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Background: Decreased respiratory muscle strength and muscle mass is key in diagnosing respiratory sarcopenia. However, the role of reduced diaphragm activity, expressed as the maximal level of diaphragmatic excursion (DEmax), in diagnosing respiratory sarcopenia in patients with chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to characterize patients with COPD and low DEmax and maximal inspiratory pressure (MIP), a measure of inspiratory muscle strength, and assess the role of DEmax in respiratory sarcopenia., Methods: Patients with COPD underwent spirometry, exercise tolerance (VO 2peak ) test, and MIP measurement. DEmax and sternocleidomastoid thickness at the maximal inspiratory level (TscmMIL) were assessed using ultrasound sonography., Results: Overall, 58 patients with COPD (median age, 76 years; median %FEV 1 , 51.3 %) were included, 28 of whom showed a %MIP of ≥80 %, defined as having preserved MIP. Based on the %MIP of 80 % and median value of DEmax (48.0 mm) as thresholds, the patients were stratified into four groups: both-high (n = 18), %MIP-alone low (n = 11), DEmax-alone low (n = 10), and both-low (n = 19) groups. The both-low group exhibited the lowest %FEV 1 , Δinspiratory capacity, VO 2peak , and TscmMIL, and these values were significantly lower than those of the both-high group. Except for %FEV 1 , these values were significantly lower in the both-low group than in the %MIP-alone low group despite adjusting DEmax level for body mass index., Conclusion: Measuring DEmax along with MIP can characterize patients with COPD, reduced exercise capacity, and decreased accessory respiratory muscle mass and can help diagnose respiratory sarcopenia., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: The receptor for advanced glycation end products (RAGE), a transmembrane receptor belonging to the immunoglobulin superfamily, is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with pulmonary arterial hypertension (PAH) and is implicated in the etiology of PAH. Recently, we reported that RAGE-aptamer, a short and single-stranded DNA directed against RAGE, inhibited an inappropriate increase in cultured PASMCs in PAH. The aim of this study was to determine the efficacy of RAGE-aptamer in monocrotaline-induced PAH in rats., Methods and Results: Rats were assigned to either an untreated control group, a group that received continuous subcutaneous administration of RAGE-aptamer immediately after monocrotaline injection, or a group that received control-aptamer immediately after monocrotaline injection. All rats survived 21 days after injection of monocrotaline and control-aptamer or RAGE-aptamer. Injection of monocrotaline with continuous subcutaneous delivery of control-aptamer resulted in higher right ventricular systolic pressure compared with controls. This increase was attenuated by continuous subcutaneous delivery of RAGE-aptamer. The proportion of small pulmonary arteries with full muscularization was greater in the monocrotaline and control-aptamer group than in the control group. Continuous subcutaneous delivery of RAGE-aptamer significantly reduced the percentage of small pulmonary arteries with full muscularization., Conclusions: Continuous subcutaneous delivery of RAGE-aptamer suppresses development of monocrotaline-induced PAH in rats. Inhibition of RAGE ameliorates muscularization of small pulmonary arteries. Treatment with RAGE-aptamer might be a new therapeutic option for PAH., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Objective: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been validated to diagnose several viral infections. However, its diagnostic accuracy in detecting SARS-CoV-2 in real-life clinical settings remains unclear. This study aimed to determine the diagnostic sensitivity and specificity of RT-LAMP compared to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) over the disease course of COVID-19., Methods: A total of 124 nasopharyngeal swab samples obtained from 24 COVID-19 patients were tested by RT-LAMP and RT-qPCR. Sensitivities and specificities of RT-LAMP compared with RT-qPCR were analyzed as a function of time from onset., Results: Up to the 9th day after onset, the RT-LAMP had a positivity of 92.8%, and the sensitivity and specificity compared with RT-qPCR was 100%. However, after the 10th day after onset, the positivity of RT-LAMP decreased to less than 25%, and the concordance of positivity between the two methods was below 60%. The limit of detection of RT-LAMP was 6.7 copies/reaction., Conclusions: Until the 9th day after the onset of symptoms, RT-LAMP had the same diagnostic accuracy as RT-qPCR. These findings suggest that RT-LAMP can be used as a diagnostic tool for COVID-19 as an alternative to RT-qPCR in the acute symptomatic phase of COVID-19., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pulmonary rehabilitation (PR) is recommended as an effective treatment for patients with chronic obstructive pulmonary disease (COPD). Previous meta-analyses showed that PR improves exercise capacity and health-related quality of life (HRQOL). However, they did not evaluate the effect of PR on the sensation of dyspnea. We searched six databases in May 2019 for randomized controlled trials (RCTs) that examined PR, including supervised lower limb endurance training as a minimal essential component that was continued for 4-12 weeks, in patients with stable COPD, with changes from baseline dyspnea as a primary outcome. Secondary outcomes were changes in exercise capacity, HRQOL, activity of daily life (ADL), physical activity (PA), and adverse events. We calculated the pooled weighted mean difference (MD) using a random effects model. We identified 42 studies with 2150 participants. Compared with the control, PR improved dyspnea, as shown using the British Medical Research Council (MRC) questionnaire (MD, -0.64; 95% CI, -0.99 to -0.30; p = 0.0003), transitional dyspnea index (MD, 1.95; 95% CI, 1.09 to 2.81; p = 0.0001), modified Borg score during exercise (MD, -0.62; 95% CI, -1.10 to -0.14; p = 0.01), and Chronic Respiratory Questionnaire (CRQ) dyspnea score (MD, 0.91; 95% CI, 0.39 to 1.44; p = 0.0007). PR significantly increased exercise capacity measured by the 6 min walking distance time, peak workload, and peak VO 2 . It improved HRQOL measured by the St. George's Respiratory Questionnaire and CRQ, but not on PA or ADL. These results indicated that PR programs including lower limb endurance training improve dyspnea, HRQOL, and exercise capacity in patients with stable COPD., Competing Interests: Conflict of interest Dr. Tohda Y. received lecture fees from Kyorin Pharmaceutical, Co., Ltd. and Teijin Pharma Co., Ltd., and research funding from Kyorin Pharmaceutical Co., Ltd., Meiji Seika Pharma Co., Ltd., Boehringer Ingelheim Co., Ltd., Teijin Pharma Co., Ltd., Daiichi Sankyo Co., Ltd., and Astellas, Pearl Therapeutics, Inc. The other authors report no conflict of interest., (Copyright © 2020 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive impairment of lung function and degradation of daily activity; however, this degradation has not been adequately elucidated. The objective of this study was to measure the physical activity of patients with IPF to determine its relationships with physiological parameters and survival rate., Methods: In total, 31 patients with IPF and 20 age-matched healthy participants were enrolled in this study. Physical activity was assessed using a physical activity monitor. The relationships among physical activity, physiological data, questionnaire-based patient-centered data, and survival were examined., Results: Physical activity, expressed as daily activity energy expenditure (AEE), was significantly lower, and the percentage of sedentary time was significantly longer in patients with IPF than in healthy participants. Moreover, AEE was moderately correlated with body-mass index, forced vital capacity, diffusing capacity of carbon monoxide, and partial arterial pressure of oxygen. Relatively strong correlation was also observed between AEE and the 6-min walk distance, but not with daily dyspnea, depression, and health-related quality of life scores. Prognostic analysis indicated that daily AEE was a significant predictor of survival., Conclusions: Patients with IPF were significantly inactive compared with age-matched healthy participants. In patients with more impaired physiological functions, the lower the physical activity was, the more was the sedentary time increased. Furthermore, lower daily physical activity resulted in significantly worse survival., (Copyright © 2017 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: The characteristics of phenotypes of elderly patients with asthma are unknown. The aim of this study was to classify these phenotypes using lung function tests and images from high-resolution computed tomography (HRCT), and to identify associations between clinical characteristics and phenotypes., Methods: A cross-sectional study was conducted in 165 elderly patients (>65 years of age) who underwent a multidimensional assessment of clinical and functional status and comorbidity. The patients were divided into three phenotypes: (1) asthma-predominant, (2) asthma-obstructive airway disease (OAD) overlap without emphysema, and (3) asthma-OAD overlap with emphysema (asthma-emphysema overlap) based on chest HRCT. A receiver operating characteristic (ROC) curve was constructed to evaluate the cutoff for differentiating between the two OAD phenotypes. Multivariate analysis was also used to distinguish between these two phenotypes., Results: The phenotypes were asthma-predominant in 48 patients (29%), asthma-OAD without emphysema in 36 (22%), and asthma-emphysema in 81 (49%). Patients with asthma-emphysema were more frequent smokers. In multivariate analysis, smoking status (odds ratio 2.92: 95% CI 1.21-7.00, P = 0.03) and % predicted FEV1 ≤70% (odds ratio 3.18: 95% CI 1.13-8.92, P = 0.03) differed significantly between the asthma-emphysema and asthma-OAD without emphysema phenotypes., Conclusions: Half of elderly patients with asthma are characterized by asthma-emphysema overlap. Our results showed that elderly patients with asthma who are smokers and have moderate or severe OAD are also likely to have emphysema., (Copyright © 2015 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Advanced glycation end products (AGE) and their receptor (RAGE) interaction elicit inflammatory and proliferative reactions in arteries, thus playing a role in cardiovascular disease. We have recently found that high-affinity DNA aptamer directed against AGE (AGE-aptamer) prevents the progression of experimental diabetic nephropathy by blocking the harmful actions of AGEs in the kidney. However, effects of AGE-aptamer on vascular injury remain unknown. In this study, we examined whether and how AGE-aptamer inhibits neointimal hyperplasia in balloon-injured rat carotid arteries., Methods: Male Wistar rats (weighting ca. 400 g at 11 weeks old) were anesthetized with sodium pentobarbital. The left common carotid artery was balloon-injured 3 times with 2F Fogaty catheter inserted through the femoral artery. Then the rats received continuous intraperitoneal infusion (3 μg/day) of either AGE-aptamer or control-aptamer by an osmotic mini pump for 2 weeks. 14 days after the procedure, the left common carotid arteries were excised for morphometric, immunohistochemical and western blot analyses., Results: Compared with control-aptamer, AGE-aptamer significantly suppressed neointima formation after balloon injury and reduced AGE accumulation, oxidative stress generation, proliferation cell nuclear antigen-positive area, macrophage infiltration, RAGE and platelet-derived growth factor-BB (PDGF-BB) expression levels in balloon-injured carotid arteries., Conclusion: The present study suggests that AGE-aptamer could prevent balloon injury-induced neointimal hyperplasia by reducing PDGF-BB and macrophage infiltration via suppression of the AGE-RAGE-mediated oxidative stress generation. AGE-aptamer might be a novel therapeutic strategy for suppressing neointima formation after balloon angioplasty., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pigment epithelium-derived factor (PEDF) has anti-inflammatory and anti-thrombogenic properties both in cell culture and animal models. Although adipose triglyceride lipase (ATGL) and laminin receptor (LR) are two putative receptors for PEDF, which receptor mainly mediates the beneficial effects of PEDF is largely unknown. In this study, we addressed the issue. siRNA raised against LR (siLR) and siATGL transfection dramatically decreased LR and ATGL levels in human cultured myeloma cells, respectively. Ten nM PEDF significantly reduced vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels in siCon- or siATGL-transfected myeloma cells, whereas PEDF increased rather than decreased these gene expressions in siLR-transfected cells. Neutralizing antibody directed against LR (LR-Ab) or LR antagonist actually bound to LR and reduced mRNA levels of VEGF, MCP-1, ICAM-1 and PAI-1 in myeloma cells. Further, pre-treatment of LR-Ab or LR antagonist suppressed the binding of PEDF to LR and resultantly blocked the effects of PEDF in myeloma cells. In addition, high concentration of LR agonist mimicked the actions of PEDF on these gene expressions in myeloma cells. This study indicates that PEDF causes anti-angiogenic, anti-inflammatory and anti-thrombogenic reactions in myeloma cells through the interaction with LR. Target domain of LR agonist and antagonist might be involved in the PEDF-signaling to gene suppression in myeloma cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10(-7) M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Objective: The effects of ambulatory oxygen for idiopathic pulmonary fibrosis (IPF) patients without resting hypoxemia have not been elucidated. The purpose of this study was to assess the effect of ambulatory oxygen on dyspnea in IPF patients without resting hypoxemia but with desaturation on exertion., Methods: This was a double-blind, placebo-controlled, randomized crossover trial of ambulatory oxygen versus ambulatory air. Patients with IPF who had a partial pressure of arterial oxygen (PaO2) between 60 mm Hg and 80 mm Hg at rest, and desaturation of 88% or less in a room-air 6-min walk test were eligible. Patients underwent a standardized 6-min walk test and a 6-min free walk test under each ambulatory gas. Oxygen and air were provided at 4 L/min intranasally. Dyspnea was evaluated immediately, 1, and 2 min after the tests., Results: Twenty patients (16 men), with a mean age of 73.5 (SD 4.1) years, % predicted forced vital capacity (FVC) of 71.0 (13.3) %, % predicted diffusion capacity for carbon monoxide (DLco) of 57.0 (13.3) %, and PaO2 of 72.5 (5.4) mm Hg were recruited. No significant differences in dyspnea were observed between ambulatory oxygen and air at each time point. However, some patients showed improvement in dyspnea with oxygen on an individual basis., Conclusions: Since oxygen provides no additional benefit over air in terms of exertional dyspnea for IPF patients without resting hypoxemia, routine prescription of ambulatory oxygen is not recommended. However, assessment on an individual basis is necessary. Trial registration. UMIN Clinical Trial Registry; No.:UMIN000005098; URL:http://www.umin.ac.jp/ctr/., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Caveolin-1 is highly expressed in prostate cancer cells, and is implicated in disease progression. Here, we identified protein phosphatase 5 (PP5) as a novel cellular binding partner of caveolin-1 using a pull-down approach in combination with mass spectrometry-based proteomic analyses. In situ proximity ligation assays demonstrated co-localization and physical interaction of caveolin-1 and PP5 in the cytoplasm of PC-3 human prostate cancer cells. Using yeast two-hybrid analysis, we found that caveolin-1 interacted with the catalytic domain of PP5. We also found that PP5 activity was elevated about 1.7-fold in the presence of 2 μM caveolin-1, and that the scaffolding domain of caveolin-1 is required for this activation. Our results suggest that caveolin-1 is a novel physiological activator of PP5., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme-rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O(2) surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from -0.465 to -0.392 V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [Fe(II)(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O(2) initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Recent studies show that COPD patients exhibit low-grade systemic inflammation, and that plasma fibrinogen and high neutrophil counts are related to faster declines in lung function. We examined correlations between serum biomarkers and the decline of lung function in COPD patients., Method: Baseline levels of 9 serum biomarkers (TIMP-1, alpha1-antitrypsin, MMP-9, TNF-alpha, TGF-beta, IL-6, IL-8, neutrophil elastase and CRP), fibrinogen and white blood cell counts (WCC) were measured in 96 COPD patients. Lung function was measured at the time of blood sampling and every 3-6 months during the observation period (median 25.0 months)., Results: Twenty patients were rapid decliners of lung function and 53 patients were non-decliners. Neutrophil counts, serum CRP and MMP-9 were significantly higher in the rapid decliners (p<0.05). The annual change of % predicted FEV(1) was inversely correlated with MMP-9 (r=-0.288; p<0.01) and CRP (r=-0.354; p<0.005) (partial correlation coefficients adjusted for age, sex, cardiovascular disease, smoking history, and baseline % predicted FEV(1)). The remaining biomarkers were not correlated with the annual change of % predicted FEV(1)., Conclusion: Serum CRP and MMP-9 levels were related to FEV(1) decline. These markers are good candidates as predictors for rapid decline of FEV(1) in COPD patients. Additional long-term and larger size studies of COPD patients could help determine the exact roles for these biomarkers.

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.

O2-dependent reactions of the ferric and ferrous forms of alpha-hydroxyheme complexed with water-soluble rat heme oxygenase-1 were examined by rapid-scan stopped-flow measurements. Ferric alpha-hydroxyheme reacted with O2 to form ferric verdoheme with an O2-dependent rate constant of 4x10(5) M(-1) s(-1) at pH 7.4 and 9.0. A decrease of the rate constant to 2.8x10(5) M(-1) s(-1) at pH 6.5 indicates that the reaction proceeds by direct attack of O2 on the pi-neutral radical form of alpha-hydroxyheme, which is generated by deprotonation of the alpha-hydroxy group. The reaction of ferrous alpha-hydroxyheme with O2 yielded ferrous verdoheme in a biphasic fashion involving a new intermediate having absorption maxima at 415 and 815 nm. The rate constants for this two-step reaction were 68 and 145 s(-1). These results show that conversion of alpha-hydroxyheme to verdoheme is much faster than the reduction of coordinated iron (<1 s(-1)) under physiological conditions [Y. Liu, P.R. Ortiz de Montellano, Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1, J. Biol. Chem. 275 (2000) 5297-5307], suggesting that, in vivo, the conversion of ferric alpha-hydroxyheme to ferric verdoheme precedes the reduction of ferric alpha-hydroxyheme.

This study determined the effect of the adenovirus E1A gene on nitric oxide (NO) production in alveolar epithelial (A549) cells. E1A-positive A549 cells (E1A transfectants), E1A-negative A549 cells (control transfectants) and untransfected A549 cells were placed in 96-well tissue culture plates. After stimulation with lipopolysaccharide (LPS) or cytokine mixture (CM), the biochemical reaction products of NO (nitrite and nitrate) in the culture medium were measured by chemiluminescence. The inducible (iNOS) and the endothelial (eNOS) isoforms of nitric oxide synthase (NOS) protein expression were examined by Western blotting. iNOS mRNA expression was examined by Northern blotting and RT-PCR. CM-induced NO production by E1A-positive A549 cells was significantly lower than that of E1A-negative cells (p < 0.0001). LPS stimulation failed to enhance NO production in both cell types. CM induced iNOS protein expression in E1A-negative A549 cells, but not in E1A-positive cells. eNOS protein expression was constitutive and was not affected by CM stimulation, LPS stimulation or E1A. CM induced iNOS mRNA expression in E1A-negative A549 cells, but not in E1A-positive cells. In conclusion, the adenovirus E1A gene suppressed NO production through transcriptional control of the iNOS gene in A549 cells. This inhibition of NO production may enable the virus to persist in human tissue, since NO is an antiviral effector of the innate immune system.

We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron. With a concentrated solution of the heme-enzyme complex, however, another species of HA binding appeared, in which the oxygen atom of HA is coordinated to the iron. This species showed an unusual low-spin signal which is similar to that of the ferric hydroperoxide species in the heme oxygenase reaction.

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets., (Copyright 2003 Elsevier B.V.)

The present study was designed to investigate whether heparin-binding epidermal growth factor-like growth factor and its related peptides are expressed in response to gastrin in rat stomach. Rat gastrin-17I (2.5 nmol/kg/hour) or gastrin-17I plus gastrin receptor antagonist, L-740,093 (2.0 mg/kg/hour), was injected intravenously into male Sprague-Dawley rats. RNA was extracted from oxyntic mucosa, and heparin-binding epidermal growth factor-like growth factor and related peptide gene expression was estimated using a ribonuclease protection assay. The level of transforming growth factor-alpha mRNA did not change at any time point during the experiment. In contrast, the levels of heparin-binding epidermal growth factor-like growth factor and amphiregulin mRNA were significantly increased within 3 hours following gastrin infusion and reached maximum levels 6 and 12 hours later, respectively. Continuous infusion of gastrin significantly increased oxyntic mucosal proliferation. Gastrin receptor antagonist significantly inhibited gastrin-induced heparin-binding epidermal growth factor-like growth factor and amphiregulin gene expression and gastrin-induced oxyntic mucosal proliferation. These findings indicate that heparin-binding epidermal growth factor-like growth factor and amphiregulin genes are induced by gastrin and that they play a role in the trophic action of gastrin on oxyntic mucosa.

We examined the biological action and expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric mucosal cell line, RGM1. HB-EGF stimulated DNA synthesis of RGM1 cells in a dose-dependent manner. Mitogenic effect of HB-EGF was as potent as that of other known mitogens for gastric epithelial cells, such as hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha. Northern blot analysis showed that RGM1 cells as well as rat gastric mucosal tissue expressed a 2.5-kilobase transcript of HB-EGF. Not only HB-EGF and TGF-alpha but also HGF caused a rapid induction of HB-EGF mRNA in the cells. Treatment with heparitinase which destroys heparan sulfate proteoglycan (HSPG) or with chlorate which inhibits sulfation of HSPG diminished [3H]thymidine incorporation of RGM1 cells in serum-free medium. In addition, a synthetic peptide corresponding to the heparin-binding domain of HB-EGF inhibits the DNA synthesis of RGM1 cells in serum-free medium in a dose-dependent manner. These results suggest that HB-EGF is an autocrine and paracrine growth factor for gastric epithelial cells and may play significant roles in mucosal repair of the stomach in cooperation with other growth factors.

Helicobacter pylori (H. pylori) induces hyperproliferation of the gastric mucosa. This study was designed to clarify whether H. pylori infection is involved in the gene expression of hepatocyte growth factor (HGF), a potent stimulator of cell proliferation in gastric mucosa. Levels of HGF mRNA were determined by a reverse transcription-polymerase chain reaction in endoscopic gastric biopsy specimens from 9 control subjects and 9 patients with H. pylori infection. In patients with H. pylori infection, levels of HGF mRNA in gastric mucosa were significantly higher than those in control subjects. HGF mRNA levels in patients with H. pylori infection were correlated with the severity of gastric mucosal inflammation. Our observations indicate that H. pylori infection increases the expression of HGF gene in gastric mucosa probably through the mucosal inflammation.

The developmental expression of the glucose-dependent insulinotropic polypeptide (GIP) gene was investigated in rat intestine. Steady state levels of GIP mRNA were determined in the intestine during fetal and postnatal development by double ribonuclease protection assays. GIP mRNA could be detected as early as day 20 of embryonic development and very low levels remained until postnatal day 3. The GIP mRNA levels increased markedly in the period between days 3 and 5 of postnatal life and then gradually increased toward adult levels. Since intron 1 of the GIP gene contains putative TATA and CCAAT boxes, and some potential cis-acting promoter elements, we examined whether or not another transcript starting from exon 2 of the GIP gene is expressed during development of rat intestine. Ribonuclease protection assays suggested that although an abbreviated transcript might exist starting from exon 2, it appears to be minor and its relative abundance is unchanged during development or following intraduodenal glucose stimulation. These observations suggest that GIP may play an important role in early postnatal development probably associated with suckling.

The rat glucose-dependent insulinotropic peptide (GIP) gene has been isolated and characterized. The gene spans approximately 8.2 kilobase pairs (kb) and the GIP mRNA (0.8 kb) is encoded by six exons. The 42 amino acid hormone is encoded by exons 3 and 4. The exon-intron organization of the rat GIP gene revealed that the splice acceptor site for intron 2 is 24 nucleotides downstream compared to the comparable splice acceptor site in the human gene. This intron sliding results in an 8 amino acid deletion in the amino terminal extension of the prepropeptide. Primer extension analysis and RNase protection assay demonstrated the existence of multiple closely spaced sites for transcriptional initiation. Both the 5'-flanking region and intron 1 contain TATA and CCAAT boxes consistent with initiation of gene transcription, although a TATA box in intron 1 is functionally inactive in adult rats in spite of its reasonable location.

We previously demonstrated that extremely high amounts of N-terminal big gastrin (G-34) fragments are excreted in human urine and three of them are N-terminal octa-, nona-, and decapeptide of G-34. Our subsequent examination revealed that there exists a considerable amount of another N-terminal G-34 fragment in urine, less hydrophobic than the three peptides. We purified this fragment from urine of an achlorhydric patient and determined the structure: less than Glu-Leu-Gly-Pro-Gln-Gly. The purification was carried out by Sep-Pak C18 cartridges, Sephadex G-25, and reverse phase HPLC. The structure was determined by a combination of amino acid analysis, amino acid sequence analysis, and mass spectral analysis. N-terminal hexapeptide of G-34 is the second richest component of urinary N-terminal G-34 fragments next to N-terminal octapeptide of G-34 in normal subjects.

The concentrations and molecular forms of urinary and plasma gastrin from normal subjects were studied by radioimmunoassays using two region-specific antisera. Urinary concentration of NH2-terminal big gastrin (G-34) immunoreactivity was several hundred times as great as that of COOH-terminal gastrin immunoreactivity. Fractionation of urine extract showed a broad giant peak of NH2-terminal G-34 immunoreactivity (gastrin fragments "U") eluting in a later position than G-34(1-17) by Sephadex G-50 column chromatography. HPLC revealed that urinary NH2-terminal G-34 immunoreactivity was composed of four fragments including G-34(1-8), G-34(1-9), and G-34(1-10). Sephadex G-50 column chromatography of plasma extract revealed two or three peaks of NH2-terminal G-34 immunoreactivity, and a major peak eluted in the same position as urinary gastrin fragments "U". These results and data on renal clearances suggest that most of all gastrin fragments "U" in plasma are excreted in urine without renal reabsorption, whereas almost all of plasma COOH-terminal gastrin peptides including G-34 and little gastrin (G-17) are removed and metabolized in the kidney.

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.

We previously demonstrated that there existed extremely abundant NH2-terminal big gastrin immunoreactivity (NT G-34-IR) in human urine. This report describes the purification and sequence of NT G-34-IR from the urine of an achlorhydric patient. The purification was carried out by a combination of Sep-Pak C18 cartridges, Sephadex G-25, and HPLC steps using a radioimmunoassay specific for NH2-terminus of G-34 and ultraviolet absorption at 214 nm as monitors. Three peptides were isolated. The amino acid analysis, mass spectrometry, and sequence analysis confirmed the structures of urinary NT G-34 fragments being less than Glu-Leu-Gly-Pro-Gln-Gly-Pro-Pro, less than Glu-Leu-Gly-Pro-Gln-Gly- Pro-Pro-His, and less than Glu- Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu. NH2-terminal octapeptide of G-34 was the main component of urinary NT G-34-IR.