Light-inducible pigmentation in Portulaca callus; selection of a high betalain producing cell line

Summary We have established a unique betalain pigmentation system in callus cultures that originated from seedlings of Portulaca sp. Jewel. Within three different Jewel lines examined, one line (JR) was clearly superior with regard to callus growth rate and pigment formation. Furthermore, after ten cycles of selection of deeply colored callus patches, the selected clones contained on an average four times the amount of betalain as compared to the non-selected mother line. The colorization was induced by light, but disappeared in the dark. Pigment synthesis was detectable within 30 h after irradiation and showed positive correlation with irradiation periods.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - HPLC high performance liquid chromatography     

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Evaluation of extensional and torsional stiffness of single actin filaments by molecular dynamics analysis

It is essential to investigate the mechanical behaviour of cytoskeletal actin filaments in order to understand their critical role as mechanical components in various cellular functional activities. These actin filaments consisting of monomeric molecules function in the thermal fluctuations. Hence, it is important to understand their mechanical behaviour on the microscopic scale by comparing the stiffness based on thermal fluctuations with the one experimentally measured on the macroscopic scale. In this study, we perform a large-scale molecular dynamics (MD) simulation for a half-turn structure of an actin filament. We analyse its longitudinal and twisting Brownian motions in equilibrium and evaluated its apparent extensional and torsional stiffness on the nanosecond scale. Upon increasing the sampling-window durations for analysis, the apparent stiffness gradually decreases and exhibits a trend to converge to a value that is close to the experimental value. This suggests that by extrapolating the data obtained in the MD analysis, we can estimate the experimentally determined stiffness on the microsecond to millisecond scales. For shorter temporal scales, the apparent stiffness is larger than experimental values, indicating that fast, local motions of the molecular structure are dominant. To quantify the local structural changes within the filament on the nanosecond scale and investigate the molecular mechanisms, such as the binding of the actin-regulatory proteins to the filaments, it is preferable to analyse the mechanical behaviour on the nanometre and nanosecond scales using MD simulation.     

A new class of pentapeptide KISS1 receptor agonists with hypothalamic–pituitary–gonadal axis activation

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂ (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.     

Substrate specificity of tuliposide-converting enzyme,a unique non-ester-hydrolyzing carboxylesterase in tulip: Effects of the alcohol moiety of substrate on the enzyme activity

6-Tuliposides A (PosA) and B (PosB) are glucose esters accumulated in tulip (Tulipa gesneriana) as major defensive secondary metabolites. Pos-converting enzymes (TgTCEs), which we discovered previously from tulip, catalyze the conversion reactions of PosA and PosB to antimicrobial tulipalins A (PaA) and B (PaB), respectively. The TgTCEs, belonging to the carboxylesterase family, specifically catalyze intramolecular transesterification, but not hydrolysis. In this report, we synthesized analogues of Pos with various alcohol moieties, and measured the TgTCE activity together with a determination of the kinetic parameters for these analogues with a view to probe the substrate recognition mechanism of the unique non-ester-hydrolyzing TgTCEs. It was found that d-glucose-like structure and number of the hydroxyl group in alcohol moiety are important for substrate recognition by TgTCEs. Among the analogues examined, 1,2-dideoxy analogues of PosA and PosB were found to be recognized by the TgTCEs more specifically than the authentic substrates by lowering K_m values. The present results will provide a basis for designing simple, stable synthetic substrate analogues for crystallographic analysis of TgTCEs.     

A novel proapoptotic gene PANO encodes a post-translational modulator of the tumor suppressor p14ARF

The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene.     

Growth and nitrogen use in Xanthium canadense grown in an open or in a dense stand

Plants develop branches profusely when grown solitarily, while less so when grown in a dense stand. Such changes in architecture are associated with changes in dry mass allocation and nitrogen use. Here, we studied what traits in plant growth and nitrogen use were influenced by different light climates in the stand. Annual plants (Xanthium canadense) were grown solitarily or in a dense stand. Dry mass growth was analyzed as the product of the net assimilation rate (NAR) and leaf area (LA). Nitrogen use efficiency (NUE) was analyzed as the product of nitrogen productivity (NP) and the mean residence time (MRT) of nitrogen. These growth variables were further factorized into their components. Solitary plants maintained a high NAR, whereas plants in the dense stand decreased the NAR due to mutual shading. Plants in the dense stand developed a larger LA with a higher specific leaf area than solitary plants. Solitary plants had higher NUE due to higher NP. A temporal increase in NUE was attributed to the increase in MRT of nitrogen. Light climate was different between solitary and dense-stand plants, but they took up a comparable amount of nitrogen and used it differently in response to the given light climate. NUE was thus demonstrated to be a useful tool for analyzing the mechanism leading to different N use in plant growth.     

Relaxation-expansion model for self-driven retinal morphogenesis: a hypothesis from the perspective of biosystems dynamics at the multi-cellular level

The generation of complex organ structures such as the eye requires the intricate orchestration of multiple cellular interactions. In this paper, early retinal development is discussed with respect to the structure formation of the optic cup. Although recent studies have elucidated molecular mechanisms of retinal differentiation, little is known about how the unique shape of the optic cup is determined. A recent report has demonstrated that optic-cup morphogenesis spontaneously occurs in three-dimensional stem-cell culture without external forces, indicating a latent intrinsic order to generate the structure. Based on this self-organizing phenomenon, we introduce the "relaxation-expansion" model to mechanically interpret the tissue dynamics that enable the spontaneous invagination of the neural retina. This model involves three consecutive local rules (relaxation, apical constriction, and expansion), and its computer simulation recapitulates the optic-cup morphogenesis in silico.     

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.     

Inputting information about amino acid residues in protein bioinformatics: a case study on predicting helical regions with a neural network

The best way to introduce information about amino acid residues into calculations of protein bioinformatics was examined. That was done for predicting helical regions with a neural network. Several fundamental and instructive ways for information processing were developed and are described.