Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Particle size specific distribution of perfluoro alkyl substances in atmospheric particulate matter in Asian cities.

Seasonal and local characteristics of perfluorinated alkylated substances (PFASs) were examined using size-segregated particles including an ultrafine range. The examination included sampling and analysis of ambient particles collected at four sites located in different environments in three different countries, Japan (Kanazawa and Okinawa), Hong Kong and India. To minimize the evaporation artefacts derived from PFASs during the sampling, an air sampler that permitted particles smaller than 0.1 µm (PM0.1) to be separated at a moderate pressure drop (<5-15 kPa), was used for all of the air sampling procedures. In the case of Kanazawa, a local city in Japan, the concentration of PFASs was found to be dominated by carboxylates, especially PFOA, PFNA and PFDA regardless of the particle size and sampling period. Ultrafine particles were found to be the largest contributor to the mass fraction of PFCAs, while the maximum PFOS mass fractions were determined to be in the coarse-sized fractions. The seasonal difference in the total PFAS concentration can be largely attributed to precipitation. The results were basically similar for all sites that were examined. The type of land use may be a more influencing factor on the mass fraction of the PFASs than the country of origin. The dependency of PFAS mass fraction on the specific surface of the particle suggests that ultrafine PFAS particles are segregated, not only by gas deposition but could also be segregated by a mechanism involving compositional dependence or the primary source of the particles. Other possible sources of PFASs, other than from traffic are also possible.

Retention of tocilizumab and anti-tumour necrosis factor drugs in the treatment of rheumatoid arthritis.

OBJECTIVES: The retention of the anti-rheumatic agent tocilizumab (TCZ) has not been well documented in patients with rheumatoid arthritis (RA). We conducted an observational study to compare the retention of TCZ and anti-tumour necrosis factor (TNF) drugs in the treatment of patients with RA. METHOD: We reviewed continuation rates and causes of discontinuation of biological agents (biologics) by assessing medical records of patients with RA who were administered biologics at our institute from September 1999 to April 2012, using the Osaka University Biologics for Rheumatic Diseases (BiRD) registry. RESULTS: A total of 401 patients were included. TCZ, infliximab (IFX), etanercept (ETN), and adalimumab (ADA) were administered to 97, 103, 143, and 58 patients, respectively. There were some differences between the baseline characteristics of the groups. The median duration (range) of TCZ, IFX, ETN, and ADA administration was 2.5 (0.1-12.6), 1.9 (0.0-7.7), 2.9 (0.0-11.3), and 1.3 (0.0-3.4) years, respectively. Continuation rates for TCZ and ETN were significantly higher than those for IFX and ADA. Multivariate analyses showed that discontinuation due to lack or loss of efficacy was significantly less common in the TCZ group than in the other groups. Discontinuation due to overall adverse events was not significantly different between treatment groups. CONCLUSION: TCZ and ETN show better retention than IFX or ADA in the treatment of RA.

Development of double-decker pulse radiolysis.

Double-decker pulse radiolysis (DDPR), which utilizes double-decker electron beams, was investigated to develop a new pulse radiolysis with a high time resolution. The double-decker electron beams were generated by injecting two UV pulses into a photocathode radio-frequency gun. In the pulse radiolysis, one electron beam was used as a pump beam, and the other was converted to a probe pulse. Finally, as its first application, the DDPR was successfully used for observing solvated electrons in water, with a 10%-90% rise time of 8.6 ps.

Effect of GDF-5 and BMP-2 on the expression of tendo/ligamentogenesis-related markers in human PDL-derived cells.

OBJECTIVES: The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers. MATERIALS AND METHODS: Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control. RESULTS: Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal ligament-derived cells. CONCLUSION: Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament-derived cells. The expression of scleraxis was higher in STRO-1-negative fraction, suggesting more differentiated state of the cells.

A doxycycline loaded, controlled-release, biodegradable fiber for the treatment of aortic aneurysms.

The pathogenesis of aortic aneurysm (AA) is characterized by degradation of extracellular matrix with increased matrix metalloproteinases (MMPs) and inflammatory reaction. Doxycycline (DOXY) has been reported to control the extension of AA by regulation of MMP. However, systemic administration may cause adverse side effects. In this study, we demonstrated the possibility of local administration of DOXY controlled-release biodegradable fiber (DCRBF) for AA in mice. DCRBF was fabricated by biodegradable polymer (polylactic acid; PLA) mixed with DOXY using an electrospinning technique. DCRBF was cocultured with SMCs, macrophages and aortic tissue, and placed on an abdominal aortic aneurysm which induced apolipoprotein E-deficient mice. We evaluated gene and protein expression of proteases, elastin and inflammatory markers. In the presence of DCRBF, MMP-12 was significantly decreased, TGF-ß1 and Lox were significantly increased in SMC gene expression, MMP-9 and -12 significantly decreased gene expression of macrophages. The DCRBF preserved elastin content and decreased MMP-2 and -9 in aortic tissue. In addition, IGF-1 and TIMP-1 were significantly increased and IL-6 and TNF-α were significantly decreased with DCRBF in vivo. In conclusion, our results suggested that local administration of DCRBF may become a promising alternative therapeutic strategy for AA.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Serum levels of soluble vascular endothelial growth factor receptor-2 in patients with systemic sclerosis.

BACKGROUND: Although vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (KDR) signalling may play a major role in the microangiopathy of systemic sclerosis (SSc), serum levels of soluble KDR (sKDR) in this disease have not yet been determined. OBJECTIVES: To evaluate the possibility that serum sKDR levels can be a specific disease marker of SSc. METHODS: Serum sKDR levels of 42 patients with SSc, 10 patients with Raynaud's phenomenon (RP) and 22 healthy controls were measured with specific enzyme-linked immunosorbent assays. Quantitative real-time polymerase chain reaction (PCR) was performed to determine KDR mRNA levels. RESULTS: In females, the serum sKDR levels were significantly higher in patients with SSc, especially limited cutaneous SSc, than in patients with RP or healthy controls. Quantitative real-time PCR with RNA from skin sections revealed that KDR mRNA levels were also increased in the skin of patients with SSc with elevated serum sKDR levels. A significantly lower prevalence of pulmonary fibrosis, higher percentage vital capacity, and a higher incidence of telangiectasia were seen in female patients with SSc with elevated serum sKDR levels than those with normal levels. CONCLUSIONS: These results suggest that the skin can be one of the sources of elevated serum sKDR levels, and that serum sKDR levels are useful for diagnosis and may be a marker of microangiopathy in patients with SSc, especially females. The VEGF-A/KDR signalling system may be involved in the pathogenesis of the disease.

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

Quantification of hardness, elasticity and viscosity of the skin of patients with systemic sclerosis using a novel sensing device (Vesmeter): a proposal for a new outcome measurement procedure.

OBJECTIVES: No objective method to measure skin involvement in SSc has been established. We developed a novel method using a computer-linked device to simultaneously quantify physical properties of the skin such as hardness, elasticity and viscosity. METHODS: Skin hardness was calculated by measuring the depth of an indenter pressed onto the skin. The Voigt model was used to calculate skin elasticity, viscosity, visco-elastic ratio and relaxation time by analysing the waveform of skin surface behaviour. The results were compared with the modified Rodnan skin score (mRSS) obtained at 17 sites on the bodies of 20 SSc patients and 20 healthy controls. A functional assessment questionnaire was administered to determine how skin hardness represents a patient's disability. We also examined intra- and inter-observer variability to determine the reliability of this method. RESULTS: The crude hardness obtained with this device correlated well with the standard hardness specified by the American Society for Testing and Materials (ASTM, r = 0.957). A close relationship between hardness and total mRSS was also observed (r = 0.832). Skin elasticity correlated positively, and relaxation time negatively with mRSS. Functional disability correlated more closely with skin hardness (r = 0.643) than with mRSS (r = 0.517). Intra- and inter-observer variabilities were 7.63 and 19.76%, respectively, which were lower than those reported for mRSS. CONCLUSIONS: Increases in hardness and elasticity as well as shortening of relaxation time constitute objective characteristics of skin involvement in SSc. The system devised by us proved to be able to assess skin abnormalities of SSc with high reliability.

Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models.

Polyoxomolybdates (PMs) as discrete molybdenum-oxide cluster anions have been investigated in the course of study of their medical applications. Here, we show the significant antitumour potency of the polyoxomolybdate [Me(3)NH](6)[H(2)Mo(V)(12)O(28)(OH)(12)(Mo(VI)O(3))(4)].2H(2)O (PM-17), which is a photo-reduced compound of [NH(3)Pr(i)](6)[Mo(7)O(24)].3H(2)O. The effect of PM-17 on the growth of cancer cell lines and xenografts was assessed by a cell viability test and analysis of tumour expansion rate. Morphological analysis was carried out by Hoechst staining, flow-cytometric analysis of Annexin V staining, terminal deoxynucleotidyl transferase-mediated 'nick-end' labelling staining, and electron-microscopic analysis. Activation of autophagy was detected by western blotting and fluorescence-microscopic analysis of the localisation of GFP-LC3 in transfected tumour cells. PM-17 inhibited the growth of human pancreatic cancer (AsPC-1) xenografts in a nude mice model, and induced morphological alterations in tumour cells. Correspondingly, PM-17 repressed the proliferation of AsPC-1 cells and human gastric cancer cells (MKN45) depending on the dose in vitro. We observed apoptotic patterns as the formation of apoptotic small bodies and translocation of phosphatidylserine by Hoechst staining and flow-cytometric analysis following Annexin V staining, and in parallel, autophagic conformation by the formulation of autophagosomes and localisation of GFP-LC3 by electron- and fluorescence-microscopic analysis.

Biotransformation and cytotoxicity of a brominated flame retardant, tetrabromobisphenol A, and its analogues in rat hepatocytes.

The metabolism and cytotoxic effects of tetrabromobisphenol A (TBBPA), a phenolic flame retardant, and its analogues were studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria, respectively. The exposure of hepatocytes to TBBPA caused not only concentration (0.25-1.0 mM)- and time- (0-3 h) dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation. TBBPA at a weakly toxic level (0.25 mM) was metabolized to monoglucuronide and monosulfate conjugates: the amounts of glucuronide rather than sulfate conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In comparative effects based on cell viability, mitochondrial membrane potential and some toxic parameters, bisphenol A (BPA) was less toxic than TBBPA and tetrachlorobisphenol A (TCBPA), which are not significant differences in these parameters. In mitochondria isolated from rat liver, TBBPA and TCBPA caused an increase in the rate of State 4 oxygen consumption in the presence of succinate, indicating an uncoupling effect and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner (5-25 microM). Taken collectively, our results indicate that (i) mitochondria are target organelles for TBBPA, which elicits cytotoxicity through mitochondrial dysfunction related to oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (ii) the toxicity of TBBPA and TCBPA is greater than that of BPA, suggesting the participation of halogen atoms such as bromine and chlorine in the toxicity.

Laparoscopic gastrectomy with regional lymph node dissection for upper gastric cancer.

BACKGROUND: The technique and results of laparoscopic gastrectomy in 110 patients with gastric cancer located in the upper third of the stomach are presented. METHODS: Proximal gastrectomy was performed for lesions in the upper third of the stomach, and total gastrectomy for those that spread over both the upper and middle third. D1 and D2 lymph node dissection was undertaken in patients with T1 or T2 lesions. Anastomosis of the oesophagus was performed intracorporeally using a conventional circular stapling device or a laparoscopic linear stapler. RESULTS: Median operating time was 247 min for proximal gastrectomy and 285 min for total gastrectomy; median blood loss was 207 and 334 ml respectively. A median of 23 lymph nodes was harvested from patients in the proximal gastrectomy group and 34 from those having a total gastrectomy. There was minimal morbidity and fast recovery after surgery. Postoperative recurrence occurred in only one patient, giving a recurrence rate of 0.9 per cent. CONCLUSION: Laparoscopic gastrectomy for upper gastric cancer appears to be a safe and curative procedure.

Brain catecholamine alterations and pathological features with aging in Parkinson disease model rat induced by Japanese encephalitis virus.

We analyzed two disease model groups with rats infected by Japanese encephalitis virus (JEV), a 90-day group and a 180-day group after JEV infection. The time measured by the modified pole test showed that motor activities in these two groups were slower than those of age-matched control groups. Striatal dopamine (DA) levels were significantly decreased in all JEV-infected rats. Norepinephrine concentration in brain regions in the 180-day group was significantly decreased in the medulla oblongata and hypothalamus as compared with the control and 90-day group. Tyrosine hydroxylase-positive neurons were significantly decreased in both JEV-infected rat groups. These results suggest that DA decrease and pathological changes in JEV-infected model rats persist for a long time, at least up to 180 days, and this model will be useful for the evaluation of new anti-parkinsonian agents.

Antitumor activity of polyoxomolybdate, [NH3Pri]6[Mo7O24].3H2O, against, human gastric cancer model.

Polyoxometalates are negatively charged inorganic compounds which contain metal ions such as tungsten, molybdenum, vanadium etc. and which make clusters with the surrounding oxygen atoms. [NH3Pri]6[Mo7O24].3H2O (PM-8) was found to be a significant antitumor polyoxomolybdates. It had already been reported that the PM-8 suppressed the growth of Co-4 human colon cancer, MX-1 human breast cancer and OAT human lung cancer xenografted in nude mice. However, the mechanism of the antitumor activity has not been clarified. In this study, the antitumor activity of one of the metal oxide clusters (polyoxometalates), hexabis(isopropylammonium) heptamolybdate trihydrate, [NH3Pri]6[Mo7O24].3H2O (PM-8) were shown in an MTS assay. DNA ladder formation and detection of apoptotic bodies in nuclei were revealed that antitumor activity of PM-8 in MKN45 cells was due to apoptosis. It is concluded that the observation of significant tumor growth suppression of PM-8 in MKN45-bearing mice results from the induction of apoptosis. PM-8 shows promise as a novel anti-cancer agent.

Anticancer activity of polyoxomolybdate.

Anticancer polyoxomolybdates have been investigated for medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH3Pri]6[Mo7O24].3H2O (PM-8) has been recognized as one of significant antitumoral polyoxomolybdates. PM-8 had shown the growth suppression against several tumors, for examples, Co-4, human colon cancer, MX-1, human breast cancer, and OAT, human lung cancer. PM-8 showed the tumor growth suppression for MKN-45 human gastric cancer in tumor bearing mice. PM-8 inhibited the cell growth of AsPC-1 which depended on the dose with showing DNA ladder formation and DNA fragmentation, and positive Hoechst staining indicating apoptosis. The ratio of apoptotic cells on flow cytometry analysis were 35%, and 57% with treatment of PM-8 after 48, and 72 h, respectively. One of the anti-tumor activity of PM-8 result from the activation of apoptotic pathway. It is thought that polyoxomolybdates will be applied as a novel anti-tumor agent especially against cancers which are difficult to be treated clinically.

Effects of tetrabromobisphenol A, brominated flame retardant, in ICR mice after prenatal and postnatal exposure.

Tetrabromobisphenol A (TBBPA), brominated flame retardant, is produced in the largest amounts globally for use in plastics or building materials. TBBPA has been detected in sediment, air at the dismantling plant or human serum samples. In the present study, we examined the effects of prenatal and postnatal exposure to TBBPA in mice. TBBPA (99.1% pure) in diet was administered to pregnant ICR mice at doses of 0% (control), 0.01%, 0.1% or 1.0% from gestational day 0 to weaning at postnatal day 27. The average daily food intake and body weight of dams showed no significant differences between the control and treated groups. There were no dose-related effects on reproductive data. Serum concentrations of total-cholesterol and liver weights of treated dams and offspring were higher than those of the control mice. Histological findings in treated dams or offspring showed the increase of focal necrosis of hepatocytes and inflammatory cell infiltration in the liver, and increase of dilation or atrophy of renal tubules and cyst in the kidney. TBBPA was developed as a new, safe class of flame retardant and was not highly toxic. However, the present data suggested that TBBPA caused a lipid metabolic disorder and hepatic or kidney lesion, under these conditions.