Porcine Dental Epithelial Cells Differentiated in a Cell Sheet Constructed by Magnetic Nanotechnology.

Magnetic nanoparticles (MNPs) are widely used in medical examinations, treatments, and basic research, including magnetic resonance imaging, drug delivery systems, and tissue engineering. In this study, MNPs with magnetic force were applied to tissue engineering for dental enamel regeneration. The internalization of MNPs into the odontogenic cells was observed by transmission electron microscopy. A combined cell sheet consisting of dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) (CC sheet) was constructed using magnetic force-based tissue engineering technology. The result of the iron staining indicated that MNPs were distributed ubiquitously over the CC sheet. mRNA expression of enamel differentiation and basement membrane markers was examined in the CC sheet. Immunostaining showed Collagen IV expression at the border region between DEC and DMC layers in the CC sheet. These results revealed that epithelial-mesenchymal interactions between DEC and DMC layers were caused by bringing DECs close to DMCs mechanically by magnetic force. Our study suggests that the microenvironment in the CC sheet might be similar to that during the developmental stage of a tooth bud. In conclusion, a CC sheet employing MNPs could be developed as a novel and unique graft for artificially regenerating dental enamel.

Hematological and morphological investigation of thrombogenic mechanisms in the lungs of phenylhydrazine-treated rats.

Abnormality in hematological condition including hemolytic disorders has been suggested one of the risk factor of pulmonary thrombosis. We previously reported that phenylhydrazine (PHZ) could induce acute thrombosis in the rat lung. In this study, time-related hematological and histopathological changes were evaluated in PHZ-treated rats to reveal the pathogenesis of pulmonary thrombosis in hemolytic condition. Male Sprague-Dawley rats were administered PHZ at 40 mg/kg/day daily for up to 4 days (n=6). At 24 h after the last administration (i.e. on days 1, 2, 3, or 4), animals were euthanized and samples were subjected to hematology, light microscopy, and electron microscopy. PHZ-treated rats developed severe anemia on day 1 or later. On day 2 and after, congestion in the alveolar septa corresponding to accumulation of deformed/ghost erythrocytes in the alveolar capillaries was observed, which was the earliest change that preceded thrombus formation. Focal fibrin deposition in the alveolar septa was noted on day 3 and it expanded widely by day 4, while endothelial injury were minimally noted just on day 4. These congestive/thrombotic changes were predominant in the pulmonary capillaries. Changes in hemostatic parameters were noted only on day 4; which were prolonged prothrombin time and activated partial thromboplastin time, greatly increased plasma thrombin-antithrombin complex levels with statistical significance, and slightly decreased fibrinogen levels. In conclusion, the trigger of acute pulmonary thrombosis in PHZ-treated rats was considered to be regional stasis resulting from blockage caused by the deformed erythrocytes, and subsequent systemic hemostatic disruption.

Spontaneous amphophilic focus in the liver of a young Sprague-Dawley rat.

Altered hepatocellular focus was histopathologically observed in the liver of a 6-week-old Sprague-Dawley rat. The hepatocytes within this lesion had diffusely eosinophilic cytoplasm with scattered basophilia and slightly enlarged nuclei with prominent nucleoli. Based on these cytological characteristics, the lesion of these hepatocytes was classified as an amphophilic focus. This is the first report to describe spontaneous amphophilic focus in a young rat.

Effects of Nanpao, a Kampo medicine, on the decline in estrous cyclicity with advancing age in female rats, as measured by vaginal impedance methods.

The present study was designed to evaluate the time-dependent effects of Nanpao, a kampo medicine, on age-related changes in the estrous cycle of female rats, and to investigate the utility of measuring electrical impedance in the vagina (EIV) for studying transitional changes in the estrous cycle. Rats were allocated to 3 groups: control, Nanpao 30 mg/kg/day, and 100 mg/kg/day groups. EIV measurements and cytology samples were taken for 14 days at the age of 6 months before the initial treatment. After the start of the treatment, these data were collected at about monthly intervals until the age of 10 months in the same manner. Observations at the ages of 7 (weeks 2-3 of dosing) and 8 months (weeks 6-7 of dosing) showed that loss of a regular estrous cycle in the 100 mg/kg/day group was inhibited as compared to the control group. Moreover, at the ages of 9 (weeks 11-12 of dosing) and 10 months (weeks 17-18 of dosing), these effects were identified not only in the 100 mg/kg/day group, but also in the 30 mg/kg/day group. Since vaginal cytology and EIV gave almost concordant results as indicators of estrous cyclicity, we concluded that the measurement of EIV was capable of detecting time-dependent changes in the estrous cycle as well as observations of vaginal smears. A short period of Nanpao administration inhibited loss of regular estrous cycles, and the EIV method is a worthwhile approach to a more precise study of estrous cyclicity in rats exhibiting abnormal estrous cycles.

Effects of Nanpao, a kampo medicine, on peripheral blood flow and surface skin temperature in aged female rats.

The effects of long-term treatment with Nanpao, a kampo medicine, on cold constitution were evaluated in aged female rats. Five-month-old rats were administered Nanpao orally at doses of 0, 30, or 100 mg/kg/day. The peripheral blood flow and surface skin temperature in the hind paws were measured using a laser Doppler blood flow meter and infrared thermography, respectively. In animals treated with Nanpao, the peripheral blood flow increased dose-dependently compared to that in the control group. Moreover, the surface skin temperature after immersion in ice-cold water was higher in the Nanpao-treated groups than in the control group at all measurement times. These results suggest that Nanpao has the potential to improve cold constitution associated with decreased peripheral blood flow in women.

Anti-aging effects of nanpao, a kampo medicine, on reproductive functions in female rats.

The improvement effect of nanpao, a kampo medicine, on the age-related decline in reproductive function was evaluated in female rats given the test drug for a long-term period. Young rats were allocated to the cesarean section and natural delivery groups to examine reproductive performance (young rat groups). Five-month-old rats were allocated to the 3 groups (aged rat groups): 1--control and 2--nanpao-treated groups. They were given orally in a dose of 0, 30 or 100mg/kg/day of the test drug, respectively. In aged rats, the first mating experiment was initiated at week 21 of dosing to evaluate reproductive performance by natural delivery and the second mating experiment at week 31 of dosing was evaluated by cesarean section. In the first and second mating experiments, various reproductive functions decreased in aged rats as compared to the young rats. On the other hand, loss of regular estrous cycles, decreases in delivery and pregnancy rates and mean fetal weights were inhibited in the treated groups as compared to the control group. In addition, decreases in the numbers of mean live offspring and fetuses were inhibited in the 100 mg/kg/day group. In conclusion, nanpao maintained normal embryo-foetal development in female rats.

Time-course changes of hematology and clinical chemistry values in pregnant rats.

The aim of this study is to report how pregnancy alters hematology and clinical chemistry values in rats. Female and male Sprague-Dawley rats were mated; the day of copulation was designated as Day 0. Hematology and clinical chemistry measurements were conducted on Days 7, 14, 17 and 21 in pregnant rats. Measurements were also conducted in non-pregnant rats. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), total protein and albumin decreased on Days 7, 14, 17 and 21; sodium, chloride and glucose decreased on Days 14, 17 and 21; iron decreased on Days 17 and 21; hemoglobin content of reticulocytes (CHr), calcium, inorganic phosphorus and the albumin/globulin ratio decreased on Day 21; and total cholesterol, phospholipid and high-density lipoprotein cholesterol decreased on Day 14 in pregnant rats compared with non-pregnant rats. Reticulocyte increased on Days 7, 14 and 17; mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophil count and rate increased on Days 14, 17 and 21; platelets, fibrinogen, triglyceride and free fatty acid increased on Days 17 and 21; and activated partial thromboplastin time was prolonged on Days 17 and 21 in pregnant rats compared with non-pregnant rats. The decreased RBC, Hb, Ht, CHr and iron in pregnant rats indicated that they suffered from iron deficiency anemia. These data can be used as background information for effective evaluation in reproductive toxicology studies.

Development of enzyme immunoassay for detection of DDT.

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.

Effects of Hachimi-jio-gan (Ba-Wei-Di-Huang-Wan) on intestinal function in streptozotocin-induced diabetic rats.

We examined the effects of Hachimi-jio-gan (HJ) on the small intestinal function in streptozotocin (STZ)-induced diabetic rats. The rats had free access to pellets containing 1% HJ extract powder for 4 weeks after STZ administration. The intestinal disaccharidase (sucrase and maltase) activity was elevated in STZ-treated rats compared with control rats, whereas it was significantly reduced by HJ administration. This suggested that HJ suppresses or delays monosaccharide production in the small intestinal epithelium. In addition, the intestinal mucosal weights and DNA contents that were significantly increased in the STZ-treated rats were restrained to the control level by HJ treatment. Simultaneously, we examined the changes in the plasma levels of glucagon-like peptide 2 (GLP-2), which is a trophic factor specific for the intestine. The plasma GLP-2 levels significantly increased in the STZ-treated rats, whereas HJ decreased the plasma GLP-2 levels. Thus intestinal mucosal weights and DNA contents correlated with plasma GLP-2 levels in diabetes-associated bowel growth. These results suggest that HJ may normalize or suppress the small intestinal disaccharidase activity and the epithelial cell proliferation mediated by GLP-2 in the animal model rats.

Hepatic and intestinal changes in rats treated with T-0126, a microsomal triglyceride transfer protein (mtp) inhibitor.

In the present study, we investigated the potential toxic effects of 2-week oral treatment with T-0126, a novel microsomal triglyceride transfer protein (MTP) inhibitor, on the liver and intestine in male and female rats. Administration of T-0126 decreased serum lipids and resulted in fat accumulation in the liver and the small intestine. In addition, slight changes in the liver, including an increase in serum aminotransferase (AST and ALT) activity, presence of focal inflammatory lesions, and prolongation of PT and APTT were observed after treatment with T-0126. These changes may be related to a mechanism based on malabsorption of fat, fat-soluble antioxidants, and vitamin K, although we cannot exclude other potential mechanisms such as direct cytotoxicity of T-0126.

Effect of laughter on salivary endocrinological stress marker chromogranin A.

We investigated the effect of laughter on salivary endocrinological stress marker chromogranin A (CgA). In saliva samples collected from 11 healthy males before and after watching a comic film or a non-humorous control film, salivary CgA levels were determined by enzyme-linked immunosorbent assay (ELISA). Samples taken after watching the comic film showed increased levels of CgA. This tendency was more pronounced in individuals with lower initial levels of stress. The control samples showed no significant change in CgA levels. Stress score, subjectively evaluated using a visual analog scale, decreased significantly after watching the comic film. These findings suggest that, in addition to a stress relief effect, laughter can bring about feeling uplifted or fulfilled.

Circadian rhythm of human salivary chromogranin A.

We investigated the circadian rhythm of chromogranin A (CgA) concentrations in saliva and blood samples from 40 male college students collected at 7 : 00, 8 : 00, 10 : 30, 12 : 30, 17 : 30, and 22 : 30. CgA concentrations were determined by ELISA. Salivary CgA levels peaked upon awakening, and then quickly decreased to the nadir after 1 hour and maintained a low level throughout the day. On the other hand, plasma CgA did not show any obvious circadian rhythm. These findings suggest that salivary and plasma CgA has different routes of secretion.

Correlation between plasma glucagon-like peptide 2 levels and proliferative makers in small intestinal injury in rats induced by methotrexate administration.

Glucagon-like peptide 2 (GLP-2) is a potent intestinal epithelium-specific growth factor that has been shown to reduce the severity of inflammatory disorders of the intestine in rodent models. We examined whether a relationship exists between plasma level of GLP-2 and the degree of intestinal injury induced by chemotherapeutic agents in the rat. Methotrexate (MTX) was administrated orally for 6 consecutive days at doses of 1.25, 2.5, and 5.0 mg/kg body weight per day. Mucosal samples of rat duodenum, jejunum, and ileum were used for assessment of mucosal weight, DNA and protein content. Plasma GLP-2 levels were measured on day 8. MTX significantly reduced body weight. The values of all indices tended to decrease in all segments with increases in MTX dose. Plasma GLP-2 levels were significantly higher in the MTX 2.5 mg/kg/d group (p<0.05) and the MTX 5.0 mg/kg/d group (p<0.01) than in the control group. Correlations were found between plasma GLP-2 levels and mucosal weight, DNA and protein content. We concluded that plasma GLP-2 levels reflect the degree of intestinal injury following MTX administration in this preclinical model.

Change in salivary physiological stress markers by spa bathing.

We assessed the stress relief effect of spa bathing by measuring sensitive salivary stress markers, cortisol and chromogranin A (CgA). From 12 healthy males, saliva samples were collected immediately before and after spa bathing, and 30 min after that. Salivary cortisol and CgA levels were determined by ELISA. Salivary cortisol levels decreased after spa bathing. This tendency was more pronounced in individuals with higher levels of stress. The high-stress group showed lower salivary CgA levels after spa bathing, while the low-stress group higher salivary CgA levels in the same condition. These findings suggest that the spa bathing has a moderate affect on the stress relief.

Gene expression profiling in streptozotocin treated mouse liver using DNA microarray.

Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including liver. For analyzing changes in genes expression associated with SZ toxicity, we performed DNA microarray analyses on the liver obtained from SZ-treated mice. Eight-week-old male ICR mice were treated i.p. with 200 mg/kg of SZ, and the blood and liver were taken at 6, 24 and 48 h after the treatment. Labeled cRNA prepared from total RNA of the liver was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The number of the probe sets, which were clearly up-regulated or down-regulated, were over 100 at 6 and 24h after the SZ-treatment, and it decreased at 48 h after the treatment. Many of the up-regulated genes were categorized into cell cycle/apoptosis related genes, immune/allergy related genes and stress response/xenobiotic metabolism related genes. On the other hand, genes related to glucose, lipid and protein metabolisms were down-regulated. These changes started prior to the elevation of the serum glucose levels, indicating the direct action of SZ on the liver rather than the secondary effect of diabetes. This may be related with the previously reported hepatic changes such as lipid peroxidation, mitochondrial swelling and inhibition of hepatocyte proliferation observed before the development of hyperglycemia.

Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization.

Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.

Relationship between lifestyle scores and salivary stress markers cortisol and chromogranin A.

The authors evaluated individuals' lifestyles and investigated the relationship between lifestyle scores and the incidence of salivary stress markers cortisol and chromogranin A (CgA). Saliva samples from 173 healthy male workers were tested by enzyme-linked immunosorbent assay for salivary cortisol and CgA levels. Questionnaires concerning lifestyle were administered: respondents aged 20-39 years who had good scores for comprehensive lifestyle showed significantly lower levels of cortisol than did respondents with moderate or poor comprehensive lifestyle scores. Respondents aged 40-59 who scored better for general lifestyle than they did during the previous year showed significantly lower levels of CgA than did respondents reporting no improvement. These findings suggest that levels of salivary cortisol may be affected by current circumstances and that levels of CgA may be affected by changes in comprehensive lifestyle.

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice.

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6, 12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.

An antidiabetic thiazolidinedione induces eccentric cardiac hypertrophy by cardiac volume overload in rats.

1. To assess the involvement of volume overload in the development of cardiac hypertrophy during treatment with an antidiabetic thiazolidinedione, changes in cardiac anatomy and parameters of cardiac volume overload were evaluated in female Sprague-Dawley rats treated with the thiazolidinedione derivative T-174. 2. Two week administration of T-174 (13 and 114 mg/kg per day) increased absolute and relative heart weights by 11-24%, demonstrating the development of cardiac hypertrophy. There was no evidence of oedema in hearts from treated rats. 3. Both plasma and blood volumes were increased in T-174-treated rats without any changes in systolic blood pressure and heart rate, whereas haematocrit was decreased. In accordance with the existence of volume overload, both left ventricular end-diastolic pressure and right atrial pressure were increased. Morphometric analysis of hearts revealed that T-174 induced eccentric heart hypertrophy, as characterized by a small increase in wall thickness and a large increase in the chamber volume, which is characteristic of volume overload. Volume overload is suggested as the possible trigger mechanism because blood volume expansion preceded cardiac hypertrophy and there was a high correlation between heart weight and blood volume. 4. T-174-treated streptozotocin-induced diabetic rats also exhibited blood volume expansion and cardiac hypertrophy. 5. These findings suggest that cardiac volume overload is induced by plasma volume expansion and contributes to the development of eccentric cardiac hypertrophy during treatment with antidiabetic thiazolidinediones. Although thiazolidinediones are insulin-sensitizing agents, these cardiac effects are likely to be mediated independently of insulin.

Effect of snack eating on sensitive salivary stress markers cortisol and chromogranin A.

OBJECTIVES: To investigate the effect of snack eating on salivary cortisol and chromogranin A (CgA). METHODS: From 14∶00 to 18∶00, starting two hours after consumption of a midday meal, saliva samples were collected every 30 minutes from 15 healthy males, 7 of whom (snack group) ate a snack immediately after the sampling at 15∶00. Salivary cortisol and CgA levels were determined by ELISA. Samples were controlled according to salivary flow rates. RESULTS: For the snack group, after snack consumption, salivary cortisol increased to exceed significance (p<0.05) at 15∶30 and rose even higher at 16∶00. In the control group, there was no such change. There was no significant change in salivary CgA in either the snack group or the control groups during the sampling period. CONCLUSIONS: These findings suggest that no food should be consumed for at least 90 mins before saliva sampling for cortisol determination and that salivary CgA is probably not affected by snack eating.