Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity.     

The aphid-tending ant Lasius fuji exhibits reduced aggression toward aphids marked with ant cuticular hydrocarbons

Some aphid species are attended by ants, which protect aphids against enemies, but ants sometimes prey on the aphids they are attending depending on the resource conditions. A previous study indicated that the ant Lasius niger preys less on the aphid individuals that experienced ant attendance than on those that did not. This observation leads to the hypothesis that ants transfer some substances to the aphids they attend and selectively prey on the aphids without the substances. In this study, we focus on cuticular hydrocarbons (CHCs), which are used by ants as nestmate recognition substances, and test whether ants discriminate the aphids on the basis of CHCs. We confirmed that the ant Lasius fuji preyed less on the aphids that were attended by their nestmates than those that were not attended. Glass dummies treated with CHCs from attended aphids were attacked less by ants than those treated with CHCs from non-attended aphids. The CHC profiles of ant attended aphids resembled those of the ants, suggesting that ants’ CHCs are transferred to the aphids’ body surface through ant attendance. These results support the hypothesis that ants “mark” their attended aphids with their CHCs and the CHCs reduce ant predation intensity.     

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages

Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung.     

Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.     

In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh > t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE [(E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.     

Induction of the Na+/Pi cotransport system in the plasma membrane of Chara corallina requires external Na+ and low levels of Pi

It was shown in previous studies that the giant freshwater alga Chara corallina does not control its Na⁺‐dependent Pi uptake by monitoring the internal Pi concentration and it was hypothesized that Chara may instead detect changes in Pi supply from the environment. The present work investigated the conditions that control the induction and inactivation of high affinity Na⁺/Pi influx in Chara. Withdrawal of Pi from the external medium resulted in a gradual increase in the rate of uptake measured immediately after Pi was resupplied. The increase continued for at least 7 d of starvation. In the initial stages, 0·5 or 1 µm Pi were more effective at inducing transport activity than no Pi, suggesting that low levels of Pi are actually required for induction. The high Na⁺‐dependent Pi uptake observed in Pi‐starved cells was inactivated by treatment with as little as 1 µm Pi over 6 d. External Na⁺ plays a major role in controlling the capacity for Na⁺/Pi cotransport activity, and in the absence of Na⁺, both induction and inactivation were either delayed or abolished. Na⁺ starvation stimulated Na⁺ uptake even though there were no measurable changes in the concentrations of Na⁺, or of K⁺ or Pi in either the vacuole or cytoplasm. It was concluded that both substrate (Pi) and driver ion (Na⁺) are required at adequate concentrations for the induction of the cotransporter. In the case of Pi, it was suggested that passive leakage of Pi from the cell into the apoplast is sufficient for this purpose but that supplementation by up to 1 µm Pi is more effective at the earlier stage. A mechanism for sensing the external supply of Pi is proposed.