The effectiveness in preventing frailty of exercise intervention provided by community pharmacists to older persons with chronic conditions: A pragmatic randomized controlled trial.

BACKGROUND: Once older persons become frail, the risk of falls, bone fractures, and other problems increases. Exercise intervention is a form of prevention that has a high degree of evidence. OBJECTIVE: We investigated the effectiveness of frailty prevention consisting of exercise intervention by community pharmacists at 11 pharmacies operated by Osaka Pharma Plan. METHODS: In total, 103 older persons between 70 and 79 years of age (53 males and 50 females) who were suffering from chronic conditions and who visited one of 11 pharmacies between January and March 2021 were enrolled. They were then randomly assigned to either the Intervention group (IG: 6 pharmacies, 61 patients) who were subjected to intervention by a pharmacist, or the Usual Care group (UG: 5 pharmacies, 42 patients) who were not subjected to intervention. At the beginning of the trial and 6 month after, their muscle mass, etc. were measured using a body composition meter, and their Five-Times Sit-To-Stand Test results were also measured. Patients in the IG were provided with information by way of leaflets during the time they were guided regarding taking their medication for a period of one to six months that encouraged exercising at home. Those in the UG were given the standard guidance related to taking their medication. RESULTS: The amount of change in muscle mass was 1.08 ± 7.83% (95%CI: -1.24-3.41) in IG and - 0.43 ± 2.73% (95%CI:-1.58-0.72) in UG, indicating that there was a trend toward an increase in IG. The percent change in the Five Times Sit-To-Stand Test times at + 6 M was - 0.002 ± 0.24% (95%CI: -0.09-0.05) in IG and - 0.04 ± 0.21% (95%CI:-0.13-0.07) in UG, but in cases in which the second measured time was faster than the first measured time, the results were 65.2% for IG and 29.2% for UG, indicating a significant difference (p = 0.00563). CONCLUSION: Despite the fact that the amount of time community pharmacists can devote to providing guidance on taking medications is limited, it has been previously reported that providing information to patients causes a change in patient behavior. The results of the present study are highly significant as they suggest the possibility that this may hold true even when used to prevent frailty, based on the evidence obtained. TRIAL REGISTRATION: This trial was registered at UMIN-CRT on 1st of January, 2021. The registration number is UMIN000042571.

In vivo evaluation of pharmacokinetic drug-drug interactions between fluorinated pyrimidine anticancer drugs, 5-fluorouracil and capecitabin, and an anticoagulant, warfarin.

Warfarin is a common anticoagulant and has demonstrated interactions with several drugs. Among them, as a serious adverse event, a case of death due to the enhanced warfarin action owing to its combined use with a fluoropyrimidine anticancer drug has been reported, but the detailed mechanism has not been elucidated.Some reports have advocated that fluorinated pyrimidine anticancer drugs reduce cytochrome P450 2C9 expression, leading to the enhanced pharmacological effects of warfarin.The purpose of this study was to clarify the mechanisms of drug-drug interactions between warfarin and 5-fluorouracil (5-FU) and capecitabine in vivo using rats. Rats were administered warfarin in combination with 5-FU (15 mg/kg/d) or capecitabine (15 mg/kg/d) for 7 d. Prothrombin time (PT) and activated partial thromboplastin time were significantly prolonged in the warfarin plus 5-FU or capecitabine groups compared with those in the warfarin alone group. No significant difference was observed in the area under the plasma concentration-time curve of the warfarin alone group compared with the warfarin with 5-FU or capecitabine groups.These data suggest that the enhancement of warfarin efficacy caused by the combination of 5-FU or capecitabine was due to a pharmacological interaction rather than a pharmacokinetic interaction.

Troglitazone-Induced Autophagic Cytotoxicity in Lung Adenocarcinoma Cell Lines.

Lung cancer is the leading cause of cancer-related deaths worldwide. Troglitazone (TGZ), a peroxisome proliferator-activated receptor gamma (PPARγ) ligand, is a potential antitumor agent. However, the action mechanism of TGZ in lung adenocarcinoma cells has not been completely elucidated. To assess this mechanism and the anticancer effects of TGZ in human lung adenocarcinoma cell lines (A549 and H1975), we investigated the involvement of PPARγ, apoptosis, the mitogen-activated protein kinase (MAPK) pathway, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, and autophagy. Cell viability was measured using fluorescence-based assays. Apoptotic cells were detected by Hoechst 33342 and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining; protein expression was detected by Western blotting. TGZ inhibited cell proliferation in a dose-dependent manner in both cell lines, and the effect was not suppressed by a PPARγ inhibitor. Additionally, TGZ increased apoptotic cell number and upregulated p38 and c-Jun N-terminal kinase (JNK) phosphorylation; however, p38 and JNK inhibitors did not block TGZ-mediated inhibition of cell proliferation in either cell line. TGZ also upregulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas an ERK1/2 inhibitor enhanced TGZ-mediated cytotoxicity in A549 cells. Additionally, TGZ increased LC3-II expression, and chloroquine (an autophagy inhibitor) attenuated TGZ-mediated inhibition of cell proliferation. These findings suggest that TGZ-induced inhibition of cell proliferation is PPARγ independent. TGZ-mediated inhibition of cell proliferation was accompanied by apoptosis and independent of the MAPK signaling pathway. These results suggest that TGZ inhibits cell proliferation through autophagy-induced cytotoxicity. This study demonstrated that chemotherapy using TGZ may be effective for lung adenocarcinoma.

Telmisartan-Induced Cytotoxicity via G2/M Phase Arrest in Renal Cell Carcinoma Cell Lines.

Renal cell carcinoma (RCC) is the most common type of kidney cancer. Given that stage IV RCC is intractable, there is a need for a novel treatment strategy. We investigated the antitumor effects of telmisartan (TEL) and their underlying mechanisms in RCC, including their impact on apoptosis, Akt/mammalian target of rapamycin (mTOR) pathways, and the cell cycle using two human RCC cell lines: 786-O and Caki-2. Cell viability was detected via fluorescence-based assays. Cells were stained with Hoechst 33342 to observe chromatin condensation, and Western blotting was performed to analyze protein expression. The cell cycle was assessed using flow cytometry. Invasion and migration assays were performed using 24-well chambers. TEL induced cell death in a dose-dependent manner and increased the percentage of cells with high chromatin condensation and Bax/Bcl-2 ratio in both cell lines. TEL-induced cell death was attenuated by neither peroxisome proliferator-activated receptor-γ nor -δ inhibitors. Although TEL elevated c-Jun N-terminal kinase levels and p38 phosphorylation rates in Caki-2 cells, as well as extracellular signal-regulated kinase phosphorylation rates in 786-O cells, their inhibitors did not suppress TEL-induced cell death. TEL decreased Akt phosphorylation in 786-O cells and mTOR phosphorylation in both cell lines, increased the population of cells in the G2/M phase, and altered G2/M-related proteins in both cell lines. TEL moderately suppressed cell invasion and migration in 786-O and Caki-2 cells, respectively, and increased cell invasion in Caki-2 cells, suggesting a potential therapeutic role of TEL in RCC.

Telmisartan Exerts Cytotoxicity in Scirrhous Gastric Cancer Cells by Inducing G0/G1 Cell Cycle Arrest.

BACKGROUND/AIM: This study aimed to assess the effects of telmisartan (TEL), a potential antitumor agent, and its mechanism of action in the regulation of apoptosis, autophagy, and cell cycle in scirrhous gastric cancer (SGC). MATERIALS AND METHODS: The effect of TEL on the viability and chromatin condensation of OCUM-2M and OCUM-12 cells was assessed. Protein expression and the cell cycle were analysed using western blotting and flow cytometry, respectively. RESULTS: TEL inhibited cell proliferation in a dose-dependent manner and increased chromatin condensation and autophagy marker LC3-II levels in OCUM-12 cells. TEL also increased the proportion of cells in the G0/G1 phase transition. CONCLUSION: Apoptosis and autophagy are partially involved in the inhibitory effect of TEL on cell proliferation. Additionally, TEL caused G0/G1 cell cycle arrest. Therefore, TEL could be a promising treatment for SGC.

A plausible involvement of plasmalemmal voltage-dependent anion channel 1 in the neurotoxicity of 15-deoxy-Δ12,14 -prostaglandin J2.

INTRODUCTION: 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ) causes neuronal apoptosis independently of its nuclear receptor, peroxysome-proliferator activated receptor γ. Its membrane receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), did not also mediate the neurotoxicity of 15d-PGJ2 . In the present study, we ascertained whether membrane targets beside CRTH2 were involved in the neurotoxicity of 15d-PGJ2 . METHODS: Neuronal membrane targets for 15d-PGJ2 were separated by two-dimensional electrophoresis, identified by proteomic approach. Their localizations were detected by microscopic immunofluorescence study. Cell viability and apoptosis was evaluated by MTT-reducing activity and caspase-3 activity, respectively. RESULTS: Voltage-dependent anion channel 1 (VDAC1) was identified as one of membrane targets for 15d-PGJ2 . Modification of VDAC1 with 15d-PGJ2 was detected by pull-down assay. VDAC1 was detected in the plasma membrane and localized on the neuronal cell surface. VDAC1 was partially colocalized with membrane targets for 15d-PGJ2 . The anti-VDAC antibody significantly attenuated the neurotoxicity of 15d-PGJ2 , accompanied by the suppression of the 15d-PGJ2 -stimulated caspase-3. CONCLUSION: These findings suggested that the plasmalemmal VDAC might be involved in the neurotoxicity of 15d-PGJ2 .

Patients' Attitudes, Awareness, and Opinions About Community Pharmacies in Japan: Next Steps for the Health Support Pharmacy System.

PURPOSE: Despite the formal establishment of the Health Support Pharmacy system, few community pharmacies have transitioned to this new designation in Japan. Moreover, patients' perspectives on the usefulness of health-support pharmacies and community pharmacies have not yet been investigated. In this work, we investigated patients' attitudes, opinions, and awareness as users of member pharmacies of the Japan Federation of Democratic Medical Institutions (Min-Iren), with respect to two essential functions provided by community pharmacies-primary care and health support-to identify modern challenges facing community pharmacies. METHODS: Regular visitors to participating Min-Iren community pharmacies were asked to complete an anonymous questionnaire. Responses were compared between users of health-support pharmacies and other pharmacy types, as well as between members and non-members of "collaborating organizations" (CO). CO is organizational partners of Min-Iren whose activities support affiliated facilities. Logistic regression analysis was performed to explore the predictive value of different factors on pharmacies' primary-care and health-support functionality. RESULTS: A total of 181 Min-Iren community pharmacies (51.7%: 181/350) participated in this study, and most patients answered the questionnaire (97.7%, n=2623). Relatively few patients recognized the term "Health Support Pharmacy" (12.2%). CO members tended to have a superior understanding of a wide variety of services provided by CPs as compared to non-members. Statistically significant predictors of primary-care and health-support functionality included male gender, having a primary-care pharmacist, age ≥60 years, recognition of the term "Health Support Pharmacy" and CO membership. CONCLUSION: CO members, a class of patients with a superior awareness of health promotion, demonstrated a good understanding of the variety of services provided by community pharmacies and tended to positively rate their pharmacy. Moving forward, efforts to raise awareness about the importance of health-promotional activities among community pharmacy users should further reinforce the primary-care and health-support functions of community pharmacies.

Evaluation of molecularly imprinted polymers for chlorpromazine and bromopromazine prepared by multi-step swelling and polymerization method-The application for the determination of chlorpromazine and its metabolites in rat plasma by column-switching LC.

Monodisperse molecularly imprinted polymers (MIPs) for chlorpromazine (CPZ) and bromopromazine (BPZ), MIPCPZ and MIPBPZ, were prepared using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of MIPCPZ and MIPBPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile or a mixture of water and acetonitrile including ammonium formate as a mobile phase in reversed-phase LC. On MIPBPZ, CPZ, BPZ and imipramine (IMP) gave the maximal retention factors at a mobile-phase pH 8, while the maximal imprinting factors were obtained at a mobile-phase pH 7. Each MIP recognized a template molecule the most, while CPZ metabolites, desmethyl CPZ (DM-CPZ), CPZ sulfoxide (CPZ-SO) and 7-hydroxy CPZ (7-OH-CPZ), were moderately recognized on MIPCPZ and MIPBPZ. Furthermore, both MIPs gave the similar retention and molecular-recognition for CPZ and its metabolites. For avoiding the template-leakage problems, MIPBPZ was used as the pretreatment column for the determination of CPZ and its metabolites in rat plasma in column-switching LC with UV detection. In addition to DM-CPZ and CPZ-SO, didesmethyl CPZ (DDM-CPZ) and CPZ N-oxide (CPZ-NO) were speculated as the metabolite in rat plasma after administration of CPZ using LC-ESI-TOF-MS, while 7-OH-CPZ was not detected. The column-switching LC method was validated and applied for the determination of CPZ and its metabolites, DM-CPZ, DDM-CPZ, CPZ-SO and CPZ-NO, in rat plasma after intravenous and oral administration of CPZ using IMP as an internal standard.

In vitro and in vivo cytotoxicity of troglitazone in pancreatic cancer.

BACKGROUND: Troglitazone (TGZ) is a peroxisome proliferator-activated receptor gamma (PPARγ) agonist that has been investigated as a potential chemopreventive and chemotherapeutic agent. However, the antitumor efficacy and mechanisms of TGZ in pancreatic cancer have not been extensively investigated. This study was performed to investigate the in vitro and in vivo effects of TGZ against pancreatic cancer cell lines, as well as its action mechanisms in terms of PPARγ dependency and the Akt and mitogen-activated protein kinase (MAPK) pathways. We also evaluated the effects of TGZ on cell invasion and migration. METHODS: MIA Paca2 and PANC-1 human pancreatic cancer cell lines were used. Cell viability and caspase-3 activity were detected using fluorescent reagents, and chromatin condensation was observed after staining the cells with Hoechst 33342. Protein expression levels were detected by western blot analysis. Invasion and migration assays were performed using 24-well chambers. The in vivo antitumor effects of TGZ were investigated in nude mice inoculated with MIA Paca2 cells. Mice were orally administered TGZ (200 mg/kg) every day for 5 weeks, and tumor volumes were measured bi-dimensionally. RESULTS: TGZ showed dose-dependent cytotoxicity against both cell lines, which was not attenuated by a PPARγ inhibitor. Further, TGZ induced chromatin condensation, elevated caspase-3 activity, and increased Bax/Bcl-2 relative expression in MIA Paca2 cells. TGZ also increased phosphorylation of Akt and MAPK (ERK/p38/JNK) in both cell lines, and a JNK inhibitor significantly increased the viability of MIA Paca2 cells. TGZ moderately inhibited cell migration. Tumor growth in the MIA Paca2 xenograft model was inhibited by TGZ administration, while mouse body weights in the treated group were not different from those of the vehicle administration group. CONCLUSION: We demonstrated for the first time the in vivo antitumor effects of TGZ in pancreatic cancer without marked adverse effects. TGZ induced mitochondria-mediated apoptosis in MIA Paca2 cells, and its cytotoxic effects were PPARγ-independent and occurred via the JNK pathway. Our results indicate that TGZ is a potential approach for the treatment of pancreatic cancer and warrants further studies regarding its detailed mechanisms and clinical efficacy.

Serum lactate dehydrogenase levels as a predictive marker of oxaliplatin-induced hypersensitivity reactions in Japanese patients with advanced colorectal cancer.

OBJECTIVE: Clinical laboratory test data obtained prior to treatments were previously analyzed from the standpoint of susceptibility to hypersensitivity reactions in patients treated with the platimun anticancer agent, oxaliplatin (L-OHP). In the present study, the time course from the first to last cycle of the treatment was additionally analyzed to determine a better predictor of these reactions. METHODS: A total of 20 laboratory test data were obtained from 108 Japanese patients with advanced colorectal cancer who were treated with the L-OHP-containing regimens, FOLFOX4 and/or mFOLFOX6. The averages and variation coefficients (CV%) of the data until the last cycle of the treatment were compared between patients with hypersensitivity reactions and those without. RESULTS: The average serum lactate dehydrogenase (LDH) level was lower in patients with grade 1/2 reactions (P=0.016), whereas its CV% value was higher in patients with grade 3/4 reactions (P=0.005) than in those without reactions. An increase in serum LDH levels was observed in some patients with grade 3/4 reactions as the cycle number increased, and thereafter hypersensitivity reactions occurred. This phenomenon was not always observed, but was never detected in patients with grade 1/2 reactions. CONCLUSIONS: Serum LDH levels may be a predictive marker of hypersensitivity reactions in patients treated with L-OHP. Further extensive examinations with a larger number of patients are needed to establish a patient management strategy.

The frequency of BRO ß-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis.

We investigated the frequency of BRO ß-lactamase and its relationship to antibiotic susceptibility profiles and serum susceptibility. Moraxella catarrhalis clinical isolates (n = 197) were collected from patients with respiratory tract infections in Tokyo between November 2004 and April 2005. Phenotypic and genotypic detection of ß-lactamases was performed. The MICs of 6 antibiotics were determined by Etest, and the serum bactericidal assay was conducted by using the culture-and-spot test. Nearly all (192; 97%) of the clinical isolates were ß-lactamase producers; of these, 182 (95%) were bro-1 and 10 (5%) were bro-2 positive. MIC50, MIC90, and geometric mean MICs of penicillin, amoxicillin, cefixime, and clarithromycin for BRO-1 isolates were significantly higher than for BRO-2 isolates. The frequency of intermediate and full serum resistance was significantly higher in BRO-1 isolates than in BRO-2 isolates (P = 0.0056), but not BRO-negative isolates (P = 0.1333). We provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and ß-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant).

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens.

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing ß-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l(-1)) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

TNF-α -857C>T genotype is predictive of clinical response after treatment with definitive 5-fluorouracil/cisplatin-based chemoradiotherapy in Japanese patients with esophageal squamous cell carcinoma.

BACKGROUND: Genotypes of tumor necrosis factor alpha (TNF-α) and its surface receptors, TNFRSF1A and TNFRSF1B, have been examined in terms of the progression, metastasis, clinical efficacy, and prognosis of various cancers; however, little is known about their effects on clinical outcome in patients with esophageal squamous cell carcinoma (ESCC). In this study, TNF-α and TNFRSF1A genotypes were retrospectively evaluated in terms of predicting clinical response, long-term survival, and severe acute toxicities in 46 male Japanese ESCC patients treated with definitive 5-fluorouracil (5-FU)/cisplatin (CDDP)-based chemoradiotherapy (CRT). METHODS: A course consisted of the continuous infusion of 5-FU at 400 mg/m(2)/day for days 1-5 and 8-12, the infusion of CDDP at 40 mg/m(2)/day on days 1 and 8, and radiation at 2 Gy/day on days 1-5, 8-12, and 15-19, with a second course being repeated after a 2-week interval. The TNF-α -1031T>C (rs1799964), -863C>A (rs1800630), -857C>T (rs1799724), -308G>A (rs1800629), -238G>A (rs361525), TNFRSF1A -609G>T (rs4149570), and 36A>G (rs767455) genotypes were evaluated. RESULTS: The TNF-α -857C>T genotype was found to be predictive of clinical response, i.e., complete response or not (P = 0.010, Fisher's exact test), but had no effect on long-term survival (CC(-857) vs. CT(-857) + TT(-857), P = 0.072, Fisher's exact test, P = 0.070, Log-rank test). CONCLUSIONS: The TNF-α -857C>T genotype was found to be predictive of clinical response and was more likely to predict long-term survival in Japanese ESCC patients receiving definitive 5-FU/CDDP-based CRT. Further clinical investigations with a larger number of patients or experiments in vitro should be performed to assess the predictive value of this genotype following CRT.

L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis.

Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca(2+) channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA.

Molecular characterization of carbapenemase-producing clinical isolates of Enterobacteriaceae in a teaching hospital, Japan.

We examined the molecular characteristics of 13 phenotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates, including the relationships between plasmid-mediated quinolone-resistance genes (qnr), 6'-N-aminoglycoside acetyltransferase-encoding genes [aac(6')] and AmpC-encoding genes (pAmpC). Twelve isolates were bla(IMP-1) positive (92.3%), while one was bla(IMP-11) positive (7.7%). We detected qnr, aac(6') and pAmpC genes designated bla(ACT-1)-like in 76.9%, 100% and 53.8%, respectively, of the 13 isolates. Plasmids were transferred successfully for three of the 13 metallo-ß-lactamase (MBL)-producing isolates, and the sizes of plasmids extracted from these donors and transconjugants were deduced to be 65 kb or 70 kb. OmpC or OmpF protein expression was reduced in all Enterobacter cloacae, and one Klebsiella oxytoca lacked OmpK36. We demonstrate what appears to be the first evidence that, in Japan, Enterobacteriaceae producing MBLs carry various plasmid-mediated resistance genes, which may cause a further decrease in carbapenem susceptibility through reduction of the expression of outer-membrane proteins.

Role of Moraxella catarrhalis outer membrane protein CD in bacterial cell morphology and autoaggregation.

Moraxella catarrhalis, an important pathogen in the human respiratory tract, causes otitis media and lower respiratory tract infections. M. catarrhalis outer membrane protein CD (OMPCD) is a major heat-modifiable OMP with demonstrable potential as a vaccine candidate. The gene encoding OMPCD of M. catarrhalis strains was subjected to nucleotide sequence analysis and then inactivated by insertional mutagenesis. The ompCD mutant strains exhibited a modest growth defect in comparison with the wild-type strains. In optical microscopy and scanning/transmission electron microscopy examinations, regarding morphology, the cell size and cell wall of the ompCD mutant strains were significantly larger and thinner, respectively, than those of the wild-type strain. Furthermore, the ompCD mutant strains exhibited significant autoaggregation and increased surface hydrophobicity, in addition to a reduction in the adherence to HEp-2 cells, compared to the wild-type strains. Strains repaired by replacing the mutated ompCD gene exhibited phenotypic characteristics very similar to those of the wild-type strains. These results indicate that M. catarrhalis OMPCD, in addition to its functions related to bacterial growth and adherence to human epithelial cells, plays a very important role in bacterial physiology and pathogenesis, including aspects such as stabilizing bacterial cell morphology and preventing autoaggregation by reducing surface hydrophobicity.

Potential tumor markers of renal cell carcinoma: α-enolase for postoperative follow up, and galectin-1 and galectin-3 for primary detection.

The diagnosis of renal cell carcinoma is currently based on imaging techniques, mainly because there is no blood marker available for its detection. Thus, there is still the need for the development of novel tumor markers. We examined plasma levels of eight proteins in 15 renal cell carcinoma patients before and after surgery, and in 51 healthy controls using enzyme-linked immunosorbent assay. Plasma levels of α-enolase, calnexin, galectin-1, galectin-3 and lectin mannose-binding 2 were significantly higher in renal cell carcinoma patients than in controls (P < 0.05). Among these proteins, the sensitivities for galectin-1 and galectin-3 were higher than those for calnexin and lectin mannose-binding 2 in the specificity range from 80% to 100%. A combined use of galectin-1 and galectin-3 showed 98% specificity and 47% sensitivity. In addition, the assays showed that plasma α-enolase levels decreased significantly 4 weeks after nephrectomy (P = 0.0034), and this tendency continued until 12 weeks after nephrectomy (P = 0.0156). These findings suggest that α-enolase could be used in the postoperative follow up of renal cell carcinoma patients, whereas the combined use of galectin-1 and galectin-3 might represent a useful tool for primary detection.

Cytotoxicity of 15-deoxy-Δ(12,14)-prostaglandin J(2) through PPARγ-independent pathway and the involvement of the JNK and Akt pathway in renal cell carcinoma.

INTRODUCTION: Agonists of peroxisome proliferator-activated receptor gamma (PPARγ) have been examined as chemopreventive and chemotherapeutic agents. The aim was to investigate the cytotoxicity and action mechanisms of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), one of endogenous ligands for PPARγ, in terms of PPARγ-dependency and the mitogen-activated protein kinase (MAPK) and Akt pathway in three human renal cell carcinoma (RCC)-derived cell lines. METHODS: 786-O, Caki-2 and ACHN cells were used as human RCC-derived cell lines. Cell viability and caspase-3 activity was detected by fluorescent reagents, and chromatin-condensation was observed with a brightfield fluorescent microscope after staining cells with Hoechst33342. The expression levels of proteins were detected by Western blot analysis. RESULTS: 15d-PGJ(2) showed cytotoxicity in dose-dependent manner. 15d-PGJ(2) induced chromatin-condensation and elevated caspase-3 activity, and the cell viability was restored by co-treatment with a pan-caspase inhibitor, Z-VAD-FMK, indicating the involvement of caspase-dependent apoptosis. The cytotoxicity was not impaired by a PPARγ inhibitor, GW9662, suggesting that 15d-PGJ(2) exerted the cytotoxicity in a PPARγ-independent manner. Some antioxidants rescued cells from cell death induced by 15d-PGJ(2), but some did not, suggesting that reactive oxygen species (ROS) did not contribute to the apoptosis. 15d-PGJ(2) also increased the expression levels of phospho-c-Jun N terminal kinase (JNK) in Caki-2 cells, and decreased those of phospho-Akt in 786-O cells, indicating that the JNK MAPK and the Akt pathways participated in the anticancer effects of 15d-PGJ(2) in some cell lines. CONCLUSION: 15d-PGJ(2) exerted cytotoxic effects accompanying caspase-dependent apoptosis, and this effect was elicited in a PPARγ-independent manner in three cell lines. In addition, the JNK MAPK and Akt pathway was involved in the cytotoxicity of 15d-PGJ(2) to some extent in some cell line. Therefore, our study showed the 15d-PGJ(2) to potentially be an interesting approach for RCC treatment.

Function of adrenomedullin in inflammatory response of liver against LPS-induced endotoxemia.

Adrenomedullin (AM) is a hypotension-causing peptide that was originally isolated from human pheochromocytoma cells, and it has been found to be expressed in various organs, including the liver. As the individual physiological and pathophysiological properties of AM peptide in the liver during endotoxemia in vivo has not yet been examined, we investigated this in experimental endotoxemia using heterozygote AM-deficient (AM(+/-)) mice. The AM concentration of AM(+/-) mice was significantly lesser than that of wild-type (WT) mice in lipopolysaccharide (LPS)-induced endotoxemia. After administering LPS, the survival rate for AM(+/-) mice was significantly lower than that for WT mice. Also, expressions of IL-1ß mRNA, and TNF-α mRNA, and NF-κB p65 in the liver were markedly increased and serum ALT greatly elevated in comparison with WT mice. However, supplementation of exogenous AM reversed the deteriorations in mortality and inflammatory responses. Therefore, we conclude that AM plays an important role in regulating systemic inflammation and may be an important intrinsic factor for protecting against liver damage in LPS-induced endotoxemia.