Cause Clarification of Cysteine Oxidation by Active Species Generated during the Oxidation Process of Cinnamaldehyde and Impact on an In Chemico Alternative Method for Skin Sensitization Using a Nucleophilic Reagent Containing Cysteine.

Aldehydes comprise a major portion of skin sensitizers because they can react with both cysteine and lysine. Moreover, cinnamaldehyde (CA) is a typical moderate sensitizer and is often used in an alternative test method for skin sensitization. The amino acid derivative reactivity assay (ADRA) is an in chemico test method that evaluates the reactivity of cysteine derivatives (N-(2-(1-naphthyl)acetyl)-l-cysteine, NAC) and lysine derivatives with the test chemicals and uses CA as a proficiency substance. We found that NAC depletion for CA was only 10-20% when CA was used directly from the reagent bottle, although it increased to almost 100% when stored after being aliquoted from the reagent bottle. It was also found that this was due to the air oxidation of NAC itself rather than the reaction of NAC with CA, indicating that this result simply shows an increase in apparent reactivity. Aldehydes are known to produce active species, such as radicals, during air oxidation. Therefore, we investigated whether radicals were generated under storage conditions using the radical scavenger OH-TEMPO. LC/MS/MS analysis revealed that CA and OH-TEMPO complexes were produced during the air oxidation of CA. In the results of five aldehydes, similar to CA, active species were not generated as significantly as CA. Collectively, during the evaluation of the aldehydes, it can be seen that careful measures need to be taken to prevent the aldehydes from oxidizing during storage, indicating that assessment without preventing air oxidation carries an increased risk of overestimation compared with the intrinsic skin sensitization potency.

Comparative evaluation of reagents for measuring protein S activity: possibility of harmonization.

Patients with congenital protein S (PS) deficiency show a hereditary predisposition for thrombosis, and PS deficiency is prevalent among Japanese populations. Diagnosis is based on symptoms of thrombosis and reduced PS activity. Three reagents that use different measurement principles for determining PS activity are available in Japan. This study aimed to confirm the possibility of harmonization of these three reagents to establish a universal standard for PS activity in Japanese populations. Commercial normal plasma and plasma samples obtained from healthy individuals and healthy pregnant women were tested at three facilities using three reagents for measuring PS: STA-Staclot Protein S (STA-PS), HemosIL Protein S (Clotting) (IL-PS), and a total PS assay (SNT-PS). The within-run precision of each reagent was good, as each had a coefficient of variation of ≤ 3.8%. The dilution linearity for each reagent was also good. The correlation coefficient was 0.94 for STA-PS vs. IL-PS, 0.93 for SNT-PS vs. STA-PS, and 0.90 for SNT-PS vs. IL-PS, indicating a good correlation. Although the three reagents available in Japan for measuring PS activity use different measurement methods, each showed good performance, and large differences were not observed between the obtained values. Harmonization among them appears possible.

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Negative and positive control ranges in the bacterial reverse mutation test: JEMS/BMS collaborative study.

A large-scale study was conducted by multiple laboratories affiliated with the Japanese Environmental Mutagen Society and the Bacterial Mutagenicity Study Group to investigate possible proficiency indicators for the bacterial reverse mutation test with a preincubation procedure. Approximately 30 laboratories generated negative and positive control count data and dose-response curves of the positive control articles for the bacterial reverse mutation test, with assays conducted annually from 2013 to 2016. Overall, the majority of the negative and positive control counts for Salmonella Typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2uvrA, with and without S9 mix, were within the range of the means ±2× standard deviation. The negative counts were normally distributed (strains TA100, TA98, and WP2uvrA) or followed Poisson distribution (strains TA1535 and TA1537), and the positive control counts for all strains were approximately normally distributed. In addition, the distribution of the negative and positive control counts was relatively constant over the 4 years. The number of revertant colonies increased in a dose-dependent linear or exponential fashion up to the recommended doses for the respective positive control articles in Japan. These data are valuable for determining the acceptance criteria and an estimation of the laboratory proficiency for the bacterial reverse mutation test.

Morphological Determinants of Obstructive Hypertrophic Cardiomyopathy Obtained Using Echocardiography.

The morphological determinants of left ventricular outflow tract (LVOT) obstruction in hypertrophic cardiomyopathy (HCM) are not completely understood. We aimed to identify the anatomical risks of the obstruction using echocardiography.Fifty patients with untreated HCM were classified into two groups: those with LVOT pressure gradient (LVOTPG) ≥ 30 mmHg (obstructive HCM [HOCM] group) and those with LVOTPG < 30 mmHg (HNCM group). The echocardiographic morphological variables were analyzed to determine whether they were predictive of LVOT obstruction. Systolic anterior motions of the mitral valve were observed in 100% of patients in the HOCM group but only in 58% in the HNCM group. There were no significant differences in wall thickness, end-systolic LV dimension (LVDs), or LVOT diameter between the two groups. However, HOCM subjects had a shorter distance from papillary muscles to the inter-ventricular septum (5.97 ± 2.3 versus 9.20 ± 1.9 mm, respectively, P < 0.0001) and a longer anterior mitral leaflet (AML) length (24.7 ± 5.8 versus 20.1 ± 5.4 mm, respectively, P < 0.01) compared to the HNCM group. The AML length/LVDs ratio was significantly higher in the HOCM group compared to the HNCM group (1.02 ± 0.34 versus 0.78 ± 0.26, P < 0.01), and an LVOT obstruction was predicted with an area under the curve of 0.71 (P < 0.05). Multiple linear regression revealed that only the AML length/LVDs ratio was independently associated with LVOTPG (P < 0.01).The AML length/LVDs ratio has a significant predictive value for LVOT obstruction and a strong relationship with LVOTPGs. The AML length/LVDs ratio determines the anatomical risk of LVOT obstruction in HCM.

Humoral Hypercalcemia of Malignancy with a Parathyroid Hormone-Related Peptide-Secreting Intrahepatic Cholangiocarcinoma Accompanied by a Gastric Cancer.

Humoral hypercalcemia of malignancy (HHM) is caused by the oversecretion of parathyroid hormone-related peptide (PTHrP) from malignant tumors. Although any tumor may cause HHM, that induced by intrahepatic cholangiocarcinoma (ICC) or gastric cancer (GC) is rare. We report here a 74-year-old male who displayed HHM with both ICC and GC and showed an elevated serum PTHrP level. Treatment of the hypercalcemia with saline, furosemide, elcatonin, and zoledronic acid corrected his serum calcium level and improved symptoms. Because treatment of ICC should precede that of GC, we chose chemotherapy with cisplatin (CDDP) and gemcitabine (GEM). Chemotherapy reduced the size of the ICC and decreased the serum PTHrP level. One year after diagnosis, the patient was alive in the face of a poor prognosis for an ICC that produced PTHrP. Immunohistochemical staining for PTHrP was positive for the ICC and negative for the GC, leading us to believe that the cause of the HHM was a PTHrP-secreting ICC. In conclusion, immunohistochemical staining for PTHrP may be useful in discovering the cause of HHM in the case of two cancers accompanied by an elevated serum PHTrP level. Chemotherapy with CDDP and GEM may be the most appropriate treatment for a PTHrP-secreting ICC.

Evaluation of the repeated-dose liver, bone marrow and peripheral blood micronucleus and comet assays using kojic acid.

The repeated-dose liver micronucleus assay has the potential to detect liver carcinogens and could be integrated into general toxicological studies. To assess the performance of this assay, kojic acid was tested in 14-day and 28-day liver micronucleus assays. We evaluated the incidence of micronucleated cells in liver, bone marrow and peripheral blood and performed comet assays in both the liver and peripheral blood (comet assay was performed only for 14-days). Kojic acid, a skin-whitening agent used in cosmetic products, was orally dosed in six-week-old male rats at 250, 500 and 1000mg/kg/day for 14 days, and at 125, 250 and 500mg/kg/day for 28 days. Organ weight and histopathology were examined at the end of the experiment. Neither a clear, positive response in micronucleus (MN) incidence nor changes in the percent of tail DNA in the comet assays was noted in liver and bone marrow. An increase of relative liver weight was observed in 1000mg/kg/day for 14 days. In histopathology, minimal hypertrophy of hepatocytes was found at 1000mg/kg/day for 14 days. The results of both the micronucleus assay and the comet assay indicate that 14-day and 28-day repeated dosing of kojic acid are non-genotoxic in the liver and bone marrow. Kojic acid has been known to act as a tumor-promoter in thyroid carcinogenesis but has not been shown to have initiation activities in liver carcinogenesis. Findings in this study are consistent with the evidence that kojic acid is not an apparent initiator of liver carcinogenesis. Therefore, the liver micronucleus assay is simple and sensitive to detect genotoxic liver carcinogens.

Pathological aspects of the intraductal spread of breast cancer.

The intraductal spread of breast cancer is a major cause of local recurrence following breast-conserving therapy. To properly understand this pathology, three-dimensional (3D) cancer localization within the mammary ductal-lobular system (MDLS) is necessary. To this end we generated computer-assisted 3D reconstructions of all MDLSs using 2-mm-thick serial sections of surgically resected specimens. We then analyzed the characteristics of intraductal spread of breast cancer. In our study of quadrantectomy specimens from patients with primary invasive breast carcinoma, the intraductal spread of breast cancer was found to be continuous from the invasive tumor and spreading along the mammary glandular tree. The pattern is categorized into three types: the central type, the peripheral type, and the extensive type. The central type was found to be most common. A 3D analysis of total mastectomy specimen from a patient with primary non-invasive breast carcinoma revealed regional intraductal spread extending within and filling a single MDLS. The analysis also revealed the presence of ductal anastomoses connecting adjacent MDLSs. These ductal anastomoses were found to be an anatomical risk factor for extensive intraductal spread of breast cancer across multiple MDLSs. To minimize residual non-invasive components of breast carcinoma in the conserved breast, which is strongly associated with the outcome of local control of breast-conserving therapy, it is necessary to determine the optimum surgical margins in a flexible, patient-specific manner. This determination should be based on anatomical characteristics of the MDLS, such as those identified in the present study.

[Myeloid-derived suppressor cells in patients with breast cancer].

Myeloid-derived suppressor cells (MDSC) are one of the major cell populations responsible for regulating immune responses. These cells have been reported to accumulate in the blood, lymph nodes, and tumor sites in most patients during tumor progression and in chronic infection. They are also reported to potently suppress T-cell functions. We studied MDSC in the peripheral blood mononuclear cells(PBMC)by flow cytometry using blood samples from 29 patients with breast cancer, and from 11 healthy donors. The cell level was significantly high for patients compared to the 11 healthy donors (5. 68±6. 09% vs. 0. 91±0. 54%). MDSC was significantly higher in all of the breast cancer patients (5. 68±6. 09%), preoperative patients (5. 79±4. 92%) and recurrent disease patients (5. 59±7. 28%), compared to healthy donors, but not for postoperative patients (1. 50±0. 95%). Thus, MDSC was elevated in patients with breast cancer, but decreased to the range of healthy individuals after the removal of the tumor mass. However, MDSC increased again with recurrence. We also report that in 2 cases, MDSC in the peripheral blood and pleural effusion of patients with metastatic breast cancer decreased after chemotherapy with gemcitabine.

[One-year longitudinal change in parameters of myopic school children trained by a new accommodative training device--uncorrected visual acuity, refraction, axial length, accommodation, and pupil reaction].

PURPOSE: School children with myopia were trained using a visual stimulation device that generated an isolated blur stimulus on a visual target, with a constant retinal image size and constant brightness. Uncorrected visual acuity, cycloplegic refraction, axial length, dynamic accommodation and papillary reaction were measured to investigate the effectiveness of the training. SUBJECTS AND METHODS: There were 45 school children with myopia without any other ophthalmic diseases. The mean age of the children was 8.9 +/- 2.0 years (age range; 6-16)and the mean refraction was -1.56 +/- 0.58 D (mean +/- standard deviation). As a visual stimulus, a white ring on a black background with a constant ratio of visual target size to retinal image size, irrespective of the distance, was displayed on a liquid crystal display (LCD), and the LCD was quickly moved from a proximal to a distal position to produce an isolated blur stimulus. Training with this visual stimulus was carried out in the relaxation phase of accommodation. Uncorrected visual acuity, cycloplegic refraction, axial length, dynamic accommodation and pupillary reaction were investigated before training and every 3 months during the training. RESULTS: Of the 45 subjects, 42 (93%) could be trained for 3 consecutive months, 33 (73%) for 6 months, 23 (51%) for 9 months, and 21 (47%) for 12 months. The mean refraction decreased by 0.83 +/- 0.56 D (mean +/- standard deviation) and the mean axial length increased by 0.47 +/- 0.16 mm at 1 year, showing that the training bad some effect in improving the visual acuity. In the tests of the dynamic accommodative responses, the latency of the accommodative-phase decreased from 0.4 +/- 0.2 sec to 0.3 +/- 0.1 sec at 1 year, the gain of the accommodative-phase improved from 69.0 +/- 27.0% to 93.3 +/- 13.4%, the maximum speed of the accommodative-phase increased from 5.1 +/- 2.2 D/sec to 6.8 +/- 2.2 D/sec and the gain of the relaxation-phase significantly improved from 52.1 +/- 26.0% to 72.7 +/- 13.7% (corresponding t-test, p < 0.005). No significant changes were observed in the pupillary reaction. CONCLUSION: The training device was useful for improving the accommodative functions and accommodative excess, suggesting that it may be able to suppress the progression of low myopia, development of which is known to be strongly influenced by environmental factors.

[Therapeutic effects of vinorelbine-based chemotherapy--retrospective study of 65 patients with metastatic or relapsed breast cancer].

PATIENTS: The subjects were 65 stage IV or relapsed breast cancer patients who received vinorelbine monotherapy or combination therapy at Fukushima Medical University between May 2002 and November 2006. METHOD: Chemotherapy was divided into the following 3 groups Vinorelbine monotherapy (group A), Trastuzumab combination therapy (group B), MMVC (mitomycin C, methotrexate, vinorelbine and cyclophosphamide) therapy(group C). RESULTS: There were 33 patients in group A, 15 patients in group B, and 17 patients in group C. The percentage of patients who had received more than 3 prior chemotherapy regimens were 69. 7% in group A, 66. 7% in group B, and 82. 4% in group C. No complete responses were observed. Partial responses was observed 6 patients in group A, 5 patients in group B, and 2 patients in group C. Overall response rate was 18. 2% in group A, 33. 3% in group B, and 11. 8% in group C. Clinical benefit rate was 24. 2% in group A, 46. 7% in group B, and 23. 5% in group C. The median overall survival time was 267, 522, and 275 days in group A, B, and C respectively. DISCUSSION: Vinorelbine-based chemotherapy was considered as a new treatment option for patients with metastatic or relapsed breast cancer. It is necessary to examine the use at the early stage or more to obtain a high response rate and duration of response.

[Efficacy and safety of high-dose toremifene for hormone-responsive advanced or metastatic breast cancer patients with failed prior treatment by aromatase inhibitors].

BACKGROUND: Recently, aromatase inhibitors (AI) are widely used in postoperative adjuvant therapy for breast cancer. Nevertheless, studies of postoperative therapeutic strategies for recurrent breast cancer are insufficient. SUBJECTS AND METHOD: Data on 12 post-menopausal advanced/recurrent breast cancer patients in our department during June 2003- April 2007 were used for this study. No patient had responded to high-dose toremifene (TOR), a third-generation AI. Their therapeutic outcomes were analyzed retrospectively. The median observation period of the subjects was 16.1 months (4.0-40.9 months). Subjects were all hormone-sensitive. Overexpression of HER2 protein was found in only one case. During AI therapy immediately prior, exemestane (EXE) and anastrozole (ANA) had been given in nine and three cases, respectively. RESULTS: The complete response rate of AI therapy was 16.7% (2/12). The clinical benefit rate was 58.3% (7/12). The median of time to progression (TTP) was 33.8 weeks. Neither the presence nor absence of past history of treatment with tamoxifen (TAM) or other chemotherapies affected the anti-tumor effect. Analysis by the site of metastasis or recurrence revealed that the therapeutic effects were better for non-life-threatening cases in the lung, pleura, soft tissue, etc. The severities of adverse effects were all less than grade 2; the major ones were flushing and sweating. CONCLUSION: Results show that high-dose TOR given at an early stage can provide clinical benefits for post-menopausal advanced/recurrent breast cancer not responding to AI.

High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.

OBJECTIVE: The significant roles of brown adipose tissue (BAT) in the regulation of energy expenditure and adiposity are established in small rodents but have been controversial in humans. The objective is to examine the prevalence of metabolically active BAT in healthy adult humans and to clarify the effects of cold exposure and adiposity. RESEARCH DESIGN AND METHODS: In vivo 2-[(18)F]fluoro-2-deoxyglucose (FDG) uptake into adipose tissue was measured in 56 healthy volunteers (31 male and 25 female subjects) aged 23-65 years by positron emission tomography (PET) combined with X-ray computed tomography (CT). RESULTS: When exposed to cold (19 degrees C) for 2 h, 17 of 32 younger subjects (aged 23-35 years) and 2 of 24 elderly subjects (aged 38-65 years) showed a substantial FDG uptake into adipose tissue of the supraclavicular and paraspinal regions, whereas they showed no detectable uptake when kept warm (27 degrees C). Histological examinations confirmed the presence of brown adipocytes in these regions. The cold-activated FDG uptake was increased in winter compared with summer (P < 0.001) and was inversely related to BMI (P < 0.001) and total (P < 0.01) and visceral (P < 0.001) fat areas estimated from CT image at the umbilical level. CONCLUSIONS: Our findings, being against the conventional view, indicate the high incidence of metabolically active BAT in adult humans and suggest a role in the control of body temperature and adiposity.

Protein S and protein C gene mutations in Japanese deep vein thrombosis patients.

OBJECTIVES: Coagulation factor V Leiden has not been detected in Japanese patients suffering from thrombosis. Hitherto, the constitutional background of Japanese thrombotic patients has never been systematically examined. We have performed a systematic investigation to determine pathogenesis for deep vein thrombosis in a Japanese population. DESIGN AND METHODS: Routine coagulation and fibrinolysis tests were performed to determine the activities of protein S, protein C, antithrombin, plasminogen and fibrinogen. Gene analysis was performed in thrombotic patients having low activities of these factors. RESULTS: Our study indicates that the frequency (19/85 = 0.22) of mutations of protein S gene in the Japanese patients was 5-10 times higher than that of mutations of protein S gene in Caucasian patients, and the frequency (8/85 = 0.09) of mutations of protein C gene was almost three times higher than that of Caucasian patients. The frequency of antithrombin gene mutation was similar in both populations. CONCLUSION: Our study reinforces that the genetic anomaly in the protein S/protein C anticoagulation system is an important risk factor for thrombophilia in the Japanese population.

Effects of PSK on T and dendritic cells differentiation in gastric or colorectal cancer patients.

BACKGROUND: Vaccine therapy targeting tumor antigens recognized by cytotoxic T cells (CTL) has been tried extensively. However, in a cancer-bearing state, the Th1/Th2 balance shifts to Th2 dominance, and this has been the obstacle to vaccine therapy to induce the CTL. DC1/DC2 subsets have also been reported to control the differentiation of Th subsets. The key to tumor immunotherapy is how to activate the DC1-Th1 lineage. PATIENTS AND METHODS: Six normal adults and 14 patients with gastric or colorectal cancers, who gave informed consent, were studied. The Th1/Th2 and DC1/DC2 ratios were determined by FACS. IL-12 and IL-10 production from PBMC were measured by ELISA. RESULTS: The Th1/Th2 and DC1/DC2 ratios were all significantly lower in the patients with gastric or colorectal cancers compared to normal adults. After protein-bound polysaccharide K (PSK) therapy in cancer patients, the Th1/Th2 balance shifted to Th1 dominance and the DC1/DC2 balance to DC1 dominance. IL-10 production was significantly decreased by PSK therapy. CONCLUSION: In the cancer-bearing state, the Th1/Th2 and DC/1/DC2 balance becomes Th2- and DC2-dominant. PSK therapy results in a shift of the Th1/Th2 and DC1/DC2 balance towards Th1 and DC1 dominance. We plan to examine whether combining dendritic cells (DC) vaccination therapy with oral PSK enhances the induction of T cell and DC differentiation in cancer patients.

[A patient with metastatic breast cancer associated with retention of pleural effusion with no response to both CMF and exemestane, whose life was saved by high-dose toremifene].

The patient was a 70-year-old woman, who became aware of a mass in her right breast in 2001, but left it untreated. On March 10, 2003, she visited a nearby doctor with the chief complaint of dyspnea. Since a large painful mass was palpable in the right breast, advanced right breast cancer was suspected, and the patient was referred to our department. Examination revealed the presence of right axillary lymph node metastasis, left pleural effusion, and left atelectasis, and the patient was admitted to our hospital on an emergency basis. Two cycles of CMF were begun on April 2, but CT indicated NC to PD. Therefore, exemestane (EXE, 25 mg/day), was administered on May 13. While the size of the primary lesion was partially decreased, the tumor marker levels were increasing markedly. Administration of EXE was therefore discontinued, and toremifene (TOR, 120 mg/day), was begun. The systemic condition began to improve one month after the start of TOR administration. Two months later, the primary lesion had decreased in size. At after 9 months of TOR treatment, the size of the primary lesion and the tumor marker levels continued to decrease, and both the left pleural effusion and the left atelectasis disappeared.