Activation of p38 MAPKalpha by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)- differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPK small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 ± 20.5% in DMSO-treated THP-1 macrophages and by 20.9 ± 9% in primary monocytes (P < 0.05 each). SB-203580 (20 µM) reduced basal p38 MAPK phosphorylation by 34.7 ± 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK siRNA reduced total p38 MAPK protein by 50–60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPK siRNA reduced total FAK protein by 40–50%, and total ERK by 10–15%, but increased phosphorylated ERK 1.4 ± 0.1-fold. p38 MAPK siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAK-dependent inverse effect on p38 MAPK that might subsequently downregulate ERK. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction     

Three new flavonoids,proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana

Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1–3, and also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography method for simultaneous analysis of phenolic constituents was established and then used to investigate the phenolic profiles of the parts of the plant species, to show the presence of characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits, flower buds, and leaves. The branch extract profile also suggested the presence of alkylated ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom tyrosinase were seen, although in some cases this may have resulted from direct interaction with the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.     

Enhancement of Enantioselectivity in the Bacillus subtilis Protease-catalyzed Hydrolysis of N-free Amino Acid Esters using the Ester Grouping-modification Approach

The generality of enantioselectivity enhancement through the modification of the alcohol moiety of a substrate ester was ascertained, for in the Bacillus subtilis protease-catalyzed hydrolysis of N-unprotected amino acid esters the enantioselectivity was enhanced largely by switching the conventional methyl ester to esters with a longer alkyl chain such as the isobutyl ester (from E = 3 to E = 130–170 in the case of 4-fluorophenylalanine esters) as in the enzymatic hydrolysis mediated by Aspergillus oryzae protease. There was indeed a profound dependence of E on the nature of the ester grouping.     

Capsaicinol: synthesis by allylic oxidation and its effect on TRPV1-expressing cells and adrenaline secretion in rats

Capsaicinol is an ingredient of hot red pepper. In this study, we developed a novel method for capsaicinol synthesis and examined capsaicinol's physiological effects on capsaicin receptor (TRPV1)-related actions. Allylic oxidation of capsaicin by palladium acetate (Pd(OAc)(2)) resulted in the formation of (+/-)-capsaicinol acetate at a 7.2% yield in a single step. The effectiveness of (+/-)-capsaicinol in TRPV1 activation (EC(50)=1.1 microM) was found to be weaker than that of capsaicin (EC(50)=0.017 microM), whereas the efficacy of (+/-)-capsaicinol reached 75% of that of capsaicin. Intravenous administration of (+/-)-capsaicinol in anesthetized rats dose-dependently enhanced adrenaline secretion from the adrenal gland. The response to a 5 mg/kg-dose of (+/-)-capsaicinol was comparable to that of a 0.05 mg/kg-dose of capsaicin. The relative pungency of capsaicinol to capsaicin was coincident with the relative effectiveness in inducing these TRPV1-related actions.     

Development of visible markers for transgenic plants and their availability for environmental risk assessment

Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes--green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes--for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.     

The significance of multiple mating and male substance transferred to females at mating in the white grub beetle, <Emphasis Type="Italic">Dasylepida ishigakiensis</Emphasis> (Coleoptera: Scarabaeidae)

Males of the white grub beetle, Dasylepida ishigakiensis Niijima et Kinoshita, transfer a large amount of a colloidal substance to females during mating. In this study we investigated the effect of the male substance on the reproductive performance of mated females and the significance of multiple mating of this beetle. Females artificially separated from the males 5 min after the start of mating produced fewer eggs than those separated after 30 min or those that were undisturbed and separated spontaneously, suggesting that the male substance is used as a nutrient for egg production by the females. When females were allowed to mate with 1–4 males, multiple mating had no clear effect on reproductive performance. The amount of male substance stored in the bursa copulatrix (BC) was not significantly increased by a second mating. The functions of multiple mating of this species may be to provide a chance for females to obtain sufficient amounts of male substance when the first male to mate has only small amounts of this substance, and to increase the genetic heterogeneity of the progeny. The presence of a serine proteinase and its possible involvement in the dynamics of the BC contents are reported.     

The white-spotted longicorn beetle, <Emphasis Type="Italic">Anoplophora malasiaca</Emphasis> (Coleoptera: Cerambycidae), with a blueberry as host plant,utilizes host chemicals for male orientation

Volatile chemicals from Citrus and Salix host plants evoke orientation behavior in males of the species Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae). These chemicals are emitted from wounded branches. We hypothesized that when released, these chemicals may indicate the presence of an individual to other conspecifics. Insects that originate from different host plants may use different plant chemicals from their own host to communicate with conspecifics. To further explore this theory, we investigated this communication system in a population of A. malasiaca from a third host plant, blueberry (Vaccinium spp.). Males from a blueberry host (Vaccinium population) were attracted to the odor of wounded Vaccinium branches when released near a female model in the laboratory, as has been observed in males found on Citrus and Salix host plants. The Vaccinium branch extract that was attractive to the males was separated into six fractions, of which two were active. Three active compounds were subsequently identified: β-caryophyllene and sulfur from the hexane fraction, and (E)-phytol in the weakly polar fraction. The latter two active compounds of Vaccinium branches were different from those found in Citrus and Salix.     

Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches.     

MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.     

Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl₂. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.