Extracellular-regulated kinase activation regulates replication of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> intracellularly in primary human monocytes

Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-γ receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1β and tumor necrosis factor-α) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-α release and interleukin-1β production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.     

Phenolic glycosides from Kaempferia parviflora

Three phenolic glycosides were isolated together with two known flavonol glycosides from the H2O-soluble fraction of rhizomes of Kaempferia parviflora. Their structures were determined to be rel-(5aS,10bS)-5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-d-glucopyranoside] (1), its rel-5aS,10bR isomer (2), and (2R,3S,4S)-3-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-d-glucopyranosyl]-3'-O-methyl-ent-epicatechin-(2alpha-->O-->3,4alpha-->4)-(5aS,10bS)-5a,10b-dihydro-1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside] (3). The structures were elucidated on the basis of analyses of chemical and spectroscopic evidence.     

Apoptotic myocytes generate monocyte chemoattractant protein-1 and mediate macrophage recruitment.

The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2-1 mumol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 mumol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 +/- 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 +/- 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.     

Inhibition of recombinant human histidine decarboxylase activity in different strawberry cultivars

Strawberry fruit contains many constituents, some of which have the potential to inhibit histidine decarboxylase (HDC) activity. HDC converts l-histidine to histamine, which is associated with allergic and other biological reactions in the human body. The HDC inhibition levels were different and the component ratios varied by genotype in strawberry. Among the 11 cultivars collected locally in Japan, ‘Tokun’ had an approximately ten times higher inhibition ratio than the lowest cultivar. The reproducibility was confirmed using five cultivars under the same conditions in a glass greenhouse, suggesting that genotypic variation is a major factor of HDC inhibition. The potential inhibitors of HDC might be polyphenols because they showed moderate correlations with HDC inhibition rates. Among the polyphenols, the anthocyanin content possessed a moderate negative correlation. Ascorbic acid, which contributes to the overestimation of total polyphenol, did not independently inhibit HDC activity. These findings will support the identification of potential HDC inhibitors in strawberry and indicated that genotypic differences would make useful probes for inhibitor identification.     

Ground-surface application of pheromones through a mini-dispenser for mating disruption of the white grub beetle <Emphasis Type="Italic">Dasylepida ishigakiensis</Emphasis> (Coleoptera: Scarabaeidae)

The effect of mating disruption by the ground-surface application of 2-cm-long dispensers (mini-dispensers) of 2-butanol against the white grub beetle Dasylepida ishigakiensis Niijima et Kinoshita (Coleoptera: Scarabaeidae) was examined in sugarcane fields on the Miyako Island, Okinawa, Japan. Mating rates and male catches with sex pheromone traps were reduced to a low level comparable to that obtained from a conventional method in which rope dispensers (25 m) were hung at a height of ca. 30 cm along the sugarcane ridges. Both mating rates and male catches were reduced with increasing number of treated mini-dispensers. These results suggest that by using mini-dispensers the amounts of synthetic sex pheromone and plastic resin can be reduced to 1/10–1/5 and 1/6–1/3, respectively, of their conventional application with rope-type dispensers, without impairing the efficiency of mating disruption in this beetle. Furthermore, the amount of labor required for the application of this method is expected to be greatly reduced compared to the rope-type dispenser method.     

A jacalin‐like lectin domain‐containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant–oomycete interactions

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant–pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin‐related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg‐inoculated foxtail millet leaves. SgJRLs consist of a jacalin‐like lectin domain and an N‐terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N‐terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1‐mediated induction of defence‐related genes was suppressed by co‐expression of SG06536‐GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.     

Ketone components in the contact sex pheromone of the white-spotted longicorn beetle, Anoplophora malasiaca, and pheromonal activity of synthetic ketones

Two active fractions were found during the isolation of contact sex pheromone of female elytra of the white‐spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae), in addition to fraction of hydrocarbons that had previously been identified. One fraction was essential to evoke a series of precopulatory behaviors of males toward a glass dummy when coated together with the hydrocarbon blend. The other fraction enhanced this activity when added to the mixture. From the latter synergistic fraction, we isolated five novel compounds and identified them as 10‐heptacosanone, (Z)‐18‐heptacosen‐10‐one, (18Z,21Z)‐heptacosa‐18,21‐dien‐10‐one, (18Z,21Z,24Z)‐heptacosa‐18,21,24‐trien‐10‐one, and 12‐heptacosanone by GC‐MS and NMR analyses. A blend of four of these synthetic ketones, without 12‐heptacosanone, in the ratio and concentration found in female elytra extract (250 : 400 : 1000 : 180 ng FE^?1) showed greater synergistic effect than the natural fraction containing the ketones. This effect was canceled out by further addition of 12‐heptacosanone (100 ng FE^?1), which was still comparable to the effect of the natural ketone fraction.     

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea

Small subunit rRNA gene sequences were identified without cultivation from parabasalid symbionts of termites belonging to the hypermastigid orders Trichonymphida (the genera Hoplonympha, Staurojoenina, Teranympha, and Eucomonympha) and Spirotrichonymphida (Spirotrichonymphella), and from four yet-unidentified parabasalid symbionts of the termite Incisitermes minor. All these new sequences were analyzed by Bayesian, likelihood, and parsimony methods in a broad phylogeny including all identified parabasalid sequences available in databases and some as yet unidentified sequences probably derived from hypermastigids. A salient point of our study focused on hypermastigids was the polyphyly of this class. We also noted a clear dichotomy between Trichonymphida and the other parabasalid taxa. However, this hypermastigid order was apparently polyphyletic, probably reflecting its morphological diversity. Among Trichonymphida, Teranympha (Teranymphidae) grouped together with the members of the family Eucomonymphidae, suggesting that its family status is ambiguous. The monophyletic lineage composed by Spirotrichonymphida exhibited a narrower branching pattern than Trichonymphida. The root of parabasalids was examined but could not be discerned accurately.     

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.     

Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37°C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 ± 1.8 vs. 42.0 ± 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 ± 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 µM), an ERK activation inhibitor, increased basal phagocytosis (26.9 ± 1.8 vs. 31.7 ± 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction