Characteristics of gas exchange and morphology of a spring ephemeral, Erythronium japonicum, in comparison with a sun plant, Glycine max

Morphological characteristics and responses of gas exchanges to light intensity were examined in a typical vernal species, Erythronium japonicum Decne (E. japonicum), grown (i) on the floor of a deciduous broad-leaved Quercus mongolica forest (one of its native habitats, the Q. mongolica stand); (ii) bare land left undisturbed for 9 years after forest clearing (the bare stand); and (iii) in a sun crop, soybean, grown for 110 days in an experimental field and for 17 days in pots, in order to evaluate the adaptability of the photosynthetic process of this vernal species to its shady native habitats. The daytime solar radiation, ai and leaf temperatures and leaf–air vapor pressure difference (VPD) were significantly higher at the bare stand than at the Q. mongolica stand. When environmental factors observed at the Q. mongolica and bare stands were reproduced in an assimilation chamber, leaf temperatures of E. japonicum plants increased markedly with increased radiation, whereas those of soybean plants differed little from the respective air temperatures. The photosynthetic and transpiration rates and stomatal conductance in the former plants placed under conditions at the Q. mongolica stand increased with radiation and reached respective steady state values at maximum radiation at the site; whereas, under the conditions at the bare stand, they also increased and reached respective steady state values, but then continuously decreased to be lower than the respective value at the Q. mongolica stand. However, both rates and the conductance in the soybean plants under both conditions increased significantly with radiation and reached much higher respective values at the respective maximum radiations. Water use efficiency for E. japonicum plants was much higher under conditions at the Q. mongolica stand than at the bare stand and was practically equal to those for soybean plants under both conditions. Water potential in the leaves of E. japonicum at maximum radiation at the bare stand was one-third that of those at the Q. mongolica stand. The potential in soybean leaves differed little between both conditions and was roughly equal to the low value in E. japonicum leaves at the bare stand conditions. The stomatal densities on upper and lower leaf surfaces and the ratio of root weight to leaf area (R : L) differed little between E. japonicum plants grown at both stands as well as between young and adult soybean plants. However, the densities on the upper and lower surfaces of E. japonicum were 25% and 60% of the respective values of both soybean plants. The ratios of R : L of the E. japonicum plants were only one-quarter that of the young and adult soybean plants. The cooperation between these morphological and gas exchange characteristics in E. japonicum plants is discussed in relation to adaptation to the environment in native habitats.     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase.

The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL. His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.     

Activation mechanism of Gi and Go by reactive oxygen species.

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.     

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K_d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.     

Protective effects of probucol treatment on pancreatic beta-cell function of SZ-induced diabetic APA hamsters.

To clarify whether oxidative stress is involved in the pathogenesis of islet lesions of diabetic animals, the effects of probucol (PB), an antioxidant and anti-hyperlipidemia agent, on the islets in streptozotocin (SZ)-induced diabetic APA hamsters in the acute and chronic phases of diabetes were examined. The control (CB group) and diabetic (SZ group) hamsters were treated with PB (1% in the diet) for 4 weeks from several days after SZ injection as the acute diabetic group, or 8 weeks from 6 weeks after SZ injection as the chronic diabetic group. Glucose tolerance test revealed that PB treatment decreased the high serum glucose level after glucose injection in the diabetic APA hamsters in the acute diabetic phase. Immunohistochemistry revealed that PB treatment significantly increased the percentage of the insulin positive area in the diabetic hamsters pancreata in both the acute and chronic phases. In addition, 4-hydroxy-2-nonenal (4HNE; an oxidative stress marker) positive cells were slightly reduced by PB treatment in the acute diabetic phase. Double-immunostaining for insulin and PCNA (proliferating cell nuclear antigen) revealed that elevation of the percentage of insulin and PCNA double-positive cells against insulin-positive cells was seen in the islets of PB-treated diabetic hamsters, but the difference was not significant compared with untreated diabetic hamsters (p = 0.07). In semi-quantitative RT-PCR, the expression of two genes, Reg (Regenerating gene) and INGAP (islet neogenesis associated protein), in the diabetic APA hamsters was significantly increased compared to the control groups in both diabetic phases. PB treatment significantly reduced Reg expression in the chronic diabetic phase. These data suggest that PB treatment in SZ-injected diabetic hamsters partially restored beta-cell function through acting as an antioxidant and induced higher expression of Reg and INGAP genes in the pancreas of hamsters.     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Effects of body size and predators on intracohort competition in wild and domesticated juvenile salmon in a stream

Competition between masu salmon (Oncorhynchus masou) of wild and aquaculture origin was investigated. Fry were individually marked and released in stream enclosures with and without a piscivorous predator. The aim was to assess the effects of predators and salmon body size on survival and growth of the two types of fish under natural conditions. The presence of predaceous Japanese huchen (Hucho perryi) resulted in lower mean growth rates of surviving fry. Relatively large fish survived and grew better than relatively small fish in the absence of predators, but not in their presence. This probably indicates an indirect effect of predation risk on within-cohort competitive behavior among salmon juveniles, with larger fish forced to give up their position as superior competitors. Domesticated fish survived in larger numbers and grew much faster than wild fish, irrespective of predator presence. Comparison with similar field studies revealed a pattern that the pre-experimental environment influenced the outcome of competition between wild and domesticated juvenile salmon. Domesticated fish were superior competitors even in the absence of an initial size advantage, which commonly gives a further advantage to hatchery-raised fish in natural streams. Therefore, caution dictates to avoid the release or escape of such fish into the wild.     

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.