Spatial pattern dynamics over 10 years in a conifer/broadleaved forest,northern Japan

The present study investigated stand dynamics during 10-year period in a conifer/broadleaved mixed forest in Hokkaido, northern Japan, focusing on spatially dependent recruitment, mortality and growth of two growth-form groups, deciduous broadleaved species and the dominant evergreen conifer Abies sachalinensis. The stand-level basal area was maintained over the 10-year period, while a compositional equilibrium at the individual species level was not confirmed. Univariate and bivariate spatial analyses revealed clustering of many of the constituent species. The absence of single-species patches suggested an ambiguous mosaic formed by co-occurrence of Abies and broadleaved trees. The trend towards an aggregated distribution of Abies and broadleaved trees was caused by spatially dependent recruitment rather than mortality. New recruits of broadleaved species were spatially associated with surviving broadleaved trees, while this was not the case for Abies. The degree of competitive effects on growth was not consistent over the 10-year period. Abies showed between-groups competition, but not within-group competition. In contrast, we found asymmetric competitions between the broadleaved trees. Our results suggest that Abies is not sufficiently competitively dominant to exclude broadleaved trees, and that the co-occurrence of the two growth-form groups might be maintained.     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.     

Prediction of subcellular locations of proteins: where to proceed?

Since the proposal of the signal hypothesis on protein subcellular sorting, a number of computational analyses have been performed in this field. A typical example is the development of prediction algorithms for the subcellular localization sites of input protein sequences. In this review, we mainly focus on the biological grounds of the prediction methods rather than the algorithmic issues because we believe the former will be more fruitful for future development. Recent advances on the study of protein sorting signals will hopefully be incorporated into future prediction methods. Unfortunately, many of the state-of-the-art methods are published without sufficient objective tests. In fact, a simple test employed in this article shows that the performance of specifically developed predictors is not significantly better than that of a homology search. We suspect that this is a general problem associated with the interpretation of genome sequences, which have evolved through gene duplication and speciation.     

Determination of folates by HPLC‐chemiluminescence using a ruthenium(II)–cerium(IV) system,and its application to pharmaceutical preparations and supplements

A chemiluminescence (CL) reaction of folic acid (FA) with ruthenium (II) and cerium (IV) was applied to quantify FA‐related compounds such as FA, dihydrofolic acid, tetrahydrofolic acid, 5‐methyltetrahydrofolic acid, 5‐formyltetrahydrofolic acid and methotrexate (MTX). Among the FAs, 5‐methyltetrahydrofolic acid provided the highest CL intensity. HPLC‐CL detection of FA was applied to quantify FA in pharmaceutical preparations and supplements. Analytical samples were separated on a semi‐micro ODS column with a mixture of 20 mM phosphate buffer (pH 5.7) and acetonitrile (94 : 6, v/v %). The separated samples were mixed with a post‐column CL reagent consisting of 1.5 mM Ru(bipy)₃²⁺ and 1.0 mM Ce(SO₄)₂, then the generated CL was monitored. The calibration range for FA was 10–100 μM and the limit of detection was 1.34 μM (signal‐to‐noise ratio of 3). Repeatabilities were 4.2, 4.6 and 5.0 RSD% (10, 25, 50 μM), and the recoveries for FA supplement, vitamin B complex supplement and FA‐containing medication (tablet) were 102.4 ± 10.5, 103.3 ± 13.3 and 100.3 ± 8.5%, respectively. The described method is robust against changes in the chromatographic parameters of ± 3.3 or ± 1.5%. The measured FA content corresponded well to the labeled content of FA‐containing products (100.6–104.9%), demonstrating the precision and accuracy of this method for the evaluation of FA pharmaceutical preparations. Copyright © 2014 John Wiley & Sons, Ltd.     

Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

Background and purpose

Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.

Experimental approach

For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.

Key results

The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC₅₀ of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.

Conclusions and implications

Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.     

Molecular dynamics simulation of structural changes of lipid bilayers induced by shock waves: Effects of incident angles

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus.     

PASK (proline-alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization

Proline–alanine-rich Ste20-related kinase (PASK, also referred to as SPAK) has been linked to ion transport regulation. Here, we report two novel activities of PASK: binding to tubulin and microtubules and the promotion of microtubule assembly. Tubulin binding assay showed that full-length PASK and its kinase domain bound to purified tubulin whereas the N-terminal or C-terminal non-catalytic domains of PASK did not. The full-length PASK and its kinase domain were sedimented with paclitaxel-stabilized microtubules by ultracentrifugation. These results indicate that the kinase domain of PASK can interact directly with both microtubules and soluble tubulin in vitro. Truncated PASK lacking the N-terminal non-catalytic domain promoted microtubule assembly at a subcritical concentration of purified tubulin. FLAG–PASK expressed in COS-7 cells translocated to the cytoskeleton when the cells were stimulated with hypertonic sodium chloride, and stabilized microtubules against depolymerization by nocodazole. Our findings suggest that PASK may regulate the cytoskeleton by modulating microtubule stability.     

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.     

Hemolytic and hemagglutinating activities of Prevotella intermedia and Prevotella nigrescens

A total of 91 isolates of Prevotella intermedia or Prevotella nigrescens from subgingival sites were identified by PCR using primers specific for sequences of 16S rRNA. The hemolytic and hemagglutinating activities of the P. intermedia isolates exhibited significantly higher levels compared to those of the P. nigrescens isolates by quantitative analysis. The hemagglutinin gene (phg) was found in 23 of 26 P. intermedia isolates (88.5%), whereas it was found in only two of 44 isolates (4.5%) of P. nigrescens. The high hemolytic and hemagglutinating activities of P. intermedia may be involved in the pathogenicity of P. intermedia in the progression of periodontal disease.     

Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development.

We have isolated chick rax/rx cDNAs, cRaxL (chick Rax/Rx-like) and cRax, (chick Rax) and examined their expression patterns during early eye and brain development. The cRaxL cDNA encodes a 228 amino acid protein that is most closely related to the zebrafish Rx1 and Rx2. The cRax cDNA encodes a 317 amino acid protein, which shares higher homology with the Xenopus Rx. In addition to the homeodomain, the octapeptide and paired tail domains are conserved between the cRax and other vertebrate Rax/Rx, while cRaxL lacks the octapeptide containing N-terminal region which is conserved among all other members of the rax/rx gene family identified so far. The chick rax/rx genes are expressed in overlapping domains in the anterior neural ectoderm which corresponds to the forebrain and retina field, and later in the optic vesicle. cRax mRNA can be detected earlier than cRaxL prior to the formation of the notochord and its expression domain appears broader than that of cRaxL.