CHRK1, a chitinase-related receptor-like kinase,plays a role in plant development and cytokinin homeostasis in tobacco

CHRK1 encodes a receptor-like kinase that contains a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. In this study, we showed that CHRK1 is mainly expressed in the shoot apex region including leaf primordia and young leaves, and germinating seedlings and vascular tissues, based on GUS activity of transgenic tobacco plants carrying the CHRK1 promoter-GUS fusion gene. Transgenic tobacco plants in which CHRK1 expression was suppressed exhibited pleiotrophic developmental abnormality, including formation of proliferating shooty calli from emerging seedlings and severely altered seedling development. At the cellular level, ectopic cell proliferation, reduced cell specificity, and aberrant chloroplast development were observed. The transgenic lines contained 3-fold higher level of cytokinin than the wild-type plants. Consistently, the transgenic seedlings exhibited a typical cytokinin response in the absence of hormone, such as deetiolation under the dark. Based on these results, we propose that CHRK1 is involved in a developmental signaling pathway regulating cell proliferation/differentiation and the endogenous cytokinin levels in tobacco.     

Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: Implications of the signal divergence by cytokinin-inducible response regulators in the cytosol and the nuclei

Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L. to characterize their function in cytokinin signaling. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4–ZmRR7), 3 type-B RRs (ZmRR8–ZmRR10), and 3 HPs (ZmHP1–ZmHP3) were found in leaves. All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin. Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus. Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6). In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs. In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4. These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status.     

Differential scanning calorimetry of the effects of Ca2+ on the thermal unfolding of Pseudomonas cepacia lipase

Thermal unfolding of P. cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca2+. From the Ca2+ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa2+ < = => D + Ca2+, where N and D represent the native and denatured states, respectively, of the enzyme.     

Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.     

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.     

sigma marY1, the LTR of the gypsy-type retroelement marY1 from the basidiomycete tricholoma matsutake, allows multicopy DNA integration in Lentinula edodes

sigma marY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a sigma marY1 derivative, sigma* marY1, conferred the hygromycin-resistant phenotype stronger than the vector without sigma* marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that sigma marY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.     

Phosphorus foraging root development of Brassica rapa nothovar. grown in a phosphorus-deficient nonallophanic Andisol

Lateral root of Brassica crops firmly aggregated around Ca-alginate gel beads containing dicalcium phosphate dihydrate and -cyclodextrin (DCPD gel bead) in a phosphate (P)-deficient soil (Nanzyo et al., 2002, Soil Sci. Plant Nutr. 48, 847–853). The first aim of the present study was to identify the component in the DCPD gel beads that accounts for the special root proliferation. This P-foraging root growth was observed in plots applied with either polyolefin-coated NH₄H₂PO₄ (POC-MAP) or DCPD powder instead of the DCPD gel beads. The POC-MAP neither contains Ca, alginate nor -cyclodextrin. The DCPD powder was applied in a similar number of spots with the number of DCPD gel beads. Thus, the essential component in the DCPD gel beads for the P-foraging root growth around them was P. The second aim was to examine the effect of various inorganic P sources on the P uptake of B. rapa nothovar. While significant P uptake was obtained in the plot applied with apatite from Florida, USA, sediment origin (F-Ap), almost no P uptake was obtained in that with apatite from Quebec, Canada, igneous origin in the P-deficient nonallophanic Andisol. Hence, a P-release level from F-Ap was near the lower limit for the P uptake by the B. rapa nothovar. under the present experimental conditions. These results indicate the P foraging characteristics of the Brassica roots contribute to improve the P recovery rate in the agricultural fields with localized application of moderately-soluble P fertilizers.