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Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) is characterized by its high ability of invasiveness and metastasis, namely lacking expression of estrogen receptor (ER), progesterone receptor, and HER2. We previously demonstrated that estrogen responsive finger protein (Efp) plays a tumor-promotive role in ER-positive breast cancer, yet it remains to be addressed whether Efp contributes to TNBC pathophysiology. We here found that Efp mRNA and protein were abundantly expressed in TNBC patient-derived cells and MDA-MB-231 cells. Efp silencing significantly decreased the growth and migration of both TNBC cell models. Cell-cycle profiling showed a decrease in the S phase population upon Efp silencing. Moreover, exogenous Efp expression increased the growth, migration, and the percentages of S phase population of TNBC cells. Transcriptomic analysis in the Efp-silenced TNBC cells identified several candidate Efp targets including cell cycle-related genes CDCA7 and HELLS, whose contribution to cell growth were validated by siRNA-mediated gene silencing. These results suggest that Efp plays a tumor-promotive role in TNBC and can be a potential therapeutic target for the aggressive disease., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Noriko Gotoh reports equipment, drugs, or supplies was provided by Daiichi Sankyo Co Ltd. Noriko Gotoh has patent #2014-244956 Japan pending to Licensee. no activities to declare., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ozenoxacin, a novel non-fluorinated topical quinolone, is used for the treatment of acne vulgaris in Japan. We investigated bactericidal activity and post-antibiotic effect (PAE) of ozenoxacin against Propionibacterium acnes, a major causative bacterium of acne vulgaris. The minimum inhibitory concentrations (MICs) of ozenoxacin against 3 levofloxacin-susceptible strains (MIC of levofloxacin; ≤4 μg/mL) and 3 levofloxacin-resistant strains (MIC of levofloxacin; ≥8 μg/mL) ranged from 0.03 to 0.06 μg/mL and from 0.25 to 0.5 μg/mL, respectively. These MICs of ozenoxacin were almost the same or lower than nadifloxacin and clindamycin. The minimum bactericidal concentrations (MBCs) of ozenoxacin against the levofloxacin-susceptible and -resistant strains were from 0.06 to 8 μg/mL and from 0.5 to 4 μg/mL, respectively. These MBCs were lower than those of nadifloxacin and clindamycin. In time-kill assay, ozenoxacin at 1/4, 1 and 4 times the respective MIC against both levofloxacin-susceptible and -resistant strains showed a concentration-dependent bactericidal activity. Ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains showed more potent and more rapid onset of bactericidal activity compared to nadifloxacin and clindamycin at 4 times the respective MICs. The PAEs of ozenoxacin at 4 times the MICs against the levofloxacin-susceptible strains were from 3.3 to 17.1 h, which were almost the same or longer than nadifloxacin and clindamycin. In contrast, the PAEs were hardly induced by any antimicrobial agents against the levofloxacin-resistant strains. The present findings suggest that ozenoxacin has a potent bactericidal activity against both levofloxacin-susceptible and -resistant P. acnes, and a long-lasting PAE against levofloxacin-susceptible P. acnes., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Benzoyl peroxide (BPO), a therapeutic agent for acne vulgaris, was assessed for in vitro antimicrobial activity against Propionibacterium acnes using a novel broth microdilution testing that improved BPO solubility. We searched for a suitable culture medium to measure the minimum inhibitory concentration (MIC) of BPO against P. acnes and finally found the Gifu anaerobic medium (GAM) broth supplemented with 0.1(v/v)% glycerol and 2(v/v)% Tween 80, in which BPO dissolved up to 1250 μg/mL and P. acnes grew well. The MICs and minimum bactericidal concentrations (MBCs) of BPO against 44 clinical isolates of P. acnes collected from Japanese patients with acne vulgaris were determined by our testing method using the supplemented GAM broth. The MICs of BPO were 128 or 256 μg/mL against all isolates of P. acnes regardless of susceptibility to nadifloxacin or clindamycin. The MBCs of BPO were also 128 or 256 μg/mL against the same isolates. Moreover, BPO at the MIC showed a rapid bactericidal activity against P. acnes ATCC11827 in time-kill assay. In conclusion, we could develop a novel assay for the MIC and MBC determinations of BPO against P. acnes, which is reliable and reproducible as a broth microdilution testing and the present results suggest that BPO has a potent bactericidal activity against P. acnes., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

A type IV pilus filament, mainly composed of PilA, is retracted by the driving power generated by PilT and PilU ATPases. pilA is required for injection of type III ExoS effectors into epithelial cells thereby facilitating Pseudomonas aeruginosa penetration through the epithelial barrier by impairing the defense function of tight junctions. Here, we examined whether the pilT and pilU of the P. aeruginosa PAO1 strain are required for ExoS injection into epithelial cells. We measured the quantity of ExoS injected into epithelial cells, and found that within such cells its quantity decreased by 80% (ΔpilA strain), 75% (ΔpilT strain), and 30% (ΔpilU strain) compared with the wild-type strain. pilT deficiency decreased the disruption of human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers to the same extent as that of pilA and exoS deficiency, whereas pilU deficiency decreased disruption of the monolayers less than deficiency of the other genes. pilT and pilU deficiency decreased bacterial penetration of the Caco-2 cell monolayers to the same level as pilA and exoS deficiency. Our data showed that the pilU gene expression level was reduced in the PAO1 strain after adhesion to Caco-2 cell surfaces, but the expression levels of the pilA and pilT genes did not change. We conclude that P. aeruginosa injects ExoS into cells through the function of type IV pilus retraction, and that pilT makes a greater contribution to this process than pilU., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: Age-related macular degeneration (AMD) is the leading cause of severe visual impairment. Despite treatment, a central scotoma often remains. The size of the scotoma depends on the lesion size of the choroidal neovascular membrane and significantly affects the patient's quality of life, and the lesion size of neovascularization also affects response to treatments. The aim of this study was to identify genes associated with the neovascular lesion size in neovascular AMD., Design: A genome-wide association study (GWAS)., Participants: We included 1146 Japanese patients with neovascular AMD., Methods: We performed a 2-stage GWAS for the lesion size of AMD as a quantitative trait among 1146 (first stage: 727, second stage: 419) Japanese patients with neovascular AMD. Lesion size was determined by the greatest linear dimension measured with fluorescein angiography examination before treatment. We examined the association between the genotypic distribution of each single nucleotide polymorphism (SNP) and the trait using an additive model adjusted for age and sex. To evaluate the associations between AMD development and SNPs associated with lesion size, we also performed a case-control study by using the genotype data from these 1146 Japanese patients as case subjects and the fixed dataset from the Nagahama Study as control subjects., Main Outcome Measures: Genes associated with the lesion size in neovascular AMD., Results: In the discovery stage, rs10895322 in MMP20 showed a genome-wide significant P value of 6.95×10(-8), and rs2284665 in ARMS2/HTRA1 showed a P value of 1.55×10(-7). The associations of these 2 SNPs were successfully replicated in the replication stage, and a meta-analysis of both stages showed genome-wide significant P values (2.80×10(-9) and 4.41×10(-9), respectively). In a case-control study using 3248 Japanese subjects as controls, we could not find contribution of MMP20 rs10895322 for AMD development. Although MMP20 has been thought to be expressed only in dental tissues, we confirmed MMP20 expression in the human retina and retinal pigment epithelium/choroid with polymerase chain reaction., Conclusions: The growth of choroidal neovascularization in AMD would be affected by 2 genes: MMP20, a newly confirmed gene expressed in the retina, and ARMS2/HTRA1, a well-known susceptibility gene for AMD., (Copyright © 2015 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

In this study, supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry was applied to the profiling of several regioisomeric triacylglycerols (TAGs). SFC conditions (column, flow rate, modifier) were optimized for the effective separation of TAGs. In the column test, a triacontyl (C30) silica gel reversed-phase column was selected to separate TAG regioisomers. Multiple reaction monitoring was used to selectively quantify each TAG. Then, the method was used to perform detailed characterization of a diverse array of TAGs in palm and canola oils. Seventy TAGs (C46:0-C60:2) of these oils were successfully analyzed as a result, and twenty isomeric TAG pairs were separated well. In particular, this method provided the fast and high resolution separation of six regioisomeric TAG pairs (PPLn/PLnP, PPL/PLP, PPO/POP, SPLn/SLnP, SPO/SOP, SSO/SOS-stearic acid (S, 18:0), oleic acid (O, 18:1), linoleic acid (L, 18:2), linolenic acid (Ln, 18:3), palmitic acid (P, 16:0)) in a short time (50min) as compared to high performance liquid chromatography. We were able to demonstrate the utility of this method for the analysis of regioisomeric TAGs in edible oils., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A high-density, polymeric C18 stationary phase (Inertsil ODS-P) or a polymeric C30 phase (Inertsil C30) provided improved resolution of the isomeric fatty acids (FAs), FA methyl esters (FAMEs), triacylglycerols (TAGs), and tocopherols with an increase in pressure of 20-70MPa in reversed-phase HPLC. With respect to isomeric C18 FAMEs with one cis-double bond, ODS-P phase was effective for recognizing the position of a double bond among petroselinic (methyl 6Z-octadecenoate), oleic (methyl 9Z-octadecenoate), and cis-vaccenic (methyl 11Z-octadecenoate), especially at high pressure, but the differentiation between oleic and cis-vaccenic was not achieved by C30 phase regardless of the pressure. A monomeric C18 phase (InertSustain C18) was not effective for recognizing the position of the double bond in monounsaturated FAME, while the separation of cis- and trans-isomers was achieved by any of the stationary phases. The ODS-P and C30 phases provided increased separation for TAGs and β- and γ-tocopherols at high pressure. The transfer of FA, FAME, or TAG molecules from the mobile phase to the ODS-P stationary phase was accompanied by large volume reduction (-30∼-90mL/mol) resulting in a large increase in retention (up to 100% for an increase of 50MPa) and improved isomer separation at high pressure. For some isomer pairs, the ODS-P and C30 provided the opposite elution order, and in each case higher pressure improved the separation. The two stationary phases showed selectivity for the isomers having rigid structures, but only the ODS-P was effective for differentiating the position of a double bond in monounsaturated FAMEs. The results indicate that the improved isomer separation was provided by the increased dispersion interactions between the solute and the binding site of the stationary phase at high pressure., (Copyright © 2014 Elsevier B.V. All rights reserved.)

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan., (Copyright © 2013 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. P. aeruginosa secretes a number of toxins by a type III secretion system, and these are important in virulence. One of these toxins, ExoS can induce a cytotoxic effect and is associated with the ability to produce lung damage. ExoS is a bifunctional toxin, with N-terminal GTPase-activating protein (GAP) activity and a C-terminal ADP ribosyl transferase (ADPRT) domain. Although these two domains have numerous potential cellular targets, the overall mechanism of ExoS-induced cytotoxicity remains unclear. We carried out a yeast two-hybrid screen using the ExoS truncation mutant ExoSΔ (residue 1-388), which lacks the 14-3-3 binding site in the ADPRT domain, to identify unknown cellular targets associated with ExoS-induced cytotoxicity. We identified the mammalian factor, kinesin family member 7 (KIF7), which is involved in Hedgehog signaling, as a binding partner for ExoSΔC2. A pull-down assay revealed that ExoS bound to the truncated KIF7 gene encoding the N-terminal domain (residues 1-109) of KIF7. Yeast two-hybrid analysis showed that the ADPRT domain (residues 234-354) of ExoS bound to the truncated KIF7. Furthermore, exoS gene expression and silencing the expression of KIF7 both caused significant cytotoxicity in cultured human bronchial epithelial cells (BEAS-2B). Taken together, our results suggest that ExoS could induce cytotoxicity in BEAS-2B cells by interacting with KIF7., (Copyright © 2013 Japanese Society of Chemotherapy and The Japanese Association for Infectious Disease. Published by Elsevier Ltd. All rights reserved.)

Microglia, the resident immune cells of the central nervous system (CNS), are thought to contribute to the pathogenesis of age-related neurodegenerative disorders. It has been hypothesized that microglia undergo age-related changes in gene expression patterns that give rise to pathogenic phenotypes. We compared the gene expression profiles in microglia isolated ex vivo from the retinas of mice ranging from early adulthood to late senescence. We discovered that microglial gene expression demonstrated progressive change with increasing age, and involved genes that regulate microglial supportive functions and immune activation. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling demonstrated age-related change. In particular, expression levels of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent microglia may contribute to complement dysregulation during disease pathogenesis. Taken together, senescent microglia demonstrate age-related gene expression changes capable of altering their constitutive support functions and regulation of their activation status in ways relating to neuroinflammation and neurodegeneration in the CNS., (Published by Elsevier Inc.)

The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. We have shown that a large number of cells of the P. aeruginosa wild-type PAO1 strain invade cultured human bronchial epithelial cells (BEAS-2B). In the present study, we investigated whether P. aeruginosa pilus protein PilA might modulate cellular functions by binding to unknown factor(s) in human host cells. Using a yeast two-hybrid screen, we showed that the calcium-modulating cyclophilin ligand (CAMLG), which is involved in Ca(2+) signaling, was the major host cell PilA binding protein. Overexpression of the pilA gene in BEAS-2B cells resulted in a significant increase in cytoplasmic Ca(2+) and consequent upregulation of the activity of the nuclear factor of activated T cells, followed by induction of cyclooxygenase 2 gene expression. Infection of BEAS-2B cells with the P. aeruginosa wild-type strain, but not with the pilA gene knockout strain (ΔpilA), caused a significant increase in intracellular Ca(2+) concentration in infected cells. Therefore, we propose a novel bacterial strategy for PilA modulation of intracellular Ca(2+) signaling through intracellular PilA-CAMLG interaction.

Background: Pseudomonas aeruginosa is frequently isolated from chronic wounds and causes serious infection in immunocompromised hosts. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) is synthesized by an autoinducer synthase encoded by the bacterial lasI gene in P. aeruginosa, which regulates the production of virulence factors and biofilm formation in this bacterium. Recent studies have suggested that 3-oxo-C12-HSL contributes to the modulation of immune responses. However, the effect of this molecule on wound healing in P. aeruginosa infection remains to be elucidated., Objective: We used an animal model to study the effect of 3-oxo-C12-HSL on wound healing in skin infected with P. aeruginosa., Methods: Wounds were created on the backs of Sprague-Dawley (SD) rats and the P. aeruginosa strain PAO1 (PAO1) or its lasI deletion mutant (ΔlasI) was inoculated onto the wound surface. To examine the biological activity of 3-oxo-C12-HSL, rats were injected intraperitoneally with anti-3-oxo-C12-HSL antiserum or administered 3-oxo-C12-HSL at the wound surface. The wound tissues were harvested for analysis of the healing process and inflammatory response., Results: PAO1 inoculation significantly accelerated the wound healing and inflammatory response on day 3 post-wounding. These responses were reversed by inoculation with ΔlasI instead of PAO1 or treatment with anti-3-oxo-C12-HSL antiserum. In contrast, administration of 3-oxo-C12-HSL in the absence of PAO1 significantly promoted these responses, which were suppressed by the anti-TNF-α mAb., Conclusion: These results strongly suggest that 3-oxo-C12-HSL may be involved in healing wounds infected with P. aeruginosa through induction of inflammatory responses., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Pseudomonas aeruginosa can penetrate the extracellular mucin barrier formed by the intestinal epithelial cell layer and establish gut-derived sepsis in immunocompromised patients. We found that two efficient mechanisms, flagellar motility and mucin degradation, are needed for penetration of P. aeruginosa through the mucin barrier. Deletion of the flagellar motility-related gene, the filament protein gene fliC, the cap protein gene fliD, and the motor complex protein genes motABCD from P. aeruginosa PAO1 decreased association of P. aeruginosa with the apical surface of human epithelial colorectal adenocarcinoma (Caco-2) cells. A penetration experiment using an artificial mucin layer suggested that the decreased penetration is caused by attenuation of mucin penetration ability. Additionally, the presence of P. aeruginosa decreased the total mucin, including the secreted mucin protein MUC2, on the surface of the Caco-2 cell monolayer, regardless of flagellar motility. Construction of the PAO1 mutant series knocked out 12 putative serine protease genes and identified the mucD gene, which participated in degradation of total mucin, including MUC2. Furthermore, decreased association with the surface of the Caco-2 cell monolayer was observed in the mucD mutant, and the decrease was synergistically amplified by double knockout with fliC. We conclude that P. aeruginosa can penetrate the mucin layer using flagellar motility and mucin degradation, which is dependent on the MucD protease or the mucD gene-related protease.

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Translocation of endogenous Pseudomonas aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Comparative genome hybridization analysis of high virulent and low virulent strains allowed us to identify bacterial genes that are associated with bacterial translocation from gut in infected hosts. Here we focused on the pvdE pyoverdine synthesis gene among the identified bacterial genes, showing that the pvdE gene is required for bacterial penetration through epithelial cell monolayers and for bacterial translocation from gut to hemolymph in infected silkworms. We next revealed that mRNA expression level of the exoS gene in a pvdE-deficient mutant (ΔpvdE) after incubation with Caco-2 cells was greatly reduced as compared with that in the wild-type strain. The pvdE- and exoS-complemented ΔpvdE strains (ΔpvdE/pvdE and ΔpvdE/exoS) showed recovery of the ability of bacterial penetration through Caco-2 cell monolayers and of the ability of bacterial translocation from gut to hemolymph in infected silkworms. However, there were differences between the ability of ΔpvdE/pvdE and ΔpvdE/exoS to kill silkworms after intestinal infection and to replicate in hemolymph following direct injection into the hemolymph: ΔpvdE/pvdE could kill silkworms after intestinal infection and could replicate in hemolymph to levels similar to those of the wild-type strain, but ΔpvdE/exoS could not. Taken together, our results suggest that the virulence of the wild-strain mediated by the pvdE gene is the result of the ability to both penetrate through the intestinal epithelial cell barrier depending on ExoS and to replicate in hemolymph independently of ExoS.

We investigated the influence of the type III effector, ExoS, on the host epithelial cell response to Pseudomonas aeruginosa infection, and we found that disruption of the exoS gene caused a significant increase in the amount of interleukin-8 (IL-8) in the culture medium of Caco-2 cells. We show that IL-8 was degraded in the culture medium following infection of the cells with the wild-type (PAO1), but not the exoS knock-out (the ΔexoS) strain. Purified ExoS protein itself did not degrade IL-8. We next show that IL-8 degradation by PAO1 was inhibited by the addition of serine protease inhibitors. These results strongly suggest that a bacterial serine protease that degrades IL-8 is expressed and secreted into the culture medium of Caco-2 cells infected with PAO1, and that the expression of this protein is repressed in cells infected with the ΔexoS strain. The PAO1 genome encodes 28 different protease genes, including two serine proteases: PA3535 and mucD. PA3535 and mucD gene knock-outs were constructed (ΔmucD and ΔPA3535), and ΔmucD but not ΔPA3535 showed reduced IL-8 degradation. To understand the significance of IL-8 degradation, we next evaluated neutrophil infiltration in lungs excised from mice intranasally infected with the P. aeruginosa strains. Increased neutrophil infiltration was observed in PAO1-infected mice, but not in ΔexoS- or ΔmucD-infected mice. Taken together, our results suggest that P. aeruginosa escapes from phagocytic killing due to IL-8 degradation following the secretion of the MucD serine protease, whose expression appears to be influenced by ExoS.

Purpose: To investigate whether photodynamic therapy (PDT) outcomes of polypoidal choroidal vasculopathy (PCV) are related to baseline clinical characteristics, smoking history, or genetic factors by analyzing the retreatment-free period after the first PDT., Design: Retrospective cohort study., Participants: The study consisted of 167 patients with PCV who underwent PDT as their first treatment., Methods: We targeted 638 single nucleotide polymorphisms (SNPs) in 42 possible susceptible genes for age-related macular degeneration to evaluate their relation to the effectiveness of PDT for PCV. For this evaluation, we used 2 methods: (1) survival analysis, with the retreatment-free period as the target; and (2) logistic regression test between the need for additional therapy within 3 months after the first PDT and the genotypes, with age, gender, smoking status, and greatest linear dimension (GLD) at baseline as covariates. The contributions of smoking status and GLD at baseline for the retreatment-free period also were evaluated. Contributions of these factors to visual prognosis were evaluated for 1 year after PDT., Main Outcome Measures: Retreatment-free period after the first PDT for PCV. Secondary outcome measures included correlation of the susceptible factor to the retreatment requirement within the 3-month follow-up and the mean visual acuity change., Results: In survival analyses, SERPINF1 rs12603825 showed a significant association with the retreatment-free period after the first PDT; those patients homozygous for the minor allele A of rs12603825 received additional treatment after PDT within significantly shorter times than those with other genotypes (P = 0.0038). There was no significant difference in the retreatment-free period between baseline GLD and smoking status. Retreatment within 3 months was required significantly more in patients with the AA genotype, even after taking into consideration the effect of clinical characteristics (age, gender), baseline PCV lesion size, and smoking status (P = 0.0027). Furthermore, patients with the AA genotype showed significantly worse visual prognosis after PDT (P = 0.013)., Conclusions: Pigment epithelium-derived factor (SERPINF1 or PEDF) polymorphisms may influence the initial response to and visual prognosis after PDT for PCV. Our findings may lead to understanding the pathogenesis of PCV and modification of the effects of PDT., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

In our previous studies, we employed recycle HPLC for the separation of triacylglycerol (TAG)-positional isomers (PIs). In this study, a recycle HPLC system equipped with a polysaccharide-based chiral column was applied to the enantiomeric separation of some asymmetric TAGs having straight-chain C16-C18 acyl residues. As a result, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (rac-PPO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (rac-OOP), and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol (rac-PPL) were resolved into their respective enantiomers. However, neither 1,2-dioleoyl-3-linoleoyl-rac-glycerol (rac-OOL), consisting of only unsaturated fatty acids, nor 1,2-dipalmitoyl-3-stearoyl-rac-glycerol (rac-PPS), consisting of only saturated fatty acids, was resolved. These results suggest that the asymmetric TAGs, used in this study, having both a palmitic acid moiety and an oleic acid (or a linoleic acid) moiety at the sn-1 or sn-3 positions are resolved by the chiral column. This new chiral separation method can be used in combination with atmospheric pressure chemical ionization mass spectrometry to determine the sn-OOP/sn-POO ratio in palm oil. This method is applicable for the chiral separation of asymmetric TAGs in palm oil., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The epidermal growth factor receptor family tyrosine kinases transduce signals for cell proliferation and migration and contribute to tumorigenesis. A recent extensive research has highlighted the major roles of the negative regulators of complex epidermal growth factor receptor signaling networks. These regulators fine-tune signaling under physiological conditions. When their expression is downregulated, the resultant aberrant epidermal growth factor receptor signaling may promote cell proliferation and migration, leading to increased tumorigenesis. In this paper, I review specific feedback inhibitors that target epidermal growth factor receptors preferentially, via multiple modes of action. The inhibitors include mitogen-inducible gene-6 (Mig-6)/receptor-associated late transducer (RALT)/Gene 33, fibroblast growth factor receptor substrate 2beta (FRS2beta)/suc1-associated neurotrophic factor target-2 (SNT-2)/FRS3, suppressor of cytokine signaling 3 (SOCS3)/SOCS4/SOCS5, and leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1). Although only fragmentary evidence is available regarding these inhibitors, they might be useful as cancer biomarkers, and the development of drugs that target them would certainly advance personalized medicine in the near future.

The docking protein FRS2 alpha is an important mediator of fibroblast growth factor (FGF)-induced signal transduction, and functions by linking FGF receptors (FGFRs) to a variety of intracellular signaling pathways. We show that the carotid body is absent in FRS2 alpha(2F/2F) mice, in which the Shp2-binding sites of FRS2 alpha are disrupted. We also show that the carotid body rudiment is not formed in the wall of the third arch artery in mutant embryos. In wild-type mice, the superior cervical ganglion of the sympathetic trunk connects to the carotid body in the carotid bifurcation region, and extends thick nerve bundles into the carotid body. In FRS2 alpha(2F/2F) mice, the superior cervical ganglion was present in the lower cervical region as an elongated feature, but failed to undergo cranio-ventral migration. In addition, few neuronal processes extended from the ganglion into the carotid bifurcation region. The number of carotid sinus nerve fibers that reached the carotid bifurcation region was markedly decreased, and baroreceptor fibers belonging to the glossopharyngeal nerve were absent from the basal part of the internal carotid artery in FRS2 alpha(2F/2F) mutant mice. In some of the mutant mice (5 out of 14), baroreceptors and some glomus cells were distributed in the wall of the common carotid artery, onto which the sympathetic ganglion abutted. We propose that the sympathetic ganglion provides glomus cell precursors into the third arch artery derivative in the presence of sensory fibers of the glossopharyngeal nerve.

Objective: To describe the 3-dimensional (3-D) imaging of the pathologic changes in the foveal photoreceptor layer in eyes with central serous chorioretinopathy (CSC) using high-speed optical coherence tomography (OCT)., Design: Prospective observational case series., Participants: Twenty-seven eyes of 27 consecutive patients with various stages of CSC., Methods: A prototype high-speed OCT system was fabricated for patient examinations based on Fourier domain OCT. The system had a sensitivity of 98 dB, a tissue axial resolution of 4.3 mum, and an acquisition rate of approximately 18700 axial scans per second. Three-dimensional imaging was performed based on a raster-scan protocol., Main Outcome Measures: Anatomic features of CSC distinguished by 3-D OCT., Results: A line corresponding to backreflection from the external limiting membrane (ELM) was visible in images from eyes with all stages of CSC, including 6 in the acute, 5 in the chronic, and 9 in the recurrent phase of retinal detachment and 11 examined in the quiescent phase (including 4 reexamined after reattachment). Backreflection from the photoreceptor inner and outer segment junction (IS/OS) was missing before but present after reattachment. The ELM line bordered the photoreceptor nuclear and inner segment layers, clearly showing that CSC primarily alters the outer segment (OS) layer. Punctate areas of intense reflectivity were observed more frequently in the OS layer of detached retinas in cases of chronic or recurrent versus acute CSC (P<0.05, chi-square test). Of 11 eyes with reattached macula, 3 eyes with large defects in the subfoveal IS/OS had poor visual acuity (VA), and 8 eyes with small or no defects had good VA (P<0.05 and P<0.001, chi-square test). Decreases in foveal full and outer thickness were associated with VA loss (P<0.05 and P<0.001, Spearman rank correlation test)., Conclusions: Three-dimensional OCT imaging delineates the microstructural changes that occur within the photoreceptor layers and demonstrates the spatial relationship between the laterally spreading or scattering microstructures and the fovea in eyes with CSC. Visualization of the 3-D relationship between the ELM and each photoreceptor layer before and after macular reattachment facilitates understanding of anatomic and vision changes that result from CSC.

Objective: To demonstrate the advantages of 3-dimensional imaging of macular hole pathology using new-generation high-speed optical coherence tomography (OCT)., Design: Prospective observational case series., Participants: Twenty-one eyes from 20 consecutive patients diagnosed with a macular hole., Methods: A prototype high-speed OCT system was built based on a Fourier-domain OCT (FD OCT) technology for patient examination. The system has achieved sensitivity of approximately 98 decibels, axial resolution of approximately 4.3 mum in tissue, and an acquisition rate of approximately 18,700 axial scans per second. Three-dimensional imaging of macular hole pathology was performed based on a raster scan protocol consisting of 256x256 axial scans. All patients were imaged with 3-dimensional OCT, Stratus OCT, and OCT Ophthalmoscope C7., Main Outcome Measures: Images of macular hole pathologies obtained by 3-dimensional OCT and standard OCT instruments., Results: The 3-dimensional OCT imaging successfully generated realistic 3-dimensional images of the vitreofoveal interface and intraretinal microstructures associated with a macular hole. The 3-dimensional overview of the vitreofoveal interface was helpful in gaining an immediate understanding of the dynamic interactions of the vitreous and fovea. Observations of consecutive en face images in combination with conventional longitudinal images and of cross-sectional images in combination with sectioned volume images enabled identification of intraretinal microstructures and their 3-dimensional extension associated with a macular hole, such as subfoveal structural changes after vitreous traction, connection of the flap to intraretinal structures, the external limiting membrane (ELM) and its disruption, and elevated photoreceptor inner and outer segments delineated by the ELM. The appearance of inner-wall images of a macular hole produced by photoreceptor inner and outer segment backreflection varied throughout macular hole stages., Conclusions: Three-dimensional imaging of macular holes with high-speed OCT based on FD OCT technology offers 3-dimensional overviews that facilitate understanding of the abnormalities in the vitreofoveal interface. It also provides consecutive orthogonal images that allow much more precise and minute observation of 3-dimensionally extending intraretinal structural changes associated with a macular hole than conventional OCT imaging, especially in the photoreceptor inner and outer segments.

The control of cellular responses with fibroblast growth factors and neurotrophins is mediated through membrane-linked docking proteins, SNT (suc1-binding neurotrophic target)-1/FRS2alpha and SNT-2/FRS2beta. ERK1/2 are members of the mitogen-activated protein kinase family that regulate diverse cellular activities in response to various stimuli. Here, we demonstrate that SNT-2 does not become tyrosine phosphorylated significantly in response to EGF but forms a complex with ERK2 via the region of 186-252 amino acid residues, and the complex formation is enhanced upon EGF stimulation. SNT-2 downregulates ERK2 phosphorylation, suppresses and delays ERK2 nuclear accumulation which occurs following EGF stimulation. In contrast, the mutant SNT-2 which carries deletion of 186-252 amino acids and lacks ERK2 binding does not have these effects. These observations suggest that SNT-2 negatively regulates ERK2 signaling activated via EGF stimulation through direct binding to ERK2.

Intrinsic resistance to mureidomycin is shown in Escherichia coli. This is in contrast to Pseudomonas aeruginosa, which generally displays intrinsic resistance to a variety of antimicrobial agents, but not to mureidomycin. Isogenic efflux system mutants from both species were subjected to antibiotic susceptibility tests. These studies showed that the differences regarding the susceptibility of E. coli and P. aeruginosa to mureidomycin A and C may be explained by the expression of efflux systems that mediate resistance to mureidomycin A and C.

Overproduction of multidrug efflux systems MexAB-OprM, MexCD-OprJ, and MexXY/OprM of Pseudomonas aeruginosa caused reduction of susceptibility of the mutant, which lacked AmpC and all three systems to panipenem, meropenem, S4661 and DU6681a; meropenem, S4661 and DU6681a; and BO2727, panipenem, meropenem, S4661 and DU6681a, respectively, but not reduction of the susceptibility to imipenem and biapenem. Thus, we determined substrate specificities of these efflux systems to carbapenems.

The tripartite efflux systems MexAB-OprM and MexCD-OprJ of Pseudomonas aeruginosa each display characteristic substrate specificity against a variety of antimicrobial agents. The chimeric efflux system MexC-MexB-OprJ/DeltaMexD constructed by exchange of MexD with MexB endowed the recombinant host the same resistance profile as MexAB-OprM rather than MexCD-OprJ. The change of substrate specificity was shown to be due to extrusion from the chimeric efflux system by cellular accumulation experiments using tetracycline, erythromycin, and ethidium bromide. Thus, we conclude that MexB and MexD are primary components of the efflux system responsible for sorting extrusion substrates.

Grb2-associated binder-1 (Gab1) undergoes tyrosine phosphorylation in response to stimulation by growth factors and hormones including insulin, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). However, the HGF receptor is the only one known to associate directly with Gab1. Herein, we explore the mechanism of Gab1 phosphorylation by other receptor protein-tyrosine kinases unable to bind to Gab1 directly. The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit binds Gab1 in a phosphorylation-independent manner. Moreover, the regulatory subunit of PI3K can mediate the association of Gab1 and receptor protein-tyrosine kinases including the insulin, EGF, and NGF receptors, all of which phosphorylate Gab1. Thus, it appears that the PI3K regulatory subunit acts as an adaptor protein via a phosphotyrosyl-independent SH2 interaction, allowing Gab1 to serve as a substrate for several tyrosine kinases. This is a new role for the PI3K regulatory subunit., (Copyright 2001 Academic Press.)

We reported previously that the high susceptibility of Moraxella catarrhalis to macrolide antibiotics and other hydrophobic antimicrobial agents was related to the hydrophobicity of the cell surface. Electrophoretic analysis of lipopolysaccharide (LPS) extracted from M. catarrhalis revealed a deep rough-type profile similar to that of an LPS Re type mutant of Salmonella typhimurium, which also exhibits high susceptibility to macrolides. Moreover, treatment of 32P-labeled cells of M. catarrhalis by phospholipase C induced the release of radioactive materials. These results suggested that hydrophobic agents such as macrolides readily access the cell surface exposed by the deep rough-type LPS and phospholipids, and permeate into the cell interior through the lipid bilayer. In fact, M. catarrhalis cells rapidly accumulated large amounts of the macrolide antibiotics, erythromycin and rokitamycin, whereas no accumulation of the macrolides was observed in cells having smooth-type or Rc type LPS under the same conditions.

Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKC delta inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKC delta plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.

Pseudomonas aeruginosa encodes three sets of antibiotic extrusion proteins designated as MexA,B.OprM, MexC,D-OprJ and MexE,F-OprN regulated by the nalB, nfxB and nfxC genes, respectively. MexB,D,F, OprM, J,N and MexA,C,E function as the inner membrane pumps, the outer membrane channels and the membrane fusion proteins, respectively. To investigate the possibility of subunit interchangeability, we constructed the following combinations of chimeric pumps: MexA,D-OprM/delta MexB, MexC,B-OprM/delta MexA, and MexA,B-OprJ/delta OprM. The strains producing MexA,D-OprM/delta MexB and MexC,B-OprM/delta MexA failed to restore the antibiotic resistance shown in the strains producing the natural combinations of the subunit proteins. These results suggested that the inner membrane components cannot be interchanged. In contrast, the stains producing MexA,B-OprJ/delta OprM exhibited higher resistance to several antibiotics than the mutant lacking OprM and lower resistance than the strain overexpressing OprM. This result suggests that OprJ may complement the OprM function partially. A spectrum of antibiotics, of which the minimum inhibitory concentrations were restored partially by the complementation, was the same as the spectrum to which the nalB type mutant shows resistance. We surmised from these results that the MexA/MexB unit sustains the substrate specificity of the MexA,B-OprM machinery.

In the previous study (Sato K.-I. et al. (1997) FEBS Lett. 410, 136-140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F). We found that Y317F Shc but not Y239/240F or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns(4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2.

The effects of vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), and beta-carotene as antioxidants and their cooperative action against the oxidation of lipid in solution, membranes, and lipoproteins have been studied and reviewed. Ascorbic acid and alpha-tocopherol act as potent, and probably the most important, hydrophilic and lipophilic antioxidants, respectively. They function at their own site individually and furthermore act synergistically. beta-Carotene has lower reactivity toward radicals than does alpha-tocopherol and acts as a weak antioxidant in solution. It is more lipophilic than alpha-tocopherol and is assumed to be present at the interior of membranes or lipoproteins, which enables it to scavenge radicals within the lipophilic compartment more efficiently than does alpha-tocopherol. The cooperative interaction between vitamin C and vitamin E may be quite probable, that of vitamin C and beta-carotene is improbable, whereas that between vitamin E and beta-carotene may be possible.

Epidermal growth factor receptor (EGF-R) is a widely expressed ligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R corresponds to Y416 of v/c-src kinase, which is highly conserved and functionally important in many tyrosine kinases. To clarify the functional role of Y845, we constructed a mutant human EGF-R in which this tyrosine was replaced with phenylalanine and transfected it to NIH3T3 cells. EGF-R F845 induced EGF-dependent cellular transformation and revealed tyrosine-autophosphorylation of a 170 kDa protein, and initiated DNA synthesis similar to the wild-type EGF-R. We conclude here that Y845 is dispensable in the above mentioned functions of EGF-R tyrosine kinase.

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.