Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase.

BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.

Low Amino Acid Score of Breakfast is Associated with the Incidence of Cognitive Impairment in Older Japanese Adults: A Community-Based Longitudinal Study.

BACKGROUND: The protein digestibility-corrected amino acid score (PDCAAS) represents the degree of utilizable dietary protein, namely the protein quality. The PDCAAS of a diet is required to be evaluated on a meal-by-meal basis, as food digestion and absorption occur with each meal intake. Although a positive association between protein intake and cognitive function has been reported, no study has investigated the association between PDCAAS of a diet and cognitive function. OBJECTIVES: To investigate the relationship between PDCAAS of a diet and cognitive impairment in older adults. DESIGN: Longitudinal epidemiological study. SETTING: Community-based setting. PARTICIPANTS: We analyzed 541 community-dwellers who participated in both baseline and follow-up survey. They were 60-83 years of age without cognitive impairment at baseline. MEASUREMENTS: Cognitive impairment was defined as a Mini-Mental State Examination (MMSE) score ≤27. Individual PDCAASs were calculated for each of three regular meals from the 3-day dietary records at baseline. Participants were classified into two groups according to the sex-specific tertiles (T1-T3) of the PDCAAS for each meal (i.e., T1 as the low score group and T2-T3 as the medium and high score group). The dependent variable was cognitive impairment observed after 4 years, and the explanatory variables were the PDCAAS groups for each meal (the medium and high group as the reference) and covariates (sex, age, body mass index, education, depressive symptoms, medical history, protein intake at each meal, and the MMSE score at baseline). Multivariable logistic regression analysis was performed to evaluate the low PDCAAS group for cognitive impairment after 4 years. RESULTS: A significant association was observed only between a low PDCAAS of breakfast and the incidence of cognitive impairment (the adjusted odds ratios [95% confidence intervals] of low PDCAAS for cognitive impairment for breakfast, lunch, and dinner were 1.58 [1.00-2.50], 0.85 [0.54-1.34], and 1.08 [0.71-1.65], respectively). CONCLUSION: A lower PDCAAS of breakfast, i.e., a diet with poor quality of protein, was associated with the incidence of cognitive impairment in older adults of the community.

Neutron Laue diffraction study on the magnetic phase diagram of multiferroic MnWO4 under pulsed high magnetic fields.

We have combined time-of-flight neutron Laue diffraction and pulsed high magnetic fields at the Spallation Neutron Source to study the phase diagram of the multiferroic material MnWO(4). The control of the field-pulse timing enabled an exploration of magnetic Bragg scattering through the time dependence of both the neutron wavelength and the pulsed magnetic field. This allowed us to observe several magnetic Bragg peaks in different field-induced phases of MnWO(4) with a single instrument configuration. These phases were not previously amenable to neutron diffraction studies due to the large fields involved.

The Association between Dietary Amino Acid Intake and Cognitive Decline 8 Years Later in Japanese Community-Dwelling Older Adults.

OBJECTIVES: Previous studies have reported a relationship between low protein intake and cognitive decline and have suggested that this association may be related to specific amino acid intake. However, the effects of amino acid intake on the maintenance of cognitive function have yet to be clarified. We examined the longitudinal association between dietary amino acid intake and cognitive function in community-dwelling older adults. DESIGN: Longitudinal epidemiological study. SETTING: Community-based setting. PARTICIPANTS: This study comprised 427 study participants aged 60-82 years with no cognitive decline, defined as a Mini-Mental State Examination (MMSE) score of >27 at baseline, who also participated in a follow-up. The average and standard deviation of the follow-up period was 8.2 ± 0.3 years. MEASUREMENTS: Dietary intake was assessed using three-day dietary records at baseline. Participants were classified into quartiles (Q1-Q4) based on the intake of 19 amino acids for males and females. Next, we classified participants into Q1 and Q2-Q4 groups. Cognitive function was assessed using the MMSE both at baseline and at follow-up. Multivariable logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between the Q1 group and cognitive decline (MMSE ≤27), using the Q2-Q4 group as a reference group. Covariates were age, sex, body mass index, years of education, severity of depressive symptoms, history of lifestyle diseases (hypertension, dyslipidemia, diabetes mellitus, stroke, and ischemic heart disease), energy intake (kcal/d), protein intake (g/d), and MMSE score at baseline. RESULTS: Cognitive decline was present in 133 (31.1%) participants. After adjustment for covariates, including total protein intake, the ORs (95% CIs) for cognitive decline were 2.40 (1.21-4.75) for lysine, 2.05 (1.02-4.09) for phenylalanine, 2.18 (1.09-4.34) for threonine, and 2.10 (1.06-4.15) for alanine. CONCLUSION: The results suggest that lysine, phenylalanine, threonine, and alanine intake is important for the maintenance of cognitive function in older people, independent of total protein intake.

Oral environmental factors affecting number of microbes in saliva of complete denture wearers.

The purpose of this study was to clarify which oral environmental factors affected number of microbes in saliva in an edentulous environment. We enrolled 68 edentulous subjects in the study. Numbers of total anaerobic bacteria and Candida species in saliva were determined. Age, sex, un-stimulated salivary flow rate, pH and viscosity of saliva, histatin level in saliva, tongue coating status, tongue pressure, denture plaque status, material of denture base, duration of edentulism, frequency of self oral health care and number of cigarettes per day were also investigated as oral environmental factors. Correlation between number of total anaerobic bacteria or Candida species and each oral environmental factor was determined with the Spearman rank correlation coefficient. Stepwise logistic regression analysis was used to identify which factors were significantly associated with level of total anaerobic bacteria and Candida species. Correlation and stepwise logistic regression analyses revealed associations between un-stimulated salivary flow rate, tongue coating status, denture plaque status or frequency of self oral health care and number of total anaerobic bacteria. The correlation analysis showed a significant correlation between age and number of total anaerobic bacteria. Stepwise logistic analysis revealed associations between pH of saliva or viscosity of saliva and level of anaerobic bacteria; it also revealed associations between histatin level in saliva or un-stimulated salivary flow rate and level of Candida species. We conclude that salivary flow rate, in particular, affects number of salivary microbes in an edentulous environment.

The significance of gastrectomy in advanced gastric cancer patients with non-curative factors.

BACKGROUND: The role of gastrectomy in the treatment of advanced gastric cancer patients with non-curative factors remains controversial. We investigated prognostic factors and evaluated the role of gastrectomy in such patients. PATIENTS AND METHODS: Eighty-eight advanced gastric cancer patients with non-curative factors were prospectively studied. The patients were categorized into the following two groups: Group A: 52 patients who underwent gastrectomy and subsequently received chemotherapy, Group B: 36 patients who received chemotherapy alone. RESULTS: The median survival times of group A and B patients were 351 and 182 days, respectively (p=0.008). Multivariate analysis showed that gastrectomy was the only positive independent prognostic factor, with no effect on the results of chemotherapy. There was no significant difference in the duration of hospital stay between patients of the two groups, while significantly longer maintenance of oral intake was observed for group A. CONCLUSION: In advanced gastric cancer patients with non-curative factors, gastrectomy was beneficial for survival with longer maintenance of oral intake.

FDG-PET predicts treatment efficacy and surgical outcome of pre-operative chemoradiation therapy for resectable and borderline resectable pancreatic cancer.

BACKGROUND: The efficacy of neoadjuvant chemoradiotherapy (NACRT) for resectable and borderline resectable pancreatic cancer is important for predicting outcomes after radical surgery, but few clinical indicators predict outcome before resection. This study examined the utility of FDG-PET in predicting the efficacy of NACRT and outcome after radical surgery. METHODS: Eighty-three pancreatic cancer patients who underwent FDG-PET before and after NACRT and had positive standard uptake values (SUVs) before NACRT were enrolled in this study. Peri-operative clinical factors, including FDG-PET findings, were examined to predict the efficacy of NACRT and outcome after surgery. RESULTS: Evans grade I, IIA, IIB, III, and IV was determined in 11, 31, 27, 11, and 3 patients, respectively. The maximum SUVs after NACRT (post SUV-max) and tumor size were significantly decreased compared to pretreatment values (p < 0.001 and p = 0.007, respectively). The post SUV-max and regression index were significantly related to grade III/IV (p = 0.04 and p < 0.001, respectively), but only the regression index predicted NACRT efficacy (p = 0.002). The AUC of the regression index for the detection of grade III/IV was 0.822, and 13 of 14 grade III/IV patients were picked up using 50% as the threshold (p < 0.001). Patients with a regression index >50% had a significantly better prognosis after radical resection than patients with <50% (p = 0.032). Regression index as well as pathological lymph node status and resectability status were independent prognostic factors in multivariate analysis (exp 2.086, p = 0.043). CONCLUSION: The regression index is potentially a good indicator of the efficacy of NACRT and outcome after radical resection for pancreatic cancer.

Increased intensity of acute graft-versus-host disease after reduced-intensity bone marrow transplantation compared to conventional transplantation from an HLA-matched sibling in children.

Eight children underwent reduced-intensity stem cell transplantation (RIST) from an HLA-matched sibling. They received a fludarabine-melphalan based preparative regimen. Stem cell source was bone marrow, and GVHD prophylaxis consisted of cyclosporine A alone. Acute GVHD grade II-IV and grade III-IV were observed in four (50%) and three (37.5%), respectively, out of these eight patients. This incidence was significantly higher than that after conventional bone marrow transplantation, without severe tissue damage, in the same setting of stem cell source and GVHD prophylaxis. Although the number of patients is small, our results suggest that incidence of acute GVHD after RIST for children is significant. It should be remembered that RIST for children does not seem to be an easy transplant procedure from the viewpoint of acute GVHD, although RIST is less toxic.

Translesional synthesis on a DNA template containing N2-methyl-2'-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I.

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N2-methyl-2'-deoxyguanosine (N2-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N2-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N2-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N2-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3'-terminus of a dT:N2-Me-dG pair was only 2.1 times lower than from a dC:N2-Me-dG pair. We conclude that N2-Me-dG is a miscoding lesion capable of generating G-->A transition mutations.

Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA). METHODS: Purified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay. RESULT: AQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes. CONCLUSION: Taken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.

Novel prognostic prediction models for patients with stage IV colorectal cancer after concurrent curative resection.

BACKGROUND: We developed a prediction tool for recurrence and survival in patients with stage IV colorectal cancer (CRC) following surgically curative resection. PATIENTS AND METHODS: From January 1983 to December 2012, 113 patients with CRC and synchronous liver and/or lung metastatic CRC were investigated at the Osaka Medical Center for Cancer and Cardiovascular Diseases. All patients underwent curative resection of primary and metastatic lesions. In the group of patients who underwent surgery from 1983 to 2008, a Cox regression model was used to develop prediction models for 1-year, 3-year and 5-year cancer-specific survival (CSS) and relapse-free survival (RFS). In the other group of patients who underwent surgery from 2009 to 2012, the developed prediction model was validated. RESULTS: Univariate analysis of clinicopathological factors showed that the following factors were significantly correlated with CSS and RFS: preoperative serum carcinoembryonic antigen level, tumour location, pathologically defined tumour invasion and lymph node metastasis, and synchronous metastatic lesions. Using these variables, novel prediction models predicting CSS and RFS were constructed using the Cox regression model with concordance indexes of 0.802 for CSS and 0.631 for RFS. The prediction models were validated by external data sets in an independent patient group. CONCLUSIONS: We developed novel and reliable personalised prognostic models, integrating tumour, node, metastasis (TNM) factors as well as the preoperative serum carcinoembryonic antigen level, tumour location and metastatic lesions, to predict patients' prognosis following surgically curative resection. This individualised prediction model may help clinicians in the treatment of postoperative stage IV CRC following surgically curative resection.

A new crystal form and preliminary crystallographic data for ferricytochrome c' from Rhodospirillum rubrum.

A new crystal form of ferricytochrome c' from the photosynthetic bacteria, Rhodospirillum rubrum, has been obtained by dialysing protein solution against polyethylene glycol 4000. The crystals belong to the space group P61 (or its enantiomorph P65) with unit cell dimensions: a = b = 51.63 A and c = 155.39 A. The asymmetric unit contains one dimer molecule of 28,000 molecular weight and the solvent content of the unit cell is approximately 38%.

Unrelated donor bone marrow transplantation for 100 pediatric patients: a single institute's experience.

In all, 100 unrelated donor bone marrow transplantations (UD-BMT) were performed in our institute between October 1993 and January 2003. Of 93 evaluable patients, 73 patients had hematological malignancy, 13 had nonmalignancy and seven had lymphoproliferative disease. The estimated 9-year event-free survival (EFS) rate was 57.1+/-5.5% in all patients. In the following analyses of the patients with hematological malignancy, the standard group had significantly better EFS than the high-risk group (61.5+/-7.0 vs 35.6+/-9.7%, P=0.02), and the EFS rate of the tacrolimus (FK-506)+methotrexate (MTX)+/-methylprednisolone prophylactic group for graft-versus-host disease was superior to that of the FK-506 without MTX group (75.7+/-8.0 vs 55.8+/-7.6%, P=0.02). When we compared the EFS rates of the FK506+MTX+/-methylprednisolone (mPSL) group and the HLA-matched related donor BMT group in our institute, these were almost similar (75.7+/-8.1 vs 68.4+/-9.3%). Therefore, UD-BMT using FK-506+MTX+/-mPSL is a safe and useful method for children with hematological malignancy who require allogeneic BMT.

Cytotoxic chemotherapy successfully induces durable complete remission in 2 patients with mosquito allergy resulting from Epstein-Barr virus-associated T-/natural killer cell lymphoproliferative disease.

Recent findings indicate that Epstein-Barr virus (EBV)-infected T-/natural killer (NK) cells play an important role in the pathogenesis of mosquito allergy, and most patients with mosquito allergy die early in life if not properly treated. Over the last 7 years, we have been using combination chemotherapy and allogeneic stem cell transplantation for the treatment of EBV-associated T-/NK cell lymphoproliferative disease (LPD) in which chronic active EBV infection and mosquito allergy were included. As of this writing, we have successfully treated 2 patients with mosquito allergy with chemotherapy in which EBV-infected T-/NK cells were eradicated. The findings suggest the possible role of chemotherapy in the treatment of EBV-associated T-/NK cell LPD.

Cyber visual training as a new method for the mastery of endoscopic surgery.

BACKGROUND: We devised a new method incorporating cyber visual training whereby novices in endoscopic surgery are instructed via repetition of a video-recorded procedure. We then conducted a study designed to investigate the impact of this cyber visual training on the mastery of intracorporeal knot-tying as an endoscopic technique. METHODS: For the cyber visual training a 10-min video of the same procedure was replayed at normal, slow, and rapid speeds or presented in a series of still images. The training was undertaken by 36 medical students and 1st year trainee doctors who had had no experience of endoscopic surgery. They were divided into three groups, each of all received the same introductory lecture. Group A was only given training with the instructor for 15 min. Group B trained with the instructor for 15 min and was allowed self-training for 10 min. Group C viewed the cyber video beforehand and then underwent training with the instructor for 15 min. For all participants, the time required to complete a knot-tying task was measured and the level of endoscopic skill before and after the training was assessed using a virtual reality system the minimally Invasive Surgical Trainer-Virtual Reality (MIST-VR), in terms of the following three parameters: time, errors, and efficiency of hand movements. The Steel-Dwass test was used to evaluate the differences among the three groups in task performance. RESULTS: Group C completed the knot-tying task faster than group A (p = 0.0375), but there were significant differences between groups A and B and groups B and C. There were no significant differences in the parameters assessed using the MIST-VR. CONCLUSIONS: Our new concept of cyber visual training is effective for mastering the knot-tying technique. This type of training should be widely applicable to other procedures, such as dissection, clipping, and hemostasis.

Epidermodysplasia verruciformis: viral particles in early malignant lesions.

Two cases of epidermodysplasia verruciformis were studied histologically and electron microscopically. Four lesions examined were histologically benign, and had viral particles morphologically similar to that of common human warts. Two lesions on the forehead and the face were histologically in the stages of malignant transformation, the intraepidermal epithelioma and the early invasive squamous cell carcinoma. Similar viral particles were also observed in the upper layers of these 2 lesions. These results suggest that at least some of the virus-induced lesions of epidermodysplasia verruciformis actually become malignant.

N-copine: a novel two C2-domain-containing protein with neuronal activity-regulated expression.

Neuronal activity is often associated with changes in gene expression. By a two-dimensional cDNA-display system, restriction landmark cDNA scanning, we identified a novel gene whose expression in the hippocampus was up-regulated by kainate stimulation. The mRNA expression was detected only in brain and up-regulated by the stimulation evoking CA3-CA1 long-term potentiation. The encoded protein contains two copies of C2-domain, known as the Ca2+-binding domain of PKC-gamma, and shows 49% identity with human copine I. We designated this protein N-copine (neuronal-copine). N-copine may have a role in synaptic plasticity.

Covalently bound flavin in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.

Na(+)-translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS-PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow-green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. N-Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.