Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca2+-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca2+ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.
Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.
We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.
We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.
Mid1 is a putative stretch-activated Ca2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype. To identify a protein regulating Mid1 or regulated by Mid1, we isolated a multicopy suppressor that rescues the mid1-1 mutant from mating pheromone-induced death and found that it encodes a truncated Spa2 protein lacking an amino-terminal region responsible for interaction with components of the mitogen-activated protein kinase cascades. One of these SPA2 alleles was SPA2ΔN, whose product lacked the region from Ser5 to Leu230. SPA2ΔN on a multicopy plasmid (YEpSPA2ΔN) complemented the mid phenotype but not another phenotype, low Ca2+ accumulation, of the mid1-1 mutant. Neither SPA2ΔN on a low-copy plasmid nor wild-type SPA2 on a multicopy plasmid had suppressive activity. The SPA2 gene is involved in the formation of a pointed mating projection, and cells of the spa2Δ mutant lacking Spa2 are viable and develop a peanut shell-like structure when exposed to mating pheromone. Like the spa2Δ mutant, the mid1-1 spa2Δ double mutant and the mid1-1/YEpSPA2ΔN strain developed the peanut shell-like structure. The mid1-1 spa2Δ double mutant did not have the mid phenotype, indicating that SPA2 is epistatic to MID1. Overexpression of Spa2ΔN abolished the localization of Spa2-green fluorescent protein to the tip of the mating projection. These results suggest that the Spa2ΔN protein interferes with the localization of the normal Spa2 protein and thereby prevents cells from entering the mating process. Therefore, we suggest that Mid1 function is influenced by Spa2 function through polarized morphogenesis.
Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation.
Four Psychrobacter strains, JCM 18900, JCM 18901, JCM 18902, and JCM 18903, related to either Psychrobacter nivimaris or Psychrobacter cibarius, were isolated from frozen marine animals. The genome information of these four strains will be useful for studies of their physiology and adaptation properties to frozen conditions.
Cytophaga fermentans strain JCM 21142T is a marine-dwelling facultative anaerobe. The draft genome sequence of this strain revealed its diverse chemoorganotrophic potential, which makes it capable of metabolizing various polysaccharide substrates. The genome data will facilitate further studies on its taxonomic reclassification, its metabolism, and the mechanisms pertaining to bacterial gliding.
Here, we report the draft genome sequences of two type strains of Lactobacillus, Lactobacillus farraginis JCM 14108T and Lactobacillus composti JCM 14202T, isolated from the compost of distilled shōchū residue. Their genome information will be useful for studies of ecological and physiological functions of these Lactobacillus species.
Inflammation and angiogenesis are important contributors to vascular disease. We evaluated imaging both of these biological processes, using Arg–Gly–Asp (RGD)-conjugated human ferritin nanoparticles (HFn), in experimental carotid and abdominal aortic aneurysm (AAA) disease.
Macrophage-rich carotid lesions were induced by ligation in hyperlipidemic and diabetic FVB mice (n=16). AAAs were induced by angiotensin II infusion in apoE−/− mice (n=10). HFn, with or without RGD peptide, was labeled with Cy5.5 and injected intravenously for near-infrared fluorescence imaging.
RGD-HFn showed significantly higher signal than HFn in diseased carotids and AAAs relative to non-diseased regions, both in situ (carotid: 1.88±0.30 vs. 1.17±0.10, p=0.04; AAA: 2.59±0.24 vs. 1.82±0.16, p=0.03) and ex vivo. Histology showed RGD-HFn colocalized with macrophages in carotids and both macrophages and neoangiogenesis in AAA lesions.
RGD-HFn enhances vascular molecular imaging by targeting both vascular inflammation and angiogenesis, and allows more comprehensive detection of high-risk atherosclerotic and aneurysmal vascular diseases.
Vascular disease; Nanoparticles; Inflammation; Angiogenesis; Atherosclerosis; Aneurysms; RGD; Ferritin
Paenibacillus pini strain JCM 16418T is a cellulolytic bacterium isolated from the rhizosphere of pine trees. Here, we report the draft genome sequence of this strain. This genome information will be useful for studies of rhizosphere bacteria.
Bacillus strains JCM 19045, JCM 19046, and JCM 19047 are alkaliphiles that produce β-cyclodextrin from starch. They are related to Bacillus xiaoxiensis and Bacillus lehensis. The genome information for these three strains will be useful for studies of the physiological role of cyclodextrin and cyclodextrin production.
Prognosis of osteosarcoma (OS) with distant metastasis and local recurrence is still poor. Y-box binding protein-1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and translation and its high expression of YB-1 protein was observed in OS, however, the role of YB-1 in OS remains unclear.
Y-box binding protein-1 expression in OS cells was inhibited by specific small interfering RNAs to YB-1 (si-YB-1). The effects of si-YB-1 in cell proliferation and cell cycle transition in OS cells were analysed in vitro and in vivo. The association of nuclear expression of YB-1 and clinical prognosis was also investigated by immunohistochemistry.
Proliferation of OS cell was suppressed by si-YB-1 in vivo and in vitro. The expression of cyclin D1 and cyclin A were also decreased by si-YB-1. In addition, si-YB-1 induced G1/S arrest with decreased cyclin D1 and cyclin A in OS cell lines. Direct binding of YB-1 in OS cell lines was also observed. Finally, the nuclear expression of YB-1 was significantly related to the poorer overall survival in OS patients.
Y-box binding protein-1 would regulate cell cycle progression at G1/S and tumour growth in human OS cells in vitro and in vivo. Nuclear expression of YB-1 was closely associated with the prognosis of OS, thus, YB-1 simultaneously could be a potent molecular target and prognostic biomarker for OS.
osteosarcoma (OS); Y-box binding protein-1 (YB-1); cell proliferation; atelocollagen
Most epithelial tissues retain stem/progenitor cells to maintain homeostasis of the adult tissues; however, the existence of a thymic epithelial cell (TEC) progenitor capable of maintaining homeostasis of the postnatal thymus remains unclear. Here, we show that a cell population expressing high levels of Meis1, a homeodomain transcription factor, is enriched in TECs with an immature cellular phenotype. These TECs selectively express genes involved in embryonic thymic organogenesis and epithelial stem cell maintenance, and also have the potential to proliferate and differentiate into mature TEC populations. Furthermore, postnatal inactivation of Meis1 in TECs caused disorganization of the thymic architecture, which ultimately leads to premature disappearance of the thymus. There was an age-associated reduction in the proportion of the TEC population expressing high levels of Meis1, which may also be related to thymic involution. These findings indicate that Meis1 is potentially involved in the maintenance of postnatal TECs with progenitor activity that is required for homeostasis of the postnatal thymus.
Olprinone decreases the cardiac preload and/or afterload because of its vasodilatory effect and increases myocardial contractility by inhibiting phosphodiesterase III.
The objective of this study was to characterize the population pharmacokinetics of olprinone after a single continuous infusion in healthy male volunteers.
We used 500 plasma concentration data points collected from nine healthy male volunteers for the study. The population pharmacokinetic analysis was performed using the nonlinear mixed effect model (NONMEM®) software.
The time course of plasma concentration of olprinone was best described using a two-compartment model. The final pharmacokinetic parameters were total clearance (7.37 mL/minute/kg), distribution volume of the central compartment (134 mL/kg), intercompartmental clearance (7.75 mL/minute/kg), and distribution volume of the peripheral compartment (275 mL/kg). The interindividual variability in the total clearance was 12.4%, and the residual error variability (exponential and additive) were 22.2% and 0.129 (standard deviation). The final pharmacokinetic model was assessed using a bootstrap method and visual predictive check.
We developed a population pharmacokinetic model of olprinone in healthy male adults. The bootstrap method and visual predictive check showed that this model was appropriate. Our results might be used to develop the population pharmacokinetic model in patients.
phosphodiesterase III inhibitor; men; pharmacokinetic model
Background and Purpose
EGFR-inhibitor Cetuximab (C225) improves the efficacy of radiotherapy in only a subgroup of HNSCC patients. Identification of predictive tumor characteristics is essential to improve patient selection.
Material and Methods
Response to C225 and/or radiotherapy was assessed with tumor growth delay assays in 4 HNSCC xenograft models with varying EGFR-expression levels. Hypoxia and proliferation was quantified with immunohistochemistry and expression of proteins involved in C225-resistance with western blot.
EGFR-expression did not predict response to C225 and/or radiotherapy. Reduction of hypoxia by C225 was only observed in SCCNij202, which was highly sensitive to C225. Proliferation changes correlated with response to C225 and C225 combined with radiotherapy, as proliferation decreased after C225 treatment in C225-sensitive SCCNij202 and after combined treatment in SCCNij185, which showed a synergistic effect to combined C225-radiotherapy. Furthermore, C225-resistant SCCNij153 tumors expressed high levels of (activated) HER3 and MET.
EGFR-expression is needed for C225-response, but is not sufficient to predict response to C225 with or without radiotherapy. However, basal expression of additional growth factor receptors and effects on proliferation, but not hypoxia, correlated with response to combined C225-radiotherapy treatment and are potential clinically relevant predictive biomarkers.
head and neck cancer; EGFR-inhibition; tumor microenvironment; radiotherapy; tyrosine kinase receptors
Recent reports on Propionibacterium acnes (P. acnes) suggest that this bacterium is prevalent in the prostate, is associated with acute and chronic prostatic inflammation, and might have a role in prostate carcinogenesis.
To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in radical prostatectomy specimens using enzyme immunohistochemistry with a novel P. acnes-specific monoclonal antibody (PAL antibody), together with an anti-nuclear factor-kappa B (NF-κB) antibody. We examined formalin-fixed and paraffin-embedded tissue sections of radical prostatectomy specimens from 28 patients with prostate cancer and 18 age-matched control patients with bladder cancer, but without prostate cancer.
Immunohistochemistry with the PAL antibody revealed small round bodies within some non-cancerous glandular epithelium and stromal macrophages in most prostate samples. Prostate cancer samples had higher frequencies of either cytoplasmic P. acnes or nuclear NF-κB expression of glandular epithelium and higher numbers of stromal macrophages with P. acnes than control samples. These parameters were also higher in the peripheral zone than in the transitional zone of the prostate, especially in prostate cancer samples. Nuclear NF-κB expression was more frequent in glands with P. acnes than in glands without P. acnes. The number of stromal macrophages with the bacterium correlated with the grade of chronic inflammation in both the PZ and TZ areas and with the grade of acute inflammation in the TZ area.
Immunohistochemical analysis with a novel monoclonal antibody for detecting P. acnes in the prostate suggested that intraepithelial P. acnes infection in non-cancerous prostate glands and inflammation caused by the bacterium may contribute to the development of prostate cancer.
Since the late 1990s, patient safety has been an important policy issue in developed countries. To evaluate the effectiveness of the activities of patient safety, it is necessary to quantitatively assess the incidence of adverse events by types of failure mode using tangible data. The purpose of this study is to calculate patient safety indicators (PSIs) using the Japanese Diagnosis Procedure Combination/per-diem payment system (DPC/PDPS) reimbursement data and to elucidate the relationship between perioperative PSIs and hospital surgical volume.
DPC/PDPS data of the Medi-Target project managed by the All Japan Hospital Association were used. An observational study was conducted where PSIs were calculated using an algorithm proposed by the US Agency for Healthcare Research and Quality. We analyzed data of 1,383,872 patients from 188 hospitals who were discharged from January 2008 to December 2010.
Among 20 provider level PSIs, four PSIs (three perioperative PSIs and decubitus ulcer) and mortality rates of postoperative patients were related to surgical volume. Low-volume hospitals (less than 33rd percentiles surgical volume per month) had higher mortality rates (5.7%, 95% confidence interval (CI), 3.9% to 7.4%) than mid- (2.9%, 95% CI, 2.6% to 3.3%) or high-volume hospitals (2.7%, 95% CI, 2.5% to 2.9%). Low-volume hospitals had more deaths among surgical inpatients with serious treatable complications (38.5%, 95% CI, 33.7% to 43.2%) than high-volume hospitals (21.4%, 95% CI, 19.0% to 23.9%). Also Low-volume hospitals had lower proportion of difficult surgeries (54.9%, 95% CI, 50.1% to 59.8%) compared with high-volume hospitals (63.4%, 95% CI, 62.3% to 64.6%). In low-volume hospitals, limited experience may have led to insufficient care for postoperative complications.
We demonstrated that PSIs can be calculated using DPC/PDPS data and perioperative PSIs were related to hospital surgical volume. Further investigations focusing on identifying risk factors for poor PSIs and effective support to these hospitals are needed.
Patient safety indicators; Perioperative care; Observational study; Hospital surgical volume
Oxidative stress and smoking contribute to endothelial dysfunction. Iron might also play a role in oxidative stress generation and endothelial dysfunction. However, the involvement of iron in smoking-induced endothelial dysfunction in healthy smokers remains unclear. Therefore, we examined here whether (1) intravenous iron infusion impaired endothelial function evaluated by flow-mediated vasodilatation (FMD) in non-smokers, and (2) deferoxamine, a potent iron chelator, ameliorated endothelial dysfunction in healthy smokers.
Eight healthy young male non-smokers (23±4 years old) received intravenous injection of saccharated ferric oxide (0.7 mg/kg body weight), while 10 age-matched healthy male smokers received deferoxamine mesylate (8.3 mg/kg body weight). At baseline, 5 and 20 minutes after treatment with iron or deferoxamine, biochemical variables were measured, including serum iron and marondialdehyde (MDA), a marker of lipid oxidation, and endothelial function was simultaneously evaluated by FMD.
Compared with non-smokers, FMD was significantly lower in smokers. Iron and MDA levels were significantly increased, whereas FMD was impaired by iron infusion in non-smokers. Conversely, deferoxamine treatment significantly decreased iron and MDA levels and restored the decreased FMD in smokers. Baseline serum iron and MDA levels in all 18 subjects (non-smokers and smokers) were correlated with each other. There was a significant inverse correlation between the changes in MDA values and FMD from baseline in 18 men. Endothelium-independent vasodilation by glyceryl trinitrate was unaltered by either treatment.
Our present study suggests that iron-evoked oxidative stress might play a role in endothelial dysfunction in healthy smokers.
Gracilibacillus boraciitolerans JCM 21714T has been characterized as a highly boron-tolerant and moderately halotolerant bacterium. Here, we report the draft genome sequence of this strain. The genome sequence facilitates an understanding of the biochemical functions of boron and provides a base to identify the gene(s) involved in the boron tolerance mechanism of the strain.
Here, we report the draft genome sequence of a fibrolytic bacterium, Clostridium straminisolvens JCM 21531T, isolated from a cellulose-degrading bacterial community. The genome information of this strain will be useful for studies on the degradation enzymes and functional interactions with other members in the community.
CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.
Here we report the draft genome sequence of Bacteroides reticulotermitis strain JCM 10512T, a xylanolytic and cellulolytic bacterium isolated from the gut of a wood-feeding termite. The genome information will facilitate the study of this strain for biomass degradation and adaptation to the gut environment.
Here, we report the draft genome sequences of Bacteroides pyogenes JCM 6294T, JCM 6292, and JCM 10003, which were isolated from a cat and swine and were recently classified into a single species, B. pyogenes. Comparative analyses of these genomes revealed the diversification of B. pyogenes strains isolated from different animals.
Here, we report the draft genome sequences of the type strains of three cellulolytic or hemicellulolytic alkaliphilic Bacillus species: Bacillus wakoensis, Bacillus akibai, and Bacillus hemicellulosilyticus. The genome information for these three strains will be useful for studies of alkaliphilic Bacillus species, their evolution, and biotechnological applications for their enzymes.
HER3 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In the present study, we investigated the capacity of the HER3 blocking antibody, U3-1287/AMG888, to modulate the in vitro and in vivo radiation response of human squamous cell carcinomas of the lung and head and neck. We screened a battery of cell lines from these tumors for HER3 expression and demonstrated that all cell lines screened exhibited expression of HER3. Importantly, U3-1287/AMG888 treatment could block both basal HER3 activity and radiation induced HER3 activation. Proliferation assays indicated that HER3 blockade could decrease the proliferation of both HNSCC cell line SCC6 and NSCLC cell line H226. Further, we demonstrated that U3-1287/AMG888 can sensitize cells to radiation in clonogenic survival assays, in addition to increasing DNA damage as detected via λ-H2AX immunofluo-rescence. To determine if U3-1287/AMG888 could enhance radiation sensitivity in vivo we performed tumor growth delay experiments using SCC6, SCC1483, and H226 xenografts. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation could decrease tumor growth in studies using single or fractionated doses of radiation. Analysis of HER3 expression in tumor samples indicated that radiation treatment activated HER3 in vivo and that U3-1287/AMG888 could abrogate this activation. Immunohistochemistry analysis of SCC6 tumors treated with both U3-1287/AMG888 and a single dose of radiation demonstrated that various cell survival and proliferation markers could be reduced. Collectively our findings suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by increasing DNA damage and cell death. These findings suggest that HER3 may play an important role in response to radiation therapy and blocking its activity in combination with radiation may be of therapeutic benefit in human tumors.