Identification, characterization and cDNA cloning of two novel proteins secreted into the external space of the regenerating leg of Periplaneta americana

We have isolated two proteins (Rap 60 and Rap 40) that were expressed specifically in regenerating legs of the American cockroach (Periplaneta americana). These proteins appear to be derived from a precursor protein with 443 amino acid residues termed regeneration-associated protein (RAP). Rap 60 and Rap 40 corresponded to Glu 223 to Ser 443 and Glu 223 to Glu 366 of RAP, respectively. Hence, Rap 40 is the N-terminal part of Rap 60. These proteins contained 13 repeats of a novel motif, [Glu/Asp-Glu/Asp-Val/Ala-Lys]. The gene encoding RAP was shown to be expressed during embryogenesis and by newly-formed epidermal cells of regenerating legs. Rap 60 and Rap 40 were secreted into the external space of regenerating leg saccules, where they accumulated, suggesting that they are components of extracellular matrix of regenerating leg saccules.     

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Genes involved in cytokinin signal transduction

Although cytokinin plays a central role in plant development, our knowledge about the signal transduction pathway initiated by this plant hormone is fragmentary. By randomly introducing enhancer elements into theArabidopsis genome throughAgrobacterium-mediated transformation, 5 cytokinin independent mutant calli (cki1-1, −2, −3, −4 andcki2) were obtained. These mutants exhibit typical cytokinin responses, including rapid proliferation, chloroplast differentiation, shoot induction and inhibition of root formation, in the absence of cytokinin. TheCKl1 gene encodes a product similar to the sensor histidine kinases of two-component systems, and its overexpression in plants induces typical cytokinin responses (Kakimoto 1996). Here I report that overexpression of this gene did not alter the auxin reqirement ofArabidopsis. Another mutant,many shoots, which was also identified on the same screening, produced many adventitious shoots on cotyledons, petioles and true leaves. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Anticancer susceptibility test method using general-purpose dissolved oxygen meter

Drug susceptibility of cell can be rapidly measured by continuously monitoring its metabolic changes. We focused on respiration volume as a signal of metabolic change, and developed a new dissolved oxygen measuring system which can detect respiration volume of cells. The main feature of the system is the use of a new type bare oxygen electrode which can easily detect the changing rate of dissolved oxygen concentration. In this study, single type electrode was used to evaluate this rapid anticancer drug susceptibility test. The result obtained was almost equivalent to that with MTT method, which is a conventional method for susceptibility test of HL-60 to various kinds of anticancer drugs. We have also developed multi-type electrode plate with oxygen electrodes embedded in the bottom of 96-well plate, with which clinical evaluation of this method can be easily made.     

In Vivo Metabolism of the Vitamin D Analog, 22-Oxacalcitriol: Evidence for Side-chain Truncation and 17-Hydroxylation

After intravenous administration of the vitamin D₃ analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24R(OH)OCT, 24S(OH)OCT, (25R)-26(OH)OCT, (25S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1,25(OH)₂ vitamin D₃] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20S(OH)-hexanor-OCT and 17,20S(OH)₂-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C₂₃-O₂₂ cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1,25-(OH)₂D₃.     

Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3– or granulocyte–macrophage colony-stimulating factor–mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)–Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB–Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB–Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.