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Background: Nitric oxide (NO) is a relevant molecule for vascular homeostasis. The level of serum NO metabolites (NOx), which consist of nitrite and nitrate, has been investigated as an alternative biomarker of NO production, but its clinical value has not yet been determined., Methods and Results: 143 patients (66 ± 12 years old) were followed up after coronary catheterization. During a median (inter-quartile range) observation period of 6.13 (3.32-9.21) years, there were 20 (14 %) all-cause deaths, including 11 (8 %) cardiovascular deaths, 17 (12 %) major adverse cardiovascular events, and 17 (12 %) hospital admissions for heart failure. Median NOx level was 34.5 μmol/L (23.9-54.3). NOx was a risk factor for all-cause death [hazard ratio (HR) by unit increase, 1.010, 95 % confidence interval (CI) 1.001-1.018; p = 0.021] and heart failure (HR 1.010, CI 1.001-1.019; p = 0.029). Even after adjustment for age, sex, coronary risk factors, C-reactive protein, log-transformed brain natriuretic peptide, estimated glomerular filtration rate, and nitrate treatment, NOx was a risk factor for all-cause death (HR 1.015, CI 1.004-1.027; p = 0.008) and admission with heart failure (HR 1.018, CI 1.005-1.018, p = 0.007)., Conclusions: An increase in serum NOx level does not herald a benign clinical course but is an independent predictor of high risk of any-cause mortality and heart failure., Competing Interests: Declaration of competing interest The authors report no relationships that could be construed as a conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: The long-term prognostic value of the derivatives of reactive oxidative metabolites (d-ROMs) oxidative stress test, which measures hydroperoxide in blood, has not been fully investigated., Methods and Results: We administered the d-ROMs test to 265 patients with cardiovascular disease (204 men, 61 women; age, 65 ± 13 years) and followed these patients for up to 10 years. During the observational period of 5.82 (2.47-8.34) years, 31 (12%) patients died, including 20 (8%) of cardiovascular death, and 33 (12%) had major adverse cardiovascular events (MACEs). Cox regression analysis revealed that patients with a d-ROMs value ≥395 U.CARR had a greater risk for all-cause mortality [unadjusted hazard ratio (95% confidence interval), 3.586 (1.772-7.257)], cardiovascular death [7.034 (2.805-17.640)], and MACEs [4.440 (2.237-8.814)] (p < 0.001 for all). In a model adjusted for age, sex, estimated glomerular filtration rate, C-reactive protein, diabetes, hypertension, hyperlipidemia, coronary artery diseases, current smoking, and log-transformed brain natriuretic peptide, all-cause death [2.311 (1.059-5.135), p = 0.036], cardiovascular death [4.398 (1.599-12.099), p = 0.004], MACEs [2.696 (1.266-5.739), p = 0.010] were still significant in patients with high d-ROMS values., Conclusion: A high d-ROMs value is an independent predictor of the long-term risk of cardiovascular mortality. A d-ROMs value of 395 U.CARR was considered to be an appropriate threshold for distinguishing prognosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Conventional diuretic therapy such as loop diuretics is a cornerstone of the treatment for heart failure (HF). Diuretic response is an important factor in determining resistance to HF therapy and has been shown to be associated with subsequent clinical outcome. Tolvaptan (TVP), a vasopressin V2 receptor antagonist, has a favorable profile in terms of rapid fluid removal and less aggravation of renal function. We hypothesized that the response to TVP might be associated with the subsequent clinical outcome., Method: In this single-center retrospective study, 148 consecutive HF patients who were administered TVP from 2014 through 2018 [age 79 (69-86) years, male 89 (60%)] were included. Ninety-six patients were divided into TVP responder [N = 39 (41%)] and non-responder groups based on the cut-off value of gained urine output (+ 93 ml/mg TVP /day) on the day after TVP was introduced., Results: Early TVP introduction (p = 0.012) and lower dose of loop diuretics (p = 0.043) were predictors of TVP responder. For 2 years after discharge, TVP responders showed more favorable outcomes regarding the primary endpoint defined as the composite of all-cause death and HF readmission (p = 0.034, log-rank test) and HF readmission (p = 0.005). A multivariable Cox model analysis revealed that TVP responder was an independent predictor of the primary endpoint (hazard ratio 0.48, p = 0.041). TVP responders had a lower number of HF readmissions over a 1-year period (p = 0.002). TVP response was independently associated with the number of HF readmissions (p = 0.015). The proportion of patients with an extended period between discharge and HF readmission after TVP administration was higher in responders than non-responders (67% vs. 23%, p = 0.006). These associations of TVP response and post-discharge outcomes were more evident in patients who continued TVP after discharge., Conclusion: TVP response can be indicative of subsequent clinical outcomes and may be informative when considering advanced care planning., Competing Interests: Declaration of Competing Interest None, (Copyright © 2021. Published by Elsevier Ltd.)

Backgrounds & Aims: Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening., Methods: We combined new and existing genotype data for 10,516 cases and 20,772 controls from 5 European and 2 East Asian cohorts., Results: We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57 th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, which each play key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, JAK-STAT signalling, and differentiation of T helper (T H )1 and T H 17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some of which are well-established in the treatment of other autoimmune disorders., Conclusions: This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders., Lay Summary: Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10,516 people with PBC and 20,772 healthy individuals recruited in Canada, China, Italy, Japan, the UK, or the USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these 'candidate genes' to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC., Competing Interests: Conflicts of interest GMH has consulted and/or been a speaker for Intercept, Genfit, Cymabay, GSK, and Falk. RNS and GFM have each received research funding from Intercept Pharmaceuticals. HJC, JJF, KU, RD, YA, YH, MK, NN, S-SK, OG, YK, MN, KT, RT, YS, ZL, BDJ, EJA, AG, MC, RA, AC, MdA, AB, JH, MARF, DS, DEJ, SF, AS, VLM, KNL, CIA, MFS, PI, KAS, XM and MN report no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021. Published by Elsevier B.V.)

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P meta =6.71E-28, OR=1.88) and TNFSF15 (P meta =5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS)., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Percutaneous coronary intervention (PCI) for true bifurcation lesions is challenging. Although the jailed balloon protection technique is an established method with which to prevent side branch occlusion during the treatment of bifurcation lesions, little is known regarding the potential risks of this technique. We describe a 71-year-old man with exertional angina pectoris who was treated with PCI for a calcified true bifurcation lesion in the left anterior descending artery and diagonal branch. After performing rotational atherectomy (1.75 mm burr) for the main vessel and pre-dilatation for both the main vessel and side branch, we performed the jailed balloon technique to protect the large diagonal branch during stent implantation. However, the jailed balloon was entrapped after main vessel stent balloon inflation. The entrapped jailed balloon was then inflated again and successfully removed after balloon deflation, but significant stent deformation was seen with intravascular ultrasound imaging. Fortunately, post-dilatation was successfully performed with a non-compliant balloon, and a final coronary angiogram showed acceptable results. This case report and literature review highlights a potential risk of the jailed balloon technique and conceivable alternatives during PCI for true bifurcation lesions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Despite the recognized clinical value of exome-based diagnostics, methods for comprehensive genomic interpretation remain immature. Diagnoses are based on known or presumed pathogenic variants in genes already associated with a similar phenotype. Here, we extend this paradigm by evaluating novel bioinformatics approaches to aid identification of new gene-disease associations., Methods: We analyzed 119 trios to identify both diagnostic genotypes in known genes and candidate genotypes in novel genes. We considered qualifying genotypes based on their population frequency and in silico predicted effects we also characterized the patterns of genotypes enriched among this collection of patients., Results: We obtained a genetic diagnosis for 29 (24%) of our patients. We showed that patients carried an excess of damaging de novo mutations in intolerant genes, particularly those shown to be essential in mice (P = 3.4 × 10(-8)). This enrichment is only partially explained by mutations found in known disease-causing genes., Conclusion: This work indicates that the application of appropriate bioinformatics analyses to clinical sequence data can also help implicate novel disease genes and suggest expanded phenotypes for known disease genes. These analyses further suggest that some cases resolved by whole-exome sequencing will have direct therapeutic implications.

Background: Non-fatal suicide attempt is the most important risk factor for later suicide. Emergency department visits for attempted suicide are increasingly recognised as opportunities for intervention. However, no strong evidence exists that any intervention is effective at preventing repeated suicide attempts. We aimed to investigate whether assertive case management can reduce repetition of suicide attempts in people with mental health problems who had attempted suicide and were admitted to emergency departments., Methods: In this multicentre, randomised controlled trial in 17 hospital emergency departments in Japan, we randomly assigned people aged 20 years and older with mental health problems who had attempted suicide to receive either assertive case management (based on psychiatric diagnoses, social risks, and needs of the patients) or enhanced usual care (control), using an internet-based randomisation system. Interventions were provided until the end of the follow-up period (ie, at least 18 months and up to 5 years). Outcome assessors were masked to group allocation, but patients and case managers who provided the interventions were not. The primary outcome was the incidence of first recurrent suicidal behaviour (attempted suicide or completed suicide); secondary outcomes included completed suicide and all-cause mortality. This study is registered at ClinicalTrials.gov (NCT00736918) and UMIN-CTR (C000000444)., Findings: Between July 1, 2006, and Dec 31, 2009, 914 eligible participants were randomly assigned, 460 to the assertive case management group and 456 to the enhanced usual care group. We noted no significant difference in incidence of first recurrent suicidal behaviour between the assertive case management group and the enhanced usual care group over the full study period (log-rank p=0·258). Because the proportional hazards assumption did not hold, we did ad-hoc analyses for cumulative incidence of the primary outcome at months 1, 3, 6, 12, and 18 after randomisation, adjusting for multiplicity with the Bonferroni method. Assertive case management significantly reduced the incidence of first recurrent suicidal behaviour up to the 6-month timepoint (6-month risk ratio 0·50, 95% CI 0·32-0·80; p=0·003), but not at the later timepoints. Prespecified subgroup analyses showed that the intervention had a greater effect in women (up to 18 months), and in participants younger than 40 years and those with a history of previous suicide attempts (up to 6 months). We did not identify any differences between the intervention and control groups for completed suicide (27 [6%] of 460 vs 30 [7%] of 454, log-rank p=0·660) or all-cause mortality (46 [10%] of 460 vs 42 [9%] of 454, log-rank p=0·698)., Interpretation: Our results suggest that assertive case management is feasible in real-world clinical settings. Although it was not effective at reducing the incidence of repetition of suicide attempts in the long term, the results of our ad-hoc analyses suggested that it was effective for up to 6 months. This finding should be investigated in future research., Funding: The Ministry of Health, Labour, and Welfare of Japan., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lime sulfide poisoning by the oral route is rarely encountered in the practice of forensic science, whereas hydrogen sulfide poisoning is seen frequently. We report here two cases of fatal lime sulfide poisoning with several related cases and in addition induced histological damage with acute inflammation in animal models under at similar concentrations. We also evaluated sulfide and thiosulfate concentrations and speculated as to the cause of pancreatic damage in these cases., (Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Background: Atopic dermatitis (AD) and Japanese cedar pollinosis (JCP) are common chronically allergic diseases associated with the activation of T-helper 2 cells. Recent studies have shown that polymorphisms in the genes for IL-4 receptor α chain (IL4RA) may contribute to susceptibility of AD and JCP, although the differences in the involvements of loci of IL4RA gene between AD and JCP are unclear. In this study, we investigated the role of polymorphisms in IL-4RA gene in conferring susceptibility to the development of AD and/or JCP using a family analysis and an association analysis in a Japanese population., Methods: Five polymorphisms in the IL-4RA gene, C-3223T, T-1914C, T-890C, Ile50Val and Glu375Ala, have been genotyped using PCR-based methods in 75 trios families, including 15 AD families, 30 JCP families, and 30 families with combination of AD and JCP in the family analysis. Forty-five AD, 60 JCP and 125 control children constituted the association study., Results: The transmission disequilibrium test showed that the allele of Ile50 was significantly transmitted to children with JCP alone (p < 0.05). Haplotype analysis showed that the -3223T/Ile50 haplotype was preferentially transmitted to both AD (p < 0.01) and JCP children (p < 0.01), while that the C-3223/Ile50 haplotype was preferentially transmitted to only JCP children (p < 0.01). The association study showed that -3223T and haplotype of -3223T/Ile50 were associated with AD children, but not with JCP. Ile50 was associated with both AD and JCP., Conclusions: Our data suggest that -3223T and the -3223T/Ile50 haplotype were risk factors for AD. Ile50 allele seems to be involved in both JCP and AD. Interactions of the IL-4RA loci may play a role both conferring susceptibility and modulating severity of AD.

Background & Aims: A recent study reported that isohumulones, the bitter component of beer, activate peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma in vitro and decrease plasma glucose and lipid levels in diabetic mice. This study was to investigate the efficacy and safety of isohumulones for subjects with prediabetes., Methods: Ninety-four subjects with prediabetes were randomly divided into four groups. A 12-week double-blind dose-finding study was performed in which subjects ingested placebo capsules or test capsules containing 16 mg, 32 mg or 48 mg of isohumulones per day., Result: After treatment, fasting blood glucose was decreased in the 32 mg and 48 mg groups after 4 weeks, but did not change in the placebo group. HbA1c was also significantly decreased after 4 weeks in the 16 mg group and after 8 weeks in the 32 mg and 48 mg groups. Body mass index (BMI) was significantly decreased in the 48 mg group as compared with the placebo group at 12 weeks. The decrease in total fat area was also significantly greater in the 48 mg group than in the placebo group at 12 weeks., Conclusion: The present study suggests that ingestion of isohumulones has beneficial effects in diabetes and obesity.

The oxygen reactivity of catecholatoiron(III) complexes has been examined using a series of catecholate ligands as the substrate. All the complexes examined here, [Fe(III)(TPA)(R-Cat)]BPh(4) (1-9) (TPA: tris(pyridin-2-ylmethyl)amine; R-Cat: substituted catecholate ligand, R=3,5-(t)Bu(2) (1), 3,6-(t)Bu2 (2), 3,5-Me2 (3), 3,6-Me2 (4), 4-(t)Bu (5), 4-Me (6), H (7), 4-Cl (8) and 3-Cl (9)), exclusively afforded the intradiol cleaving products of the catecholate ligands upon exposure to O2. It was revealed that 1-7 can be categorized into two classes based on their electrochemical properties; i.e., the complexes having the dialkyl-substituted (group A) and the mono- or non-substituted (group B) catecholate ligands. In spite of their classification, these two groups show a linear correlation between the logarithm of the reaction rate constant with O2 and the energy of the catecholate-to-iron(III) LMCT band, although 2 shows a large negative deviation from the correlation line. Based on this LMCT-energy dependent reactivity of 1 and 3-9 as well as the very low reactivity of 2, we have discussed on the mechanisms of the reaction of [Fe(III)(TPA)(R-Cat)]BPh4 with O2.

The effects of acute exercise on the protein expressions of heterotrimeric G protein alpha subunits were examined in rat adipocytes. Galphai2 protein expression was significantly reduced 0 and 3h after exercise but increased 24h after exercise, without alterations in Galphai2 mRNA expressions. The protein expressions of other alpha subunits, Galphas, Galphai1, and Galphai3, were not influenced. Both the 26S proteasome activity and polyubiquitination of Galphai2 protein were significantly increased 0 and 3h after exercise. Whereas, proteasome activity was decreased, and the polyubiquitination of Galphai2 protein was returned to the control level 24h after exercise. The reductions in Galphai2 protein expressions 0 and 3h after exercise were completely prevented by the injection either of a proteasome inhibitor or of a beta-adrenergic receptor blocker prior to exercise. Thus, acute exercise altered the expression of Galphai2 protein via mechanisms which involve the coupling of beta-adrenergic receptors to an agonist with subsequent ubiquitin-proteasome-dependent proteolysis.

Three-week exercise training decreased the steady state level of beta(2)-adrenergic receptor (beta(2)AR) mRNA in peritoneal macrophages from BALB/c mice. When peritoneal macrophages from both exercise-trained and sedentary control mice were stimulated with lipopolysaccharide (LPS), interleukin (IL)-12 mRNA and protein expression was markedly higher in trained mice than in control mice. To determine whether enhanced production of IL-12 was associated with decreased expression of beta(2)AR, we transfected the macrophage cell line, RAW264, with a eukaryotic expression vector containing beta(2)ar cDNA, establishing a cell line overexpressing beta(2)AR (RAWar). Following LPS stimulation, IL-12 mRNA and protein expression was significantly lower in RAWar cells than in RAW264 cells transfected with vector alone (RAWvec). Furthermore, when the expression of transfected beta(2)AR in RAWar cells was down-regulated by a tetracycline repressor-regulated mammalian expression system, expression of IL-12 mRNA and protein following LPS stimulation tended to return to the levels in RAWvec cells. These findings indicate that macrophage production of IL-12 following LPS stimulation is regulated by the expression level of beta(2)AR, suggesting that the down-regulation of beta(2)AR expression associated with exercise training improves IL-12-induced type 1 helper T cell-mediated immune responses.

It is well known that glucose-stimulated insulin secretion (GSIS) decreases after exercise training. In the present study, we investigated the effects of exercise training (9 weeks of running) on the activity of glucokinase (GK), the production of nitric oxide (NO), and the protein expressions of both glucose transporter-2 (GLUT-2) and NO synthase (NOS) in rat pancreatic islets. Exercise training significantly reduced GSIS, with decreases in GK activity and GLUT-2 protein expression. The NO releases and cGMP contents were higher in the islets of trained rats than in those of control rats. Exercise training enhanced cNOS activity, the protein expression of both neuronal nitric oxide synthase (nNOS) and calmodulin, and NADPH-cytochrome c reductase activity in the homogenates of islets. Thus, exercise training-induced reduction of GSIS would result from, at least in part, decreases in both glucose entry and the first step in glycolytic utilization of glucose. Moreover, exercise training could enhance the protein expression of nNOS, which in turn enhances two catalytic activities of nNOS, an NO production and a cytochrome c reductase activity.

Oxidative stress is induced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. Recently, it has become aware that susceptibility of pancreatic beta-cells to oxidative stress contributes to the progressive deterioration of beta-cell function in type 2 diabetes. A hypoglycemic sulfonylurea, gliclazide, is known to be a general free radical scavenger and its beneficial effects on diabetic complications have been documented. In the present study, we investigated whether gliclazide could protect pancreatic beta-cells from oxidative damage. One hundred and fifty microM hydrogen peroxide reduced viability of mouse MIN6 beta-cells to 29.3%. Addition of 2 microM gliclazide protected MIN6 cells from the cell death induced by H(2)O(2) to 55.9%. Glibenclamide, another widely used sulfonylurea, had no significant effects even at 10 microM. Nuclear chromatin staining analysis revealed that the preserved viability by gliclazide was due to inhibition of apoptosis. Hydrogen peroxide-induced expression of an anti-oxidative gene heme oxygenase-1 and stress genes A20 and p21(CIP1/WAF1), whose induction was suppressed by gliclazide. These results suggest that gliclazide reduces oxidative stress of beta-cells by H(2)O(2) probably due to its radical scavenging activity. Gliclazide may be effective in preventing beta-cells from the toxic action of reactive oxygen species in diabetes.

Horse heart myoglobin was reconstituted with mesohemin derivatives methylated at the 6- or 7-position to evaluate the role of the heme-6-propionate or heme-7-propionate side chain in the protein. The association and dissociation of the O(2) binding for the deoxymyoglobin with 6-methyl-7-propionate mesoheme are clearly accelerated. Furthermore, the myoglobin with 6-methyl-7-propionate mesoheme shows fast autoxidation from oxymyoglobin to metmyoglobin compared to the myoglobin with 6-propionate-7-methyl heme and the reference protein. These results indicate the 6-propionate plays an important physiological role in the stabilization of oxymyoglobin because of the formation of a salt-bridge with the Lys45. The acceleration of CO binding rate is observed for the myoglobin with 6-propionate-7-methyl mesoheme, suggesting that the replacement of the 7-propionate with a methyl group has an influence on the His93-heme iron coordination. The structural perturbation of His93 imidazole was also supported by 1H NMR spectra of cyanide and deoxy forms of the myoglobin with 6-propionate-7-methyl mesoheme. Thus, it is found that the 7-propionate regulates the hydrogen-bonding network and His93-heme iron coordination in the proximal site.

Chlorocatecholatoiron complexes, [Fe(TPA)(4Cl[bond]Cat)]BPh(4) and [Fe(TPA)(3Cl[bond]Cat)]BPh(4), (4Cl[bond]Cat and 3Cl[bond]Cat: 4- and 3-chlorocatecholates, respectively; TPA: tris(2-pyridylmethyl)amine) were isolated as intermediates for the oxygenative cleavage of chlorocatechols by nonheme iron complexes. Geometric structures of these complexes together with [Fe(TPA)(DTBC)]BPh(4) (DTBC: 3,5-di-tert-butylcatecholate) as reference were analyzed by X-ray absorption spectroscopy (EXAFS) in the solid state and in solution. Structure of the DTBC complex in the solid state was shown to be noticeably different from the other complexes as seen in the magnetic susceptibility and spectroscopic data. Electronic and magnetic properties of these complexes were studied by X-ray absorption (XANES), electronic (VIS) and ESR spectroscopies, and magnetic susceptibility. Electron transfer from the catecholate ligand to the Fe(III) center was indicated by the Fe[bond]K edge values in XANES spectra and by the LMCT bands in electronic spectra. Magnetic susceptibility and ESR data indicated that at low temperatures the complexes are in equilibrium between the low (S=1/2) and high-spin (S=5/2) ferric states with the latter component increasing with temperature. Remarkable differences between the spin states in solid and in solution were observed with the DTBC complex.

The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-kappaB by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1-like sites (U site, -1126 approximately -1120; L site, -524 approximately -518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS.

Membrane type-serine protease 1 (MT-SP1) plays potential roles in the process of invasion and metastasis of carcinomas. In the present study, we cloned a rat MT-SP1 cDNA and investigated the intestinal distribution and proteolytic properties of the enzyme. By in situ hybridization we found the prominent expression of the mRNA in the epithelial layer of the small intestinal upper villi and of the colon, where cells are loosely attached to the basement membrane. When MT-SP1 was expressed in Caco-2, a colonic carcinoma cell line, the protein was localized exclusively on the basolateral side. A secreted form of the enzyme produced in COS-1 cells digested fibronectin and laminin. These findings suggest that MT-SP1 participates in the control of intestinal epithelial turnover by regulating the cell-substratum adhesion., (Copyright 2001 Academic Press.)

Effects of acute cold stress (5 degrees C for 24 h) on the functions of peritoneal macrophages and the mechanisms for controlling host homeostasis were investigated in mice. Phagocytic activity and expression of the cell surface adhesion molecule CD11b/CD18 were markedly increased in peritoneal exudate cells by acute cold stress. These alterations were attributable to an increased number and phenotypical changes of adherent cells from acute cold-stressed mice. On the other hand, a lipopolysaccharide-induced activity of src-family tyrosine kinase Fgr, an expression of interleukin-1beta (IL-1 beta) mRNA, and a bioactivity of IL-1 in the culture supernatants of adherent cells from acute cold-stressed mice were markedly lower than those from control mice. A time course study revealed that the number of adherent cells in peritoneal exudate cells was markedly increased in mice exposed to cold for 24 h but returned to normal numbers when mice were exposed to cold for 72 h. DNA fragmentation and Annexin-V(+) cells were observed in peritoneal exudate cells from acute-cold stressed mice. Thus, cold stress activated macrophages but these macrophages were destined to be eliminated by apoptosis., (Copyright 2001 Academic Press.)

Nagase analbuminemic rats (NAR) are deficient in serum albumin due to skipping of the albumin exon H caused by a mutation in the intron HI. This mutation deletes nucleotides from +5 to +11 in the 5' splice site region, where it interacts with U1snRNA. To determine whether the mutation can be suppressed by the compensatory base substitution in U1snRNA, we constructed mutated U1snRNA genes with various degrees of complementarity to the mutated 5' splice site. Several mutated U1snRNA genes activated the mutated 5' splice site of the intron HI, when cotransfected with the albumin minigene derived from NAR. In vivo activity of these mutated U1snRNAs correlated well with the predicted thermodynamic stability. Since mutation in the 5' splice site is one of common causes of genetic defects in human (5), these data indicate that NAR is a good model system to examine the possibility of gene therapy using a mutated U1snRNA., (Copyright 1998 Academic Press.)

Ovarian endometrial cysts, one of the typical manifestations of endometriosis, are generated by the retention of cyclic hemorrhages and are classified as tumor-like lesions rather than neoplasms. Clonality analysis provides important information about the histogenesis and progression of neoplastic diseases. As it is generally accepted that most neoplasms are monoclonal in origin, however, the clonality of endometrial cysts remains uncertain. Using the human androgen receptor gene (HUMARA) as an X-linked polymorphic marker, we examined the clonal status of epithelial cells in endometrial cysts. We separated 21 fresh epithelial cell samples from 11 endometrial cysts and found that all were monoclonal in the methylation pattern of the HUMARA alleles. Moreover, in each of the five cysts from which epithelial cells were sampled from multiple and distant areas, the methylation patterns of all samples from a single cyst were identical. These data indicate that endometrial cysts are monoclonal in origin and suggest their neoplastic potentiality.

Laminin-rich extracellular matrix, EHS-gel, has been demonstrated to keep a high level of liver-specific gene expression in cultured rat hepatocytes. To obtain information about the effect of EHS-gel on liver-specific functions, gene expression of liver-enriched transcription factors in rat hepatocytes was investigated. The apolipoprotein A-I and albumin mRNA levels were higher in hepatocytes cultured on EHS-gel than in those cultured on type I collagen (TIC). The levels of mRNA for HNF-4, C/EBP alpha and C/EBP beta were also higher on EHS-gel than on TIC. The level of HNF-4 mRNA in hepatocytes on EHS-gel was almost comparable to that in liver. The HNF-3 alpha mRNA level was lower on EHS-gel than on TIC. C/EBP beta mRNA was induced by dexamethasone in both EHS-gel and TIC. The induction of C/EBP alpha and HNF-4 by dexamethasone was observed only on TIC. These data suggest that EHS-gel leads hepatocytes to keep the phenotypic expression through high expression of liver-enriched transcription factors, such as HNF-4.

The effects of dietary supplementation of methionine to a 20% soybean protein isolate diet on serum lipoprotein profiles and secretion rate of VLDL in rats receiving polychlorinated biphenyls (PCB) were investigated. Serum cholesterol levels were higher in rats fed PCB or a methionine supplement than in controls. The effects of PCB and methionine were synergistic. The feeding of PCB resulted in more cholesterol in all fractions of serum lipoproteins tested, especially HDL (HDL1 and HDL2). Dietary supplementation of methionine primarily increased HDL cholesterol. The elevation of serum lipoprotein cholesterol due to PCB and/or methionine was significant in HDL1, which showed alpha-mobility. These results showed that methionine and PCB significantly influenced HDL metabolism. The secretion rate of VLDL was higher in rats fed PCB than in controls, but the addition of methionine to diets did not affect the secretion rate of VLDL cholesterol. This implies that PCB increased serum cholesterol partly through the stimulation of VLDL cholesterol secretion.

Expressions of gelsolin and alpha-actin have been investigated in a revertant cell line R1 and compared with the parental human activated Ha-ras oncogene-transformed NIH/3T3 (EJ-NIH/3T3), untransformed NIH/3T3 and partially revertant R2 cells. Gelsolin mRNA expression was strongest in R1 cells, intermediate in R2 and NIH/3T3 cells, and low in EJ-NIH/3T3 cells. Southern blot analysis gave neither signs of gross rearrangements nor amplification of the gelsolin gene. alpha-actin mRNA expression was restored in R1 cells to the level of NIH/3T3 cells. In R2 and EJ-NIH/3T3 cell lines, no alpha-actin transcript was detected. High gelsolin expression and restoration of alpha-actin expression may be associated with the acquirement of flat morphology and ordered cell growth pattern, which imply loss of tumorigenicity of R1 cells.

L-Prolyl-L-phenylalanyl-L-arginine-alpha-naphthylester (Pro-Phe-Arg-NE) was synthesized as a new substrate for use in the assay of kallikreins. An assay was developed based on the colorimetric determination of alpha-naphthol released by the enzyme reaction. With Pro-Phe-Arg--NE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001 KU. Thus use of Pro-Phe-Arg-NE provides a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25-microliter sample of human urine. Zymograms of human urinary kallikrein were prepared using Pro-Phe-Arg-NE as substrate. Six bands were separated by polyacrylamide disc gel isoelectrophoresis.

Benzoyl-L-leucyl-L-alanyl-L-arginine-alpha-naphthylester (Bz-Leu-Ala-Arg-NE) was synthesized as a new substrate for use in the assay of thrombin. In the assay alpha-naphthol released by the enzyme reaction was measured colorimetrically. With Bz-Leu-Ala-Arg-NE as substrate, the minimum detectable concentration of human thrombin was 0.0025 U. This assay using Bz-Leu-Ala-Arg-NE is a highly sensitive method for detecting prothrombin, thrombin and antithrombin III in human plasma. Prothrombin could be determined with 0.2 microliter of human plasma using Echis carinatus venom (ECV) as activator. Antithrombin III activity could be determined with 2 microliter of human plasma using human thrombin and heparin as cofactor. A zymogram of human prothrombin was prepared with Bz-Leu-Ala-Arg-NE as substrate. The preparation gave one band (pI 4.9) on polyacrylamide disc gel isoelectrophoresis.