Bisleuconothine A,an eburnane–aspidosperma bisindole alkaloid from Leuconotis griffithii

A new bisindole alkaloid, bisleuconothine A (1) consisting of an eburnane–aspidosperma type skeleton, was isolated from the bark of Leuconotis griffithii. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and X-ray analysis. Bisleuconothine A (1) showed cell growth inhibitory activity against various human cancer cell lines.     

The evolution of the regulatory mechanism of chloroplast division

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division, reminiscent of their free-living ancestor. However, chloroplasts can not divide by themselves, and the division is performed and controlled by proteins that are encoded by the host nucleus. The continuity of chloroplasts was originally established by synchronization of endosymbiotic cell division with host cell division, as seen in existent algae. In contrast, land plant cells contain multiple chloroplasts, the division of which is not synchronized, even in the same cell. Land plants have evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts (or other types of plastids) change along with their respective cellular function by changes in the division rate. We recently reported that PLASTID DIVISION (PDV) proteins, land-plant specific components of the chloroplast division apparatus, determined the rate of chloroplast division. The level of PDV protein is regulated by the cell differentiation program based on cytokinin, and the increase or decrease of the PDV level gives rise to an increase or decrease in the chloroplast division rate. Thus, the integration of PDV proteins into the chloroplast division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.Key words: chloroplast division, cell cycle, cell differentiation, cytokinin, endosymbiosis, evolution     

Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270) was obtained using sequence-based screening. From {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.Prolyl aminopeptidase (PAP) (EC 3.4.11.5), belonging to the S33 family, is an exopeptidase that catalyzes the hydrolysis of the N terminus prolyl residue of peptides or proteins. This family has catalytic Ser. To date, few applications of this enzyme for peptide synthesis have been reported. However, from the perspective of biotechnology, PAP might be a good tool for synthesizing proline-containing peptides by catalyzing aminolysis.Recently nutraceutical properties of peptides containing proline have received increasing attention. For example, prolyl hydroxyproline (Pro-Hyp) stimulates the growth of fibroblasts from mouse skin (11). Pro-Arg can protect against oxidative stress/damage and H₂O₂-induced human diploid fibroblast cell death (13). Furthermore, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro exhibit angiotensin I-converting enzyme-inhibiting activity (9). In addition to these dipeptides and tripeptides, a cyclic dipeptide (namely, diketopiperazine) containing proline shows several physiological functions. Cyclo[Pro-Pro] (cPP) exerts antibacterial activity against Micrococcus luteus and Pseudomonas aeruginosa (8). Caspase-3 activation by cyclo[Pro-Phe] in HT-29 cells has been described (3). However, its synthesis method has not been established. Enzymatic peptide synthesis presents a useful and desirable strategy because it can conduct specific reactions under milder conditions than those of chemical synthesis.Engineered endoserine proteases that have Cys substituted for catalytic Ser have also been applied for peptide synthesis since subtiligase was constructed by Abrahmsén et al. (1). Because of the weakened hydrolytic activity of the parent enzyme, it is considered that Ser/Cys-substituted protease can trap the substrate (acyl donor). Then, a nucleophilic reaction occurs between another substrate (acyl acceptor) and the trapped acyl donor (2). This is a so-called “aminolysis” reaction. Although aminolysis can conduct peptide synthesis in an aqueous solution, the problem of the necessity of using an N-protected amino acid as an acyl donor remains when using endoproteases.These problems would be solved using exoprotease as a catalyst, because N-terminal free amino groups of acyl donors are recognized by enzymes. It is rarely reported that exoprotease was applied for peptide synthesis, except in the report of Oshiro et al., in which Pro-Phe, Pro-Tyr, and Pro-Trp were synthesized (10). Recently our group reported that the Ser/Cys variant of exoprotease, aminolysin-S, has been constructed and has produced l-Phe-l-Phe ethyl ester and their derivatives from non-N-protected phenylalanine and phenylalanine ethyl ester as acyl donors in aqueous solution (12). However, aminolysin-S cannot produce proline-containing dipeptides.In this study, we describe a PAP from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270). Furthermore, synthesis of various proline peptides was attempted through catalysis by its Ser/Cys variant (scPAP14270) from proline ester and several amino acids and their esters in aqueous solution. A basic characterization to determine the effect of pH and the amount of substrate was conducted. Moreover, correlation was found between proline peptide synthesis and the log D value, which is the distribution coefficient between octanol and water, of acyl acceptors in aminolysis mediated by scPAP14270.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.     

The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria

Background  

Reminiscent of their free-living cyanobacterial ancestor, chloroplasts proliferate by division coupled with the partition of nucleoids (DNA-protein complexes). Division of the chloroplast envelope membrane is performed by constriction of the ring structures at the division site. During division, nucleoids also change their shape and are distributed essentially equally to the daughter chloroplasts. Although several components of the envelope division machinery have been identified and characterized, little is known about the molecular components/mechanisms underlying the change of the nucleoid structure.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

Effect of gap junctional intercellular communication on radiation responses in neoplastic human cells

Gap junctional intercellular communication (GJIC) is an important function of metazoan cells and is believed to have beneficial effects in anti-tumor therapy. In this study, we found that, when neoplastic human salivary gland (HSG) cells were irradiated with a 100 keV/microm carbon-ion beam, micronuclei, G(2)/M-phase arrest, and cell killing were induced and that their induction increased with dose. Treatment of confluent HSG cells with 8-Br-cAMP increased GJIC between cells. After release from this treatment, the cell cycle progress and the formation of binucleated cells were still similar to those of untreated cells. However, radiation-induced cellular damage, including micronucleus (MN) formation and G(2)/M-phase arrest of that cAMP-treated population, was less than that of the untreated population and that the surviving fraction was slightly enhanced by cAMP treatment, suggesting that increased GJIC protects HSG cells from lethal radiation damage. Moreover, when confluent HSG cells were treated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of nitric oxide (NO) free radical, MN induction and cell killing in the irradiated population were increased. Our results indicate that NO may be involved in GJIC-mediated radioprotection of HSG cells, which may have implications for radiotherapy.     

Neuroprotective role of bradykinin because of the attenuation of pro-inflammatory cytokine release from activated microglia

Bradykinin (BK) has been reported to be a mediator of brain damage in acute insults. Receptors for BK have been identified on microglia, the pathologic sensors of the brain. Here, we report that BK attenuated lipopolysaccharide (LPS)-induced release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta from microglial cells, thus acting as an anti-inflammatory mediator in the brain. This effect was mimicked by raising intracellular cAMP or stimulating the prostanoid receptors EP2 and EP4, while it was abolished by a cAMP antagonist, a prostanoid receptor antagonist, or by an inhibitor of the inducible cyclooxygenase (cyclooxygenase-2). BK also enhanced formation of prostaglandin E(2) and expression of microsomal prostaglandin E synthase. Expression of BK receptors and EP2/EP4 receptors were also enhanced. Using physiological techniques, we identified functional BK receptors not only in culture, but also in microglia from acute brain slices. BK reduced LPS-induced neuronal death in neuron-microglia co-cultures. This was probably mediated via microglia as it did not affect TNF-alpha-induced neuronal death in pure neuronal cultures. Our data imply that BK has anti-inflammatory and neuroprotective effects in the central nervous system by modulating microglial function.