Development of a new assay system for bladder cancer using interactions between cytochromes P450 and serum.

To overcome bladder cancer, one of the most common cancer and deadly cancers in the world, early diagnosis and treatment interventions are crucial. The development of efficient diagnostic methods is required. Previously, we developed a cytochrome P450 (P450 or CYP) inhibition assay that detected alterations in the quality and quantity of P450 relevant substances in the serum, caused by inflammation and exposure to endogenous or exogenous substances. Since bladder cancer is known to alter the expression levels of P450s in patients, we tested whether the P450 inhibition assay could distinguish between the sera of patients with bladder cancer and healthy individuals. When assays were performed using sera recovered from mice with bladder cancer and control mice, significant differences were observed in the inhibition rates of CYP2A13, CYP2C18 and CYP2E1. Moreover, the results of the assay using human clinical samples revealed that the P450 inhibition assay can detect bladder cancer with an area under the receiver operating characteristic (ROC) curve of 0.867-0.950. These findings demonstrated that the P450 inhibition assay can aid the future development of liquid biopsy-based diagnostic methods for bladder cancer.

Diagnosis of Parkinson's disease by investigating the inhibitory effect of serum components on P450 inhibition assay.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and diagnostic methods and biomarkers for patients without subjective motor symptoms have not yet been established. Previously, we developed a cytochrome P450 inhibition assay that detects alterations in metabolite levels associated with P450s caused by inflammation and exposure to endogenous or exogenous substances. However, it is unknown whether the P450 inhibition assay can be applied in PD diagnosis. Here, we determined whether the P450 inhibition assay can discriminate sera between patients with PD and healthy individuals. The results of the assay revealed that the P450 inhibition assay can discriminate PD with an area under the receiver operating characteristic curve (AUC) value of 0.814-0.914 in rats and an AUC value of 0.910 in humans. These findings demonstrate that the P450 inhibition assay can aid in the future development of liquid biopsy-based diagnostic methods for PD.

Importance of transmembrane helix 4 of l-alanine exporter AlaE in oligomer formation and substrate export activity in Escherichia coli.

AlaE is the smallest amino acid exporter identified in Escherichia coli. It exports l-alanine using the proton motive force and plays a pivotal role in maintaining intracellular l-alanine homeostasis by acting as a safety valve. However, our understanding of the molecular mechanisms of substrate export by AlaE is still limited because structural information is lacking. Due to its small size (149 amino acid residues), it has been speculated that AlaE functions by forming an oligomer. In this study, we performed chemical cross-linking and pull-down assays and showed that AlaE indeed generates homo-oligomers as a functional unit. Previous random mutagenesis experiments identified three loss-of-function AlaE point mutations in the predicted transmembrane helix 4 (TM4) region, two of which are present in the GxxxG motif. When alanine-scanning mutagenesis was applied to the TM4 region, the AlaE derivatives that had amino acid substitutions around the GxxxG motif showed low l-alanine export activities, indicating that the GxxxG motif in TM4 plays an important role in substrate export. However, these AlaE variants with low activity could still form oligomers. We therefore concluded that AlaE forms homo-oligomers and that the GxxxG motif in the TM4 region plays an essential role in AlaE activity but is not involved in AlaE oligomer formation.

Intestinal Clostridium species lower host susceptibility to enterohemorrhagic Escherichia coli O157:H7 infection.

Susceptibility to enterohemorrhagic Escherichia coli (EHEC) infection varies among humans. The intestinal microbiota seems to play an essential role in host defense against EHEC; thus, we hypothesized that indigenous bacteria, such as Clostridium ramosum and Clostridium perfringens, could influence the susceptibility to EHEC infection. To evaluate the effect of indigenous bacteria on EHEC infection, germ-free mice were precolonized with each indigenous bacterium, and then infected with EHEC O157:H7. Precolonization with C. ramosum or C. perfringens completely prevented death from EHEC infection througout a test period. Precolonization with C. ramosum also reduced the level of secreted Shiga toxin (Stx) 2 and prevented histopathological changes in the kidneys in a similar way to precolonization with Bifidobacterium longum, which is used as a model for preventing EHEC infection. In contrast, the mice precolonized with C. perfringens showed mild renal injuries. When evaluated using an in vitro co-culturing system, again C. ramosum inhibited the growth and Stx production of EHEC more potently than C. perfringens. These results indicate that C. ramosum and C. perfringens suppressed EHEC infection; however, the extent of their preventive effects differed. Therefore, the susceptibility to EHEC infection and its severity can depend on the functional bacteria present in the intestinal microbiota of individuals.

Suppressive effect of an analog of the antimicrobial peptide of LL­37 on colon cancer cells via exosome­encapsulated miRNAs.

Antimicrobial peptides (AMPs) are multifunctional factors with an important role in the innate immune system. Our previous studies revealed that the human cathelicidin LL­37 and its analog, FF/CAP18, limit the proliferation of colon cancer cell lines. In the present study, the exosomes released by HCT116 cells treated with FF/CAP18 were analyzed. After the treatment, exosomes were isolated from the culture supernatant by ultrafiltration and using the miRCURY™ Exosome Isolation Kit. Membrane vesicles 40­100­nm expressing CD63 and CD81 were identified before and after FF/CAP18 treatment. Exosome concentration in the culture supernatant was increased after treatment with FF/CAP18. Exosomes formed in HCT116 cells treated with FF/CAP18 induced growth suppression of the cells in a dose­dependent manner. By contrast, the exosomes formed in non­treated HCT116 cells did not affect cell viability. Microarray analysis of miRNA expression indicated that FF/CAP18 treatment induced increases in the expression of three miRNAs (miR­584­5p, miR­1202 and miR­3162­5p) in both HCT116 cells and exosomes. These results suggest that FF/CAP18 treatment increases exosome formation, and that exosome­encapsulated miRNAs suppress HCT116 cell proliferation. Exosomal miRNAs are considered to be involved in the dissemination of cell signals to control local cellular microenvironments. The present findings suggest that FF/CAP18 regulates cancer growth by modulating cell­to­cell communication. AMPs localize in the cytoplasm of cancer cells and enhance the expression of growth­suppressing miRNAs. These miRNAs are also transported to other cancer cells via exosomes. Therefore, transportation of these miRNAs has the potential to suppress cancer growth. AMPs exert their effects directly by targeting cancer cells and indirectly via exosomes.

Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less ß-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of ß-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no ß-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent KD, 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response.

Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system.

Functional structure and antimicrobial activity of persulcatusin, an antimicrobial peptide from the hard tick Ixodes persulcatus.

BACKGROUND: Antimicrobial peptides (AMPs) are considered promising candidates for the development of novel anti-infective agents. In arthropods such as ticks, AMPs form the first line of defense against pathogens in the innate immune response. Persulcatusin (IP) was found in the Ixodes persulcatus midgut, and its amino acid sequence was reported. However, the complete structure of IP has not been identified. We evaluated the relation between structural features and antimicrobial activity of IP, and its potential as a new anti-methicillin-resistant Staphylococcus aureus (MRSA) agent. METHODS: The structure of IP was predicted using homology modeling and molecular dynamics. IP and other tick AMPs were synthesized using a solid-phase method and purified by high-performance liquid chromatography. Methicillin-susceptible S. aureus (MSSA) and MRSA were used for the minimum inhibitory concentration (MIC) test and short-time killing assay of IP and other tick peptides. The influence of IP on mammalian fibroblasts and colon epithelial cells and each cell DNA and its hemolytic activity towards human erythrocytes were also examined. RESULTS: In the predicted IP structure, the structure with an S-S bond was more stable than that without an S-S bond. The MIC after 24 h of incubation with IP was 0.156-1.25 µg/mL for MSSA and 0.625-2.5 µg/mL for MRSA. Compared with the mammalian antimicrobial peptide and other tick peptides, IP was highly effective against MRSA. Moreover, IP showed a dose-dependent bactericidal effect on both MSSA and MRSA after 1 h of incubation. IP had no observable effect on mammalian cell growth or morphology, on each cell DNA and on human erythrocytes. CONCLUSIONS: We predicted the three-dimensional structure of IP and found that the structural integrity was maintained by three S-S bonds, which were energetically important for the stability and for forming α helix and ß sheet. IP has cationic and amphipathic properties, which might be related to its antimicrobial activity. Furthermore, the antimicrobial activity of IP against MRSA was stronger than that of other antimicrobial peptides without apparent damage to mammalian and human cells, demonstrating its possible application as a new anti-MRSA medicine.

Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 µmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.