as an inherent protective mechanism against age-associated tumorigenesis.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore-induced volatiles in plants

We compared volatiles from lima bean leaves (Phaseolus lunatus) infested by either beet armyworm (Spodoptera exigua), common armyworm [Mythimna (Pseudaletia) separata], or two-spotted spider mite (Tetranychus urticae). We also analyzed volatiles from the leaves treated with jasmonic acid (JA) and/or methyl salicylate (MeSA). The volatiles induced by aqueous JA treatment were qualitatively and quantitatively similar to those induced by S. exigua or M. separata damage. Furthermore, both S. exigua and aqueous JA treatment induced the expression of the same basic PR genes. In contrast, gaseous MeSA treatment, and aqueous JA treatment followed by gaseous MeSA treatment, induced volatiles that was qualitatively and quantitatively more similar to the T. urticae-induced volatiles than those induced by aqueous JA treatment. In addition, T. urticae damage resulted in the expression of the acidic and basic PR genes that were induced by gaseous MeSA treatment and by aqueous JA treatment, respectively. Based on these data, we suggest that in lima bean leaves, the JA-related signaling pathway is involved in the production of caterpillar-induced volatiles, while both the SA-related signaling pathway and the JA-related signaling pathway are involved in the production of T. urticae-induced volatiles.     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) ( EC 2.3.1.153 ) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases ( St-Pierre et al . (1998 ) Plant J. 14, 703–713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.     

Emericella appendiculata, a new species from Chinese soil

Emericella appendiculata, a new species isolated from soil of the Pamire Plateau, is described and illustrated. It is characterized by grayish green non-ostiolate ascomata surrounded by a thick layer of hülle cells, membranaceous peridium, prototunicate asci, violet-brown, lenticular ascospores which are ornamented by two stellate equatorial crests, capitate convex surfaces, and long filiform appendages, and anAspergillus anamorph with biseriate conidiogenous cells.