DNA methylation of GJA1, BMP2 and BMP4 in a human cementoblast cell line induced by lipopolysaccharide.

AIM: To examine DNA methylation of GJA1, BMP2 and BMP4 in human cementoblasts (HCEM) induced by lipopolysaccharide (LPS). METHODOLOGY: HCEM were cultured in osteoinduction medium. After 24 h, Escherichia coli LPS (1 µg/mL) was added to the medium, which was changed every 2-3 days. Untreated samples were used as controls. Messenger RNA was extracted after 4 weeks, and quantitative real-time polymerase chain reaction (qRT-PCR) for GJA1, BMP2, BMP4 and DNMT1 was performed. Genomic DNA was extracted after 4 weeks, and quantitative methylation-specific polymerase chain reaction was carried out for GJA1, BMP2 and BMP4. To detect mineralization, alizarin red and alkaline phosphatase staining were performed. The cells were also treated with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza) and examined. The significance of differences amongst groups was assessed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with P < 0.05 being significant. RESULTS: Decreased expression of mRNA was seen in GJA1, BMP2 and BMP4 after 4 weeks (P < 0.05). DNA hypermethylation was detected in GJA1, BMP2 and BMP4 (P < 0.05). Alizarin red staining and alkaline phosphatase staining revealed decreased mineralization levels in HCEM stimulated with LPS. 5Aza abolished the effects of DNA methylation in HCEM stimulated with LPS. CONCLUSIONS: These results suggest that long-term LPS stimulation induces DNA methylation of GJA1, BMP2 and BMP4 in HCEM.

Response of porcine epithelial rests of Malassez to stimulation by interleukin-6.

AIM: To investigate the proliferation and migration of epithelial cell rests of Malassez (ERM) after stimulation with IL-6. METHODOLOGY: Porcine-derived ERM were seeded on Dulbecco's modified Eagle's Medium, and IL-6 (100 pg mL-1 ) was incorporated into the culture medium. The WST-1 assay was performed to evaluate cell proliferation, and absorption was measured at 450 nm. A wound-healing assay and immunofluorescence assay for integrin α3 were conducted to investigate migration. The Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni correction were used to analyse data of WST-1 and wound-healing assays. RESULTS: Cell proliferation following the stimulation by IL-6 increased over time, with a significant increase being observed at 6 h (P < 0.05), but not in a concentration-dependent manner. Cell proliferation was significantly greater in IL-6-treated ERM than in nontreated ERM (P < 0.05). The results of the wound-healing assay revealed earlier closure in IL-6-treated ERM (P < 0.05). In the immunofluorescence assay, integrin α3 was detected at the edge of cell processes adjacent to the wound area. A neutralized antibody abrogated the effects of the IL-6 stimulation in cell proliferation and migration. CONCLUSION: IL-6 promoted the proliferation and migration of porcine ERM in vitro.

Coping strategies and risk of cardiovascular disease incidence and mortality: the Japan Public Health Center-based prospective Study.

AIMS: Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. METHODS AND RESULTS: The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000-04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50-79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality.The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73-1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55-0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64-0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01-4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07-11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03-1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40-0.99), respectively. CONCLUSION: An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Actin stabilization induces apoptosis in cultured porcine epithelial cell rests of Malassez.

AIM: To test whether actin stabilization by jasplakinolide induces inhibition of cell viability and apoptosis in epithelial cell rests of Malassez (ERM). METHODOLOGY: ERM derived from porcine were spread in a 96-well dish (5 × 10(4) /well) using Dulbecco's modified Eagle's medium. The actin-specific stabilization reagent, jasplakinolide, was incorporated into the culture medium and incubated for 24 h. To evaluate cell viability, the WST-1 assay was carried out and absorption (450 nm) was measured. To detect apoptotic cells, monoclonal antibody to single-strand DNA (ssDNA) was used and absorption (405 nm) was measured. Actin stabilization and apoptosis induced by jasplakinolide were morphologically investigated by staining with Alexa Fluor 568 phalloidin and observed under a fluorescent microscope. As a negative control, DMSO was used instead of jasplakinolide. Differences between the jasplakinolide-treated group and the control group were analysed statistically using the Student's t-test. RESULTS: Cell viability decreased in a concentration-dependent manner, and cell viability in the jasplakinolide-treated ERM was lower than that in nontreated ERM (n = 16, P < 0.01). Apoptotic cells in the jasplakinolide-treated ERM were more frequently detected compared to that in nontreated ERM (n = 16, P < 0.01). Morphologically, shrinkage, irregular forms and fragmentation of nuclei suggesting apoptotic bodies were observed in jasplakinolide-treated ERM, whilst actin filaments were extended in non-treated ERM. CONCLUSION: Actin stabilization by jasplakinolide inhibited cell viability and induced apoptosis in epithelial cell rests of Malassez.

Evaluation of oro-facial function in young subjects with temporomandibular disorders.

The aim of this study was to evaluate bite force (BF) and oro-facial functions at different dentition phases (initial-mixed, intermediate-mixed, final-mixed and permanent dentition) in children and adolescents diagnosed with temporomandibular disorders (TMDs). The sample was selected from four public schools in Piracicaba, São Paulo, Brazil. Of the 289 participants recruited, aged 8-14 years old, 46 were placed into the TMD group. TMD was diagnosed using Axis I of the Research Diagnostic Criteria for Temporomandibular Disorders (2011). Oro-facial functions were evaluated using the Nordic Orofacial Test-Screening (NOT-S), which involves both an interview and a clinical examination. BF was measured using a digital gnathodynamometer. Age and body mass index (BMI) were also considered. The data were analysed by the following tests: Kolmogorov-Smirnov test, Student's t-test, Spearman and Pearson coefficients, Qui-square test, Fisher's exact or binomial test, as indicated. Moreover, univariate and multivariable logistic regression were applied. For the TMD group, scores associated with NOT-S interview and NOT-S total were higher than for the control group (P = 0.033 and P = 0.0062, respectively). No differences in BF between genders or groups (P > 0.05) were detected. Variables included in the multivariate logistic regression were BMI and NOT-S total. Based on this analysis, NOT-S total was associated with TMDs. Reported sensory function was the specific domain within NOT-S interview that established the significant difference between the groups (P = 0.021). The TMD group also had a greater number of alterations in the face-at-rest domain of the NOT-S exam (P = 0.007). Concluding, it did not detect an association between TMDs and either dentition phase or BF. Instead, BF correlated with age and BMI. Oro-facial dysfunction was associated with TMD in the studied sample, but this association may be bidirectional, requiring further researches.

Changes in the oral-health-related quality of life of Brazilian children after an educational preventive programme: an 1-month longitudinal evaluation.

OBJECTIVE: This 1-month longitudinal study assessed whether the oral status and the oral-health-related quality of life (OHRQoL) of children changed after four sessions of an educational preventive programme. STUDY POPULATION AND METHODS: Fifty Brazilian students (11-12 year old) were examined for signs and symptoms of gingivitis using the Community Periodontal Index and two questions about gingival bleeding. The OHRQoL was measured using the Brazilian Portuguese version of the Child Oral Impacts on Daily Performances (Child-OIDP). Higher scores indicated worse OHRQoL. The results were analysed using the Shapiro-Wilk, Chi-square, Wilcoxon signed-rank and Mann-Whitney tests. The magnitude of the mean change was calculated using the effect size. RESULTS: Twenty-four percentage of children had more than six sites with bleeding at follow-up compared with 58% at baseline. There was a significant decline in the intensity and extension of impacts at follow-up. A significant improvement in the clinical status and oral hygiene was observed for both transitional categories. There was a significant decline in the Child-OIDP scores of those reporting 'much improved'. A significant improvement in the global ratings of oral health was observed at follow-up. CONCLUSIONS: In the studied sample, an improvement occurred with respect to the severity of disease, intensity and extension of impacts and global ratings of oral health after 1-month follow-up. These results suggest that improving the global transition in health by enhancing coping and management skills while inducing slight changes in the clinical status and the specific aspects of health compromised by the disease is possible.

Cell type-dependent effects of corticosteroid on periostin production by primary human tissue cells.

Overproduction of periostin, an IL-13-inducible matricellular protein, despite corticosteroid treatment is thought to be involved in the chronicity of allergic inflammation seen in corticosteroid-refractory tissue fibrosis. Therefore, we hypothesized that some tissue cells must produce periostin in a corticosteroid-insensitive manner. Here, we show that IL-4 and IL-13 each induced comparable levels of periostin production by primary normal human fibroblasts and microvascular endothelial cells derived from lung and skin. Dexamethasone, a corticosteroid, completely inhibited IL-4/13-induced, but did not affect TGF-ß-induced, periostin production by fibroblasts. In contrast, dexamethasone synergistically enhanced IL-4/13-induced periostin production by microvascular endothelial cells. TGF-ß did not induce periostin production by microvascular endothelial cells. Our novel findings suggest that IL-4/13-induced microvascular endothelium-derived and/or TGF-ß-induced fibroblast-derived periostin might play a pivotal role in corticosteroid-refractory tissue fibrosis, leading to chronic allergic inflammation in the lung and/or skin.

Efficacy and safety of mesh non-fixation in patients undergoing laparo-endoscopic repair of groin hernia: a systematic review and meta-analysis.

PURPOSE: To examine updated evidence on the efficacy and safety of mesh non-fixation in patients undergoing laparo-endoscopic repair of groin hernias. METHODS: We searched MEDLINE, Cochrane Central Library, Embase, ClinicalTrials. gov, and ICTRP databases to identify randomized controlled trials. The primary outcomes were recurrence, chronic pain, and return to daily life. The certainty of evidence (CoE) was assessed by grading recommendations, assessments, developments, and evaluations. We performed a subgroup analysis based on the surgical type. This study was registered with PROSPERO (CRD 42022368929). RESULTS: We included 25 trials with 3,668 patients (4,038 hernias) were included. Mesh non-fixation resulted in little to no difference in hernia recurrence (relative risk [RR]:1.40, 95% confidence interval [CI]:0.59-3.31; I2 = 0%; moderate CoE) and chronic pain (RR:0.48, 95% CI:0.13-1.78; I2 = 77%; moderate CoE), but reduced return to daily life (mean difference [MD]: - 1.79 days, 95% CI: - 2.79 to -0.80; I2 = 96%; low CoE). In subgroup analyses, the transabdominal preperitoneal approach (TAPP) (MD: - 2.97 days, 95% CI: - 4.87 to - 1.08; I2 = 97%) reduced return to daily life than total extraperitoneal inguinal approach (MD: - 0.24 days, 95% CI - 0.71 to 0.24; I2 = 61%) (p = 0.006). CONCLUSIONS: Mesh nonfixation improves the return to daily life without increasing the risk of hernia recurrence or chronic pain. Surgeons and patients may discuss mesh nonfixation options to accommodate a patient's desired return to daily life. Further trials focusing on TAPP are required to confirm these findings.

Evaluation of masticatory performance and bite force in children with sleep bruxism.

The aim of this study was to evaluate the association between masticatory performance (MP) and bite force (BF) in children with sleep bruxism (SB) during the mixed dentition stage, considering also the occlusal characteristics. The sample was composed by 52 healthy children of both genders, aged 6-10 years. From those, 22 presented signs and symptoms of SB and 30 were the controls. SB diagnosis consisted of both parental report and presence of tooth wear. MP was evaluated by the individual's ability to communicate an artificial chewable test material for determining the median particle size (X50) and distribution of particles in the different sieves (b). BF was measured using a digital gnathodynamometer with fork strength of 8 mm. The results were submitted to descriptive statistics, Mann-Whitney and chi-square tests, Spearman's correlation and multiple logistic regression. Mean BF and X50 did not differ between groups with and without SB. A significant negative correlation was observed between BF and X50 only in the group of children with SB. Moreover, the logistic regression model showed an association between the presence of SB and higher b index. The other independent variables included in the model showed no association with SB. BF did not differ between children with and without SB. Besides, higher BFs in children with SB meant better MP; however, they were more likely to present chewed particles retained in the larger aperture sieves, consequently requiring more chewing cycles to break down the test material in smaller particles.

Effects of olmesartan on renal and cardiovascular outcomes in type 2 diabetes with overt nephropathy: a multicentre, randomised, placebo-controlled study.

AIMS/HYPOTHESIS: The renal and cardiovascular protective effects of angiotensin receptor blocker (ARB) remain controversial in type 2 diabetic patients treated with a contemporary regimen including an angiotensin converting enzyme inhibitor (ACEI). METHODS: We examined the effects of olmesartan, an ARB, on primary composite outcome of doubling of serum creatinine, endstage renal disease and death in type 2 diabetic patients with overt nephropathy. Secondary outcome included composite cardiovascular outcomes, changes in renal function and proteinuria. Randomisation and allocation to trial group were carried out by a central computer system. Participants, caregivers, the people carrying out examinations and people assessing the outcomes were blinded to group assignment. RESULTS: Five hundred and seventy-seven (377 Japanese, 200 Chinese) patients treated with antihypertensive therapy (73.5% [n = 424] received concomitant ACEI), were given either once-daily olmesartan (10-40 mg) (n = 288) or placebo (n = 289) over 3.2 ± 0.6 years (mean±SD). In the olmesartan group, 116 developed the primary outcome (41.1%) compared with 129 (45.4%) in the placebo group (HR 0.97, 95% CI 0.75, 1.24; p = 0.791). Olmesartan significantly decreased blood pressure, proteinuria and rate of change of reciprocal serum creatinine. Cardiovascular death was higher in the olmesartan group than the placebo group (ten vs three cases), whereas major adverse cardiovascular events (cardiovascular death plus non-fatal stroke and myocardial infarction) and all-cause death were similar between the two groups (major adverse cardiovascular events 18 vs 21 cases, all-cause deaths; 19 vs 20 cases). Hyperkalaemia was more frequent in the olmesartan group than the placebo group (9.2% vs 5.3%). CONCLUSIONS/INTERPRETATION: Olmesartan was well tolerated but did not improve renal outcome on top of ACEI. TRIAL REGISTRATION: ClinicalTrials.gov NCT00141453.

Prevention of excessive collagen accumulation by human intravenous immunoglobulin treatment in a murine model of bleomycin-induced scleroderma.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrotic changes in skin and other organs involving excessive collagen deposition. Here we investigated the effect of intravenous immunoglobulin (IVIG) on fibrosis in a murine model of bleomycin (BLM)-induced scleroderma. Scleroderma was induced in C3H/He J mice by subcutaneous BLM injections daily for 35 days. The collagen content in skin samples from the BLM-injected group (6·30 ± 0·11 mg/g tissue) was significantly higher than the PBS group (5·80 ± 0·10 mg/g tissue), and corresponded with dermal thickening at the injection site. In contrast, mice treated with IVIG for 5 consecutive days after initiating BLM injection showed lesser collagen content significantly (IVIG group, 5·61 ± 0·09 mg/g tissue; BLM vs. IVIG). In order to investigate the cellular and protein characteristics in the early stage of the model, the skin samples were obtained 7 days after the onset of experiment. Macrophage infiltration to the dermis, monocyte chemoattractant protein (MCP-1)-positive cells, and increased TGF-ß1 mRNA expression were also observed in the BLM group. IVIG inhibited these early fibrogenic changes; MCP-1 expression was significantly lesser for the IVIG group (1·52 ± 0·19 pg/mg tissue) than for the BLM group (2·49 ± 0·26 pg/mg tissue). In contrast, TGF-ß1 mRNA expression was significantly inhibited by IVIG. These results suggest that IVIG treatment may inhibit macrophage recruitment to fibrotic sites by down regulating MCP-1 and TGF-ß production, and thus could be a potential drug for managing fibrotic disorders such as SSc.

High incidence of Langerhans cells in lymph nodes of athymic nude mice: an ultrastructural and morphometric study.

Cervical and axillary lymph nodes of athymic nude mice were compared morphometrically and ultrastructurally with those of normal littermates. The lymph nodes of the nude mice were larger and the volumes of the follicle, paracortex, and medulla of the lymph nodes were all larger. The paracortex of nude mice had few lymphocytes and a large number of interdigitating cells (IDC) and Langerhans cells (Lc). The results suggest that the high incidence of Lc and IDC in the lymph nodes of nude mice is due to cell accumulation and not lymphocyte depletion.

Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increased serum concentrations in postmenopausal women with osteoporosis.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family of proteins and plays an important role in the negative regulation of osteoclastic bone resorption. Whether OPG/OCIF circulates in human blood and how its level changes under pathological conditions is not known. To address these issues, a panel of monoclonal antibodies was generated against recombinant OPG/OCIF and screened for reactivity with solid-phase monomeric and homodimeric forms of the recombinant protein. Antibodies that showed high affinity for both forms of OPG/OCIF and those that selectively recognized the homodimer were identified, enabling development of two types of sensitive enzyme-linked immunosorbent assay (ELISA): one that detects both forms of OPG/OCIF equally and one specific for the homodimer. Characterization of circulating OPG/OCIF with these ELISAs revealed that the protein exists in human serum mainly in the monomeric form. The serum concentration of OPG/OCIF increased with age in both healthy Japanese men and women, and was significantly higher in postmenopausal women with osteoporosis than in age-matched controls. Within the osteoporotic group, serum OPG/OCIF concentrations were higher in patients with low bone mass. Serum OPG/OCIF concentrations were also significantly increased in those postmenopausal women with a high rate of bone turnover, as determined by increased serum bone-specific alkaline phosphatase and urinary excretion of pyridinoline and deoxypyridinoline. The results suggested that circulating OPG/OCIF levels are regulated by an age-related factor(s) and that the increased serum concentration may reflect a compensative response to enhanced osteoclastic bone resorption and the resultant bone loss rather than a cause of osteoporosis.

Role of sympathetic activity in blood pressure reduction with low calorie regimen.

To investigate the effects of a low calorie regimen on sympathetic function and its relation to blood pressure response, 22 untreated obese essential hypertensive patients (50 +/- 2 years, body mass index 29 +/- 1 kg/m2) were hospitalized and a diet was prescribed of 2,000 kcal/day for 5 days (control period) followed by 800 kcal/day for 21 days without changing salt intake (8-10 g/day). The dose of intravenous phenylephrine infusion needed to elevate systolic blood pressure 20 mm Hg (CD20) and the 24-hour urinary excretion of norepinephrine (UNE) were measured. During the low calorie period, blood pressure normalized in 14 patients (responder group, 124 +/- 3/79 +/- 4 mm Hg) and eight remained hypertensive (poor responder group, 158 +/- 6/103 +/- 3 mm Hg). At the control period, blood pressure and body mass index were similar, but the responder group had higher UNE (134 +/- 15 micrograms/day) and CD20 (127 +/- 11 micrograms) than the poor responder group (89 +/- 6 micrograms/day and 79 +/- 13 micrograms, respectively). During the low calorie period, both UNE (87 +/- 15 micrograms/day) and CD20 (74 +/- 10 micrograms) decreased in the responder group; no change was seen in the poor responder group. Changes in UNE and systolic blood pressure were correlated (r = 0.6, p less than 0.05). In conclusion, suppression of sympathetic activity plays a role in blood pressure reduction during moderate caloric restriction.

Transforming growth factor-beta stimulates, and glucocorticoids and epidermal growth factor inhibit brain natriuretic peptide secretion from cultured human amnion cells.

We previously reported the massive secretion of brain natriuretic peptide (BNP) from human amnion cells and suggested the possible role of BNP in the maintenance of human pregnancy. In this study, to elucidate the regulatory mechanism of BNP secretion from amnion cells, we measured the BNP level in the culture medium of amnion cells by RIA after incubation in the presence of various substances. Among the agents examined, cortisol (1 x 10(-7) to 1 x 10(-6) mol/L), dexamethasone (1 x 10(-8) to 1 x 10(-6) mol/L), and epidermal growth factor (EGF; 2 x 10(-11) to 2 x 10(-8) mol/L) inhibited BNP secretion from the cultured amnion cells in a dose-dependent manner. By contrast, transforming growth factor-beta (TGF beta; 4 x 10(-11) to 4 x 10(-9) mol/L) caused a 3- to 5-fold increase in BNP secretion. TGF beta-augmented BNP secretion was abolished by the addition of cortisol or EGF to the culture medium. Moreover, in this study, we revealed the presence of bioactive TGF beta in human amniotic fluid (approximately 4 x 10(-10) mol/L). The present finding of tight regulation of BNP secretion from amnion cells by cortisol, EGF and TGF beta, all at the concentrations physiologically present in human amniotic fluid, implies a physiological role of BNP secretion from amnion cells in the pregnant uterus.

Blood pressure response to hyperinsulinemia in salt-sensitive and salt-resistant rats.

We investigated the role of insulin in salt-sensitive hypertension in Dahl salt-sensitive and salt-resistant rats. The rats were kept in metabolic cages, and sodium intake and urinary sodium excretion were measured. In salt-sensitive rats receiving a 0.3% NaCl diet, sodium retention was significantly greater at weeks 1 and 2 in rats that received an insulin infusion than in those receiving a saline infusion. Mean arterial blood pressure and plasma norepinephrine levels were significantly higher at week 3 in insulin-treated rats than in saline-treated rats (mean arterial pressure, 137 +/- 3 mm Hg versus 119 +/- 3 mm Hg, p < 0.05; plasma norepinephrine, 0.40 +/- 0.02 ng/ml versus 0.27 +/- 0.01 ng/ml, p < 0.05). Insulin did not influence sodium retention, mean arterial pressure, or plasma norepinephrine in salt-resistant rats. Coadministration of an alpha-blocker (bunazosin, 10 mg/kg per day for 3 weeks) in salt-sensitive rats abolished the insulin-induced elevations in mean arterial pressure and sodium retention. When salt-sensitive rats were fed a low salt diet (0.03% NaCl), insulin did not raise mean arterial pressure. Thus, insulin elevated blood pressure only in the salt-sensitive model. The sympathetic nervous system and sodium retention in the early phase of insulin overload may contribute to elevation of mean arterial pressure in this model.

Cloning and expression of a down-regulated gene (TrEnodDR1) of white clover responded by the nod genes derived from Rhizobium leguminosarum bv. trifolii strain 4S.

The nodulation genes of Rhizobium leguminosarum bv. trifolii 4S (strain 4S) were cloned into cosmid vector pLAFR1 named pC4S8 which was contained nodNMLFEDABCIJ and a part of nodT as an insert. The pC4S8 was transferred to strain H1, Sym plasmid (pRt4Sa) cured strain of strain 4S, and isolated as Tc resistant and nodulation restored mutant, strain H1(pC4S8). During infection process of this strain, visible symbiotic features, such as root hair curling (Hac), root hair deformation (Had) and infection thread formation (Inf) were also restored. The nodule forming ability of strain H1(pC4S8) was increased 3-4 times in nodule number than that of strain 4S. Then, to investigate the effect of Rhizobium nod genes on the host plant (Trifolium repens L.) gene expression, cDNAs which were responded to the inoculation of rhizobia were differentially screened based on the presence or absence of nod genes treated with strains H1(pC4S8) or H1, respectively. The cDNA, TrEnodDR1 (Trifolium repens early nodulin down regulation 1) gene was isolated from cDNA library prepared from white clover seedlings treated with nod- strain H1, but didn't exhibit in nod+ treated cDNA library, as a down-regulated gene. Expression analysis of TrEnodDR1 was performed in various tissues of white clover, it is suppressed in root nodule and also strongly suppressed by the inoculation of rhizobia in the seedlings. It is discussed that TrEnodDR1 gene is suppressed when the white clover comes into symbiosis with rhizobia.

Detection of adult T-cell leukemia-derived factor/human thioredoxin in human serum.

Adult T-cell leukemia (ATL)-derived factor (ADF), originally defined as an inducer of interleukin-2 receptor/alpha-chain (IL-2R/p55) of human T-lymphotropic virus type I (HTLV-I) positive T cells, is a human homologue of redox-active coenzyme thioredoxin (Trx) of Escherichia coli. In this study, an enzymatic assay system based on the dithiol-dependent insulin-reducing activity of ADF/Trx was established (insulin-reducing assay) to determine the amount of ADF/Trx in human serum using NADPH and Trx reductase purified from human placenta. Insulin-reducing activity was detected in all of the serum samples from healthy volunteers (n = 30) screened by this assay, with a mean +/- SD of 10.9 +/- 2.4 U/l. This mean value corresponds with the concentration of 223 ng recombinant ADF/Trx (rADF/Trx)/ml. Human serum is known to contain several redox-active proteins with ADF/Trx motifs. To differentiate the contribution of these proteins and ADF/Trx to the insulin-reducing activity, the anti-rADF/Trx monoclonal antibody (mAb)-conjugated affinity column-depleted sera obtained from an identical source was used for analysis. The affinity column-depleted sera demonstrated a loss of over 99% of the original activity, while control column depleted sera lost less than 4%. Furthermore, the amount of affinity-purified ADF/Trx molecules eluted from the same column almost corresponded with the amount estimated by the insulin-reducing activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of haemoconcentration induced by big endothelin-1 and endothelin-1 in mice.

1. The profile of haemoconcentration induced by big endothelin-1(big ET-1), a precursor of endothelin-1 (ET-1), was compared with that induced by endothelin-1 in mice. 2. ET-1(1.5 nmol kg-1, i.v.) increased haematocrit in mice, which reached a maximum at 5 min and then returned to the control value within 30 min after the administration, this occurred at the same time as changes in the plasma immunoreactive endothelin-1 and rat atrial natriuretic peptide (rANP)-like activities (IR-ET-1 and IR-rANP, respectively). 3. Big ET-1(2.5-15 nmol kg-1, i.v.) also caused a significant and dose-dependent increase in haematocrit, that lasted over 3 h although elevated plasma IR-ET-1 and IR-rANP had almost been restored to the initial levels within 10 min after big ET-1 injection. 4. A metalloproteinase inhibitor, phosphoramidon (10 mg kg-1, i.v.), which inhibits the activity of endothelin converting enzyme (ECE), delayed the onset of big ET-1-induced haemoconcentration, but failed to alter the maximal value and the duration of the haemoconcentration. 5. Pretreatment with phosphoramidon (10 mg kg-1, i.v.) did not affect the big ET-1-induced change in plasma IR-ET-1, while significant delay of the disappearance of plasma IR-rANP and significant suppression of a sustained increase in tissue IR-ET-1 were observed. 6. These results suggest that ET-1, not in plasma but in tissue, plays an important role in the pathogenesis of big ET-1-induced long-lasting haemoconcentration, in which unknown factors besides rANP are involved.

Woodfruticosin (woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental antitumor activity.

Woodfruticosin (woodfordin C) (WFC), a new inhibitor of DNA topoisomerase II (topo-II), was isolated from methanol extract of Woodfordia fruticosa Kurz (Lythraceae) and studied for in vitro and in vivo antitumor activities in comparison with Adriamycin (ADR) and etoposide (ETP), well known inhibitors of topo-II. The inhibitory activity against DNA topo-II shown by WFC was much stronger than that shown by ETP or ADR. WFC inhibited strongly intracellular DNA synthesis but not RNA and protein synthesis. On the other hand, WFC had a weaker growth inhibitory activity against various human tumor cells than ETP or ADR, but it showed remarkable activity against PC-1 cells and moderate activity against MKN45 and KB cells. Furthermore, WFC had in vivo growth inhibitory activity against s.c. inoculated colon38. These results indicate that the mechanism by which WFC exhibits antitumor activity may be through inhibition of topo-II.