Cell-lytic activity of tobacco BY-2 induced by a fungal elicitor from alternaria alternata attributed to the expression of a class I beta-1,3-glucanase gene

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.     

Changes of C-reactive protein levels in rainbow trout (Oncorhynchus mykiss) sera after exposure to anti-ectoparasitic chemicals used in aquaculture

Changes of serum C-reactive protein (CRP) levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin, metriphonate or potassium permanganate, which are used in aquaculture as anti-ectoparasitic chemicals. The CRP level in normal trout sera is 88+/-5 microg ml(-1) according to sandwich enzyme-linked immunosorbent assay. CRP levels increased to a maximum at six or nine days after exposure to formalin for 3.5 h at 300 ppm or 9.5 h at 30 ppm, respectively; these levels are 4.3 and 18 times higher than normal. At 18 days after treatment, the CRP level had decreased to significantly below the normal level. After exposure to metriphonate (0.4 ppm for 30 min), the CRP level increased significantly to a maximum at three days after exposure (9.9 times higher than normal), then decreased to below normal. With exposure to potassium permanganate at 40 ppm for 45 min, fish showed significantly lower CRP levels than the normal level at 14 days after exposure. Fish reared at a water temperature of 16.5-19.5 degrees C showed significantly higher CRP levels than those reared at 13 degrees C. Measurement of CRP levels in trout serum can be used as a bioindicator of the health condition of the fish.     

Rat cardiac contractile dysfunction induced by Ca2+ overload: possible link to the proteolysis of alpha-fodrin

The aim of the present study was to examine the mechanisms of Ca2+ overload-induced contractile dysfunction in rat hearts independent of ischemia and acidosis. Experiments were performed on 30 excised cross-circulated rat heart preparations. After hearts were exposed to high Ca2+, there was a contractile failure associated with a parallel downward shift of the linear relation between myocardial O(2) consumption per beat and systolic pressure-volume area (index of a total mechanical energy per beat) in left ventricles from all seven hearts that underwent the protocol. This result suggested a decrease in O(2) consumption for total Ca2+ handling in excitation-contraction coupling. In the hearts that underwent the high Ca2+ protocol and had contractile failure, we found marked proteolysis of a cytoskeleton protein, alpha-fodrin, whereas other proteins were unaffected. A calpain inhibitor suppressed the contractile failure by high Ca2+, the decrease in O(2) consumption for total Ca2+ handling, and membrane alpha-fodrin degradation. We conclude that the exposure to high Ca2+ may induce contractile dysfunction possibly by suppressing total Ca2+ handling in excitation-contraction coupling and degradation of membrane alpha-fodrin via activation of calpain.     

Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae

 The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 °C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast. Received: 11 June 1999 / Received last revision: 12 November 1999 / Accepted: 28 November 1999     

Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum

Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.     

Synchronization of Phosphorylation of a Chloroplast Protein with the Cell Division Cycle in Heterosigma akashiwo

Fumigation of plants of five species with NO₂ in darkness causedvisible injuries to leaves, with the most severe injuries inkidney bean plants and the least severe in spinach plants. Fumigationof these plants in the light caused virtually no visible injuries.NO₂-fumigated leaves accumulated nitrite in the darkness butnot in the light. The level of accumulated NO₂^– was decreasedby light much more rapidly in spinach leaves than in those ofkidney bean, with much less injury to spinach leaves than tothose of kidney bean. A larger amount of NO₂^– accumulatedin the trifoliate leaves of kidney bean plants cultivated withNO₃^– as a main source of nitrogen than in those of plantscultivated with NH₄⁺, and the former plants were more susceptibleto injury from NO₂ than the latter. Administration of NO₂^–to leaves of spinach and kidney bean plants induced the destructionof Chi in the light. The extent of the destruction of Chi wassmaller in spinach than in kidney bean, consistent with theirrespective responses to NO₂. The NO₂^–-induced destructionof Chi was inhibited to some extent by scavengers of free radicals.Activities of superoxide dismutase (SOD) were higher in leavesof spinach than in those of kidney bean. These results indicatethat NO₂^– is the toxic species generated by fumigationwith NO₂ and that spinach has a greater tolerance for NO₂ thankidney bean, probably as a result both of a higher capacityfor reduction of NO₂^– and a higher level of activity ofSOD. (Received October 4, 1991; Accepted January 31, 1992)     

Diverse roles of metal ions in acyl-transferase ribozymes

The dependence on metal ions for catalysis is one of the hallmark characteristics of ribozymes. Yet despite this universal reliance, the functional role of divalent ions in promoting RNA catalysis is manifold. In this study we elucidate some different roles metal ions play as catalytic cofactors, by comparing two functionally co-evolved acyl-transferase ribozymes. Earlier studies performed on the in vitro selected acyl-transferase ribozyme, E18 [Suga, H., Cowan, J. A., and Szostak, J. W. (1998) Biochemistry 28, 10118-10125], revealed the requirement of a fully hydrated (outer-sphere) Mg2+ ion for catalytic activity. Interestingly, one class of acyl-transferase ribozymes isolated from the same RNA pool as E18 displays a unique metal dependency and is believed to be interacting with inner-sphere coordinated Mg2+ ions. New results show that one of these inner-sphere coordinating ribozymes, HS01, assumes a cloverleaf secondary structure closely resembling E18, yet apparently facilitates a distinct catalytic mechanism. Furthermore, the nature of the RNA-metal interaction(s) in HS01 seems to be dictating a unique reaction mechanism that exhibits a titratable moiety at a near-neutral pK(a). In light of the critical role metal ions play in biochemistry and the proper function of RNAs, these results compare two distinct manners by which metals serve to promote the catalysis of the same reaction.