Plant regeneration via somatic embryogenesis from protoplasts of Uganda cherry orange (Citropsis schweinfurthii)

Protoplasts isolated from embryogenic callus of Citropsis schweinfurthii (Engl.) Swing. & M. Kell were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA, 0, 300, 600 or 900 mg l^–1 malt extract and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg l^–1 BA and 600 mg l^–1 malt extract. MT basal medium containing 5% sucrose and supplemented with 0.01 mg l^–1 kinetin was found to be a medium suitable for the development of globular somatic embryos derived from protoplasts into heart-shaped somatic embryos with cotyledon-like structures. The highest percentage of shoot formation was obtained using 0.1 mg l^–1 GA₃. A complete protoplast to-plant system was developed for C. schweinfurthii, which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃ - ME malt extract     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

Mechanism of cytotoxicity of methylmercury

Mechanism of methylmercury cytotoxicity was investigated with special reference to its preferential action on microtubules and protein biosynthesis in cultured cells. The tubulin synthesis analyzed by autoradiography of two-dimensional electropherogram using³⁵S-methionine was inhibited by 50–70% in mouse glioma cells exposed to 5×10^?6 M methylmercury for 3 h, which almost completely depolymerized microtubules. Total protein synthesis monitored by incorporation of labeled methionine into acid insoluble fraction was decreased slightly but significantly and the protein bands other than tubulin on gradient urea-PAGE gel appeared to remain unchanged under the experimental condition used. These results suggest that the inhibition of protein synthesis observed on exposure to methylmercury can be ascribed, at least partly, to a possible autoregulatory depression in tubulin synthesis owing to the increase in the pool of tubulin subunits resulted from microtubule depolymerization by methylmercury.     

Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein

Sporamin, the major soluble protein of the sweet potato tuberous root, is coded for by a multigene family. Fourty-nine essentially full-length sporamin cDNAs isolated from tuberous root cDNA library have been classified by cross hybridization, restriction endonuclease cleavage pattern and ribonuclease cleavage mapping. All the cDNAs fall into one of the two distinct homology groups, subfamilies A and B, which correspond to the polypeptide classes sporamin A and B, respectively. At least 5 different sequences are detected in both of the 22 sporamin A and 27 sporamin B cDNAs. Comparison of the nucleotide sequences of the coding region of three each of sporamin A and B subfamily members, four from cDNAs and two from genomic clones, indicates that intra-subfamily homologies (94 to 98%) are much higher than inter-subfamily homologies (82 to 84%), and there are deletions or insertions of one or two codons at three locations which characterize each subfamily. Large portions of base substitutions in the coding region accompany amino acid substitutions. In contrast to the coding region, most of the structural differences among the members in the 5 and 3 noncoding regions are deletions or insertions.     

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.     

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

Costs of parturition and rearing in female sika deer (Cervus nippon)

The costs of parturition and lactation of female sika deer on Kinkazan Island (9.6 km(2) in size), northern Japan, which live at a high density (about 50 deer/km(2)), were evaluated by comparison of body weights of 481 females measured during a 15 year study (1993-2008). Weight data were chosen from only females that did not give birth in the preceding year. The mean body weight of females that did not give birth ("yelds") was significantly lower (P < 0.001) than that of females who gave birth ("milks"); yelds' body weight was 93.1% and 83.5% that of milks in the preceding and parturition years, respectively. The yelds increased in body weight by the following March by 8.2% (P < 0.001), whereas milks did not. Among the milks, those whose fawns survived until the following May ("rearing milks") lost body weight by 14.9% (P < 0.001). Milks who lost fawns within a week after birth ("early fawn-less milks") did not lose body weight (P = 0.583), while those whose fawns died after the first autumn but died before May ("late fawn-less milks") lost body weight by 19.9% (P < 0.001). These results indicate that sika deer females do not enter estrus unless they are heavy enough, and that both parturition and rearing are costly for sika deer mothers living in high-density conditions.     

Structural basis of interleukin-5 dimer recognition by its α receptor

Interleukin-5 (IL-5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL-5 receptor (IL-5R) consists of the IL-5 receptor α subunit (IL-5RA) and the common receptor β subunit (βc). IL-5 binding to IL-5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL-5 in complex with the IL-5RA extracellular domains. The structure revealed that IL-5RA sandwiches the IL-5 homodimer by three tandem domains, arranged in a “wrench-like” architecture. This association mode was confirmed for human cells expressing IL-5 and the full-length IL-5RA by applying expanded genetic code technology: protein photo-cross-linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM-CSF•GM-CSFRA•βc structure enabled us to propose complete structural models for the IL-5 and GM-CSF receptor complexes, and to identify the residues conferring the cytokine-specificities of IL-5RA and GM-CSFRA.