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Malignant pleural effusion (MPE) can accompany advanced lung adenocarcinoma. Recent studies suggest that MPE could contain a heterogeneous subpopulation of cells with stem-like properties, such as tumorigenicity and self-renewal, indicating that they could be the source of metastasis. Although previous studies analyzed the correlation between cancer stem cell (CSC) marker expression and clinical outcomes using lung cancer tissues, investigations regarding the association of MPE with CSC marker expression are limited. We performed immunohistochemistry to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and Sal-like 4 (SALL4) in 46 cell block samples of MPE from patients with lung adenocarcinoma. ALDH1-positive and SALL4-positive cancer cells in MPE were detected in 30 (65.2%) and 21 samples (45.7%), respectively. Cluster formation was detected in 26 samples (56.5%). The number of clusters was significantly higher in ALDH1-positive/SALL4-negative samples. SALL4 expression was inversely correlated with the cluster ratio (r = −0.356) and positively associated with the Ki-67 index (r = 0.326), suggesting that MPE cells with high SALL4 expression comprised the proliferative subpopulation. In conclusion, we demonstrated that MPE contains an ALDH1-positive/SALL4-negative subpopulation exhibiting cluster formation and a SALL4-positive proliferative subpopulation.

Isao Okazaki,1,* Shinsuke Shibata,2,3,* Wataru Ando,4,* Takayo Yanagawa,5,6 Hiroaki Yokomori,7 Akira Sonoda,8 Norihiko Suzuki,9 Eigoro Yamanouchi,10 Shinya Okada,11 Shinichi Kamikura,11 Kazuaki Hachimura,11 Takashi Takaki,12 Katsuya Otori,4 Yutaka Suzuki,9 Hideyuki Okano,2 Yutaka Inagaki5,6 1Department of Internal Medicine, International University of Health and Welfare (IUHW) Hospital, Nasu-Shiobara City, Tochigi Prefecture, Japan; 2Department of Physiology, Keio University School of Medicine, Shinjuku-Ku, Tokyo, Japan; 3Electron Microscope Laboratory, Keio University School of Medicine, Shinjuku-Ku, Tokyo, Japan; 4Department of Clinical Pharmacy, Kitasato University School of Pharmacy, Minato-Ku, Tokyo, Japan; 5Center for Matrix Biology and Medicine, Tokai University Graduate School of Medicine, Isehara City, Kanagawa Prefecture, Japan; 6Department of Innovative Medical Science, Tokai University School of Medicine, Isehara City, Kanagawa Prefecture, Japan; 7Department of Internal Medicine, Kitasato University Medical Center, Kitamoto City, Saitama Prefecture, Japan; 8Collaborative Research Resources, Core Instrumentation Facility, Keio University School of Medicine, Shinjuku-Ku, Tokyo, Japan; 9Department of Surgery; 10Deparment of Radiology; 11Department of Pathology, IUHW Hospital, Nasu-Shiobara City, Tochigi Prefecture, Japan; 12Division of Electron Microscopy, Showa University School of Medicine, Shinagawa-Ku, Tokyo, Japan*These authors contributed equally to this workCorrespondence: Isao OkazakiHigashi Nippon International University, Iwaki City, Fukushima Prefecture, JapanTel +81-246-35-0001Fax +81-246-25-9188Email okazakiisao@hotmail.co.jpBackground/Aims: Matrix metalloproteinase-1 (MMP-1) is a key enzyme in collagen metabolism and tissue remodeling. We previously reported that MMP-1 expression in monocytes and other types of cells in the liver is associated with disease progression in patients with nonalcoholic steatohepatitis (NASH). To further implicate MMP-1 up-regulation in NASH pathogenesis, we examined dynamic and sequential MMP-1 expression in peripheral blood monocytes and cells in steatotic liver, and attempted to elucidate the mechanism of MMP-1 induction.Methods: Twenty-nine NASH patients and 26 non-NASH subjects were recruited in the study. Their peripheral blood mononuclear cells were isolated and analyzed by fluorescence-activated cell sorting, and liver specimens were subjected to confocal laser-scanning and immunoelectron microscopic examinations. Peripheral blood monocytic lineage cells were co-cultured with steatotic hepatocytes obtained from biopsy specimens to examine MMP-1 induction.Results: Infiltrating monocytes were the earliest to acquire an MMP-1-expressing phenotype among all investigated cell types in early-stage NASH liver. On the other hand, circulating monocytes did not express significant levels of MMP-1 mRNA or protein before infiltrating steatotic liver. Co-culture with steatotic hepatocytes induced MMP-1 expression in otherwise MMP-1-negative peripheral blood monocytic lineage cells. As disease progressed, MMP-1 was sequentially expressed by other types of cells in NASH liver. Notably, clusters of OV-6- or CK19-positive hepatic progenitor cells with a morphological feature of ductular reaction showed strong MMP-1 staining in advanced-stage NASH.Conclusion: MMP-1 up-regulation in infiltrating monocytic lineage cells represents an initial event in early-stage NASH and, together with the subsequent expression in other types of cells, modulates pathophysiological aspects of NASH.Keywords: matrix metalloproteinase-1, nonalcoholic steatohepatitis, monocytic progenitor cells, hepatic progenitor cells, liver fibrosis

Background/Aims: Nonalcoholic steatohepatitis (NASH) is prevalent in both economically developed and developing countries. Twenty percent of NASH progresses to cirrhosis with/without hepatocellular carcinoma, and there is an urgent need to find biomarkers for early diagnosis and monitoring progression of the disease. Using immunohistochemical and immunoelectron microscopic examination we previously reported that expression of matrix metalloproteinase-1 (MMP- 1) increased in monocytes, Kupffer cells and hepatic stellate cells in early stage NASH. The present study investigated whether serum MMP-1 levels reflect disease activity and pharmaceutical effects in NASH patients. Methods: We measured the serum levels of MMPs, tissue inhibitors of metalloproteinases (TIMPs), and several cytokines/ chemokines in patients with histologically proven early and advanced stages of NASH and compared them with those in healthy controls. Results: Serum MMP-1 levels in stage 1 fibrosis, but not in the more advanced fibrosis stages, were significantly higher than in healthy controls (P=0.019). There was no correlation between serum MMP-1 level and fibrosis stage. Serum MMP- 1 levels in NASH patients represented disease activity estimated by serum aminotransferase values during the followup period. In contrast, MMP-2, MMP-9 and TIMPs did not change with disease activity. Consistent with the finding that MMP-1 is expressed predominantly in monocytes and Kupffer cells, serum levels of monocyte chemotactic protein-1 and granulocyte-colony stimulating factor were significantly increased in NASH with stage 1 fibrosis. Conclusions: These results suggest that serum MMP-1 levels represent disease activity and may serve as a potential biomarker for monitoring the progression of NASH. (Clin Mol Hepatol 2018;24:61-76)

Serum and tissue miR-21 expression in patients with breast cancer (BC) is a useful biomarker for cancer diagnosis, progression, and treatment. Matrix metalloproteinase-1 (MMP-1) is also important in breast cancer carcinogenesis. However, miR-21 and MMP-1/CD63 in urine exosomes in these patients have not been examined. Urine samples were collected from patients with BC and 26 healthy females. Urinary exosomes were isolated and confirmed by western blotting with anti-CD63 antibody and electron microscopy observation. MiR-21 and MMP-1/CD63 expression was examined by quantitative RT-PCR and western blotting, respectively. Patients with very early stage breast cancer were evaluated. MiR-21 expression in the patients was 0.26 [95% CI: 0.20–0.78], which was significant lower than in the 26 controls (1.00 [95% CI: 1.01–3.37], p = 0.0947). MMP-1/CD63 expression in patients was significantly higher than in controls (1.74 [95% CI: 0.86–5.08] vs 0.535 [95% CI: −0.01–2.81], p = 0.0001). Sensitivity and specificity were 0.708 and 0.783 for miR-21 and 0.792 and 0.840 for MMP-1/CD63, respectively. Sensitivity and specificity of combined expression were 95% and 79%, respectively. The sensitivity of MMP-1/CD63 expression in urinary exosomes was better than that of miR-21 expression. Thus, miR-21 and MMP/CD63 may be useful markers for BC screening.

Human matrix metalloproteinase-1 (MMP-1) has been shown to contribute to the regression of experimental liver fibrosis when ectopically expressed in rodents. It remains unknown whether the same mechanism exists in chronic liver diseases in humans. This study was conducted to elucidate the functional contribution of MMP-1 to the pathophysiology of nonalcoholic steatohepatitis (NASH). Samples from 19 histologically proven NASH patients were examined together with normal liver specimens from four subjects. Expression of MMP-1, caveolin-1, active and latent forms of TGF-β, and type 1 procollagen was analyzed using immunohistochemical, western blotting and immunoelectron microscopic (IEM) examinations. In the early stage of NASH, MMP-1 was expressed predominantly in Kupffer cells and monocytes. Remarkable expression of MMP-1 and caveolin-1 was observed in hepatic progenitor cells (HPCs), hepatic stellate cells (HSC) and proliferating capillary endothelial cells in the advanced stage of NASH. MMP-1 immunostaining was significantly greater in advanced (p < 0.014) and early (p < 0.016) NASH than in control. IEM revealed MMP-1-immunoreactive products in HPCs/HSC/small hepatocytes. Active form of TGF-β and type I procollagen were detected at the edge of those capillary endothelial cells adjacent to the HPCs. MMP-1 expressed in HPCs/small hepatocytes/ capillary endothelial cells may contribute, at least in part, to the repair and regeneration of fibrotic liver through the caveolin-1 signal in the advanced stage of NASH.

Background: One quarter of global adults by overeating and reduced physical activity cause nonalcoholic fatty liver, and progress to nonalcoholic steatohepatitis (NASH), in which 10% of cases lead to liver cirrhosis and/or hepatocellular carcinoma. Adequate therapeutic option is urgent. We previously reported that matrix metalloproteinase-1 (MMP-1) expression in monocytes (MCs) and other types of cells in NASH livers is associated with disease progression. To test the hypothesis that bone marrow-derived monocytes (BMMCs) secrete exosomes with MMP-1 before their infiltration into steatosis liver, we examined MMP-1 mRNA and protein expression in circulating BMMCs. Methods: Twenty-nine NASH patients and 26 non-NASH patients were recruited in this study. Confocal laser scanning microscopic (CLSM) and immune-electron microscopic examinations (IEM), and fluorescence-activated cell sorting of CD34+ BMMCs were performed. Peripheral CD45+CD14+CD34+ cells were co-cultured with steatosis hepatocytes (HCs) isolated from NASH liver. Findings: IEM revealed that MCs were the earliest to acquire an MMP-1-expressing phenotype amongst all investigated cell types. CLSM showed that MMP-1 expression in Kupffer cells, hepatic stellate cells, progenitor cells, HCs, and endothelial cells were observed in subsequent to MCs. In advanced stage of NASH, OV-6- or CK19-positive hepatic progenitor cells around lethal HCs showed strong staining for MMP-1. These results suggested that MMP-1 expressed in progenitor cells may contribute to regeneration of injured liver, while MMP-1 up-regulation in hepatic stellate cells caused their activation leading to fibrosis. Unlike our initial hypothesis, circulating BMMCs did not express significant amounts of MMP-1 mRNA or protein before their infiltration into steatosis liver. By contrast, co-culture with steatosis HCs induced MMP-1 expression in otherwise MMP-1-negative CD34+ BMMCs. This phenotypic change in MMP-1 expression was the most pronounced in CD45+CD14+CD34+ cells. Interpretation: The results of the present study implicated MMP-1 expression by the monocyte progenitor cell population after their migration into steatosis liver in NASH pathogenesis. Funding Statement: This work is supported by grant no. IUHW 2016-24 and -25 from Mr. Takeshi Mori and Mrs. Eiko Mori to Dr. Okazaki. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: The study protocol was approved by the Ethics Committee of IUHW (13-B-136, February 22, 2016; 13-B-205, November 24, 2016; 13-B-229, April 27, 2017). All patients provided written formal informed consent for participation in this study.

Nonalcoholic steatohepatitis (NASH) is emerging worldwide because life-styles have changed to include much over-eating and less physical activity. The clinical and pathophysiological features of NASH are very different from those of HBV- and HCV-chronic liver diseases. The prognosis of NASH is worse among those with nonalcoholic fatty liver diseases (NAFLD), and some NASH patients show HCC with or without cirrhosis. In the present review we discuss fibrogenesis and the relationship between fibrosis and HCC occurrence in NASH to clarify the role of MMPs and TIMPs in both mechanisms. Previously we proposed MMP and TIMP expression in the multi-step occurrence of HCC from the literature based on viral-derived HCC. We introduce again these expressions during hepatocarcinogenesis and compare them to those in NASH-derived HCC, although the relationship with hepatic stem/progenitor cells (HPCs) invasion remains unknown. Signal transduction of MMPs and TIMPs is also discussed because it is valuable for the prevention and treatment of NASH and NASH-derived HCC.

Global statistics estimate that approximately 25% of patients with lung cancer are never smokers. We suggest that genes related to susceptibility to metabolic syndrome were present among those related to susceptibility to lung adenocarcinoma (AC) in never smokers. There are many questions concerning lung AC in never smokers, which is increasing in incidence, with female predominance, good prognosis, unique genes related to susceptibility and good response to treatment with specific agents. The purpose of this review was to investigate the carcinogenesis of lung AC in never smokers focusing on genes related to susceptibility to lung AC and carcinogens, including environmental factors. In order to clarify the carcinogenesis of lung AC in never smokers, the definition of never smokers, survey of environmental tobacco smoke, the presence of the physical characteristics of metabolic syndrome, and other carcinogens should be investigated for primary prevention of lung AC.

Background & Aims Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients. Methods BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of α2(I) collagen gene ( COL1A2 ) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates. Results The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli. Conclusions By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Matrix metalloproteinases (MMPs) are known to participate in cancer invasion and metastasis. Copious research on MMPs and their inhibitors has promoted the understanding of the mechanisms of cancer invasion and metastasis as well as the development of effective drugs for cancer treatment. This review discusses the classification of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in relation to tumor growth and invasion mechanisms via signal transduction, followed by the possibilities for cancer treatment. The review focuses especially on the development of anti-cancer agents in the field of MMP science.

Irrespective of initial stimuli, increased production of type I collagen is a common hallmark of fibrotic diseases in various organs including the liver. A dynamic balance between the production and degradation of collagen is seen, which is rigorously controlled by several growth factors and cytokines. Of these, transforming growth factor β (TGFβ) is the most potent factor in stimulating type I collagen gene transcription. It also regulates expression of matrix metalloproteinases and their inhibitors, and modulates inflammatory reactions by influencing T cell functions. Therefore, TGFβ is considered to be the major factor accelerating liver fibrosis. Identification and characterisation of Smad proteins, intracellular mediators of the signal transduction of TGFβ, have led to a better understanding of the precise mechanisms of TGFβ functions from the viewpoint of its intracellular signalling pathway and crosstalk with other factors. Several studies have focused on the suppression of TGFβ activation and intervention of the TGFβ/Smad signalling pathways to treat liver fibrosis. However, as generalised blockade of TGFβ activity may result in the promotion of carcinogenesis and excessive immune reactions, much attention has to be paid to selective intervention of the TGFβ/Smad signal specifically in collagen-producing cells in the fibrotic tissue. TGFβ also affects the growth and differentiation of stem and progenitor cells. From this point of view, a new concept of the treatment for liver fibrosis may arise from the discipline of stem cell biology by modulating TGFβ and Smad signalling in stem/progenitor cells. TGFβ is a member of a large family of pleiotropic cytokines that includes bone morphogenetic proteins (BMPs), activins and other related factors. Mammals have three different forms of TGFβ (β1, β2 and β3). Although the three isoforms of TGFβ are encoded by distinct genes located on different chromosomes, they have approximately 80% homology at the level of amino acid sequence. Of …

Increased sleepiness at work is increasingly being focused on as a safety and health issue. However, research on workers' sleepiness is very limited in scope and the characteristics of work organization, including the impact of job stress, have not been fully addressed. A questionnaire survey was conducted to investigate the prevalence of daytime sleepiness and its associated factors among non-shift working men at two manufacturing businesses: Company A, having a rapid rate of development and growth, with 564 workers (19-61 yr old, mean age: 32.7, response rate: 81.4%); and Company B, long established, possessing a huge production facility, with 1,654 workers (20-63 yr old, mean age: 37.1, response rate: 78.2%). The prevalence of daytime sleepiness was 11.3% in company A and 16.8% in company B. Multivariate logistic regression analysis revealed that, in company A, perceived sleepiness was associated with long sleep duration on non-working days and high cognitive demands and, in company B, with insufficient daily sleep, single, and depression. Psychosomatic exhaustion resulting from jobs requiring high adaptivity due to rapid frequency of operational change as in company A may have the potential to become an important factor in perceived sleepiness. However, in a comparatively stable work organization, as in company B, increased sleepiness may be mainly linked to factors outside work. It is suggested that not only lifestyle and sleep habits, but also the characteristics and dynamics of a work organization should be a focus of attention when planning measures to prevent sleepiness at work.

ATRA (all-trans retinoic acid), which is a major bioactive metabolite of vitamin A and a potent regulator of development and differentiation, mediates down-regulation of the human albumin gene. However, the mechanism of ATRA-mediated down-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrate that ATRA causes a marked decrease in the activity of the albumin promoter, the region between nt -367 and -167 from the transcription start site, where C/EBP (CCAAT/enhancer-binding protein)-binding sites are tightly packed, is indispensable for ATRA-mediated down-regulation. ChIP (chromatin immunoprecipitation) assays revealed that in vivo binding of C/EBPalpha to the region markedly decreases upon incubation with ATRA, whereas ATRA treatment marginally increases the recruitment of C/EBPbeta. We found that ATRA has the ability to differentially and directly induce expression of a truncated isoform of C/EBPbeta, which is an LIP (liver-enriched transcriptional inhibitory protein) that lacks a transactivation domain, and to increase the binding activity of C/EBPbeta-LIP to its response element. Overexpression of C/EBPbeta-LIP negatively regulates the endogenous expression of albumin, as well as the activity of the albumin promoter induced by C/EBP transactivators such as C/EBPalpha and full-length C/EBPbeta. In conclusion, we propose a novel model for down-regulation of the albumin gene, in which ATRA triggers an increase in the translation of C/EBPbeta-LIP that antagonizes C/EBP transactivators by interacting with their binding sites in the albumin promoter.

Background: We report a case of Epstein-Barr virus-positive NK/T-cell lymphoma primarily presenting with pericardial effusion without nasal lesions.Case: A 63-year-old man with dyspnea and 38°C fever was found in physical examination to have pericardial effusion of 1, 000ml. Tumor cells in effusion showed marked nuclear pleomorphism and variable-sized basophilic cytoplasm with azurophilic granules. Flow cytometoric and immunohistochemical analysis showed tumor cells to be positive for CD2, CD3e, CD7, CD45RA, CD45RO, CD56, perforin, T-cell intracellular antigen 1, granzyme B, but negative for CD4, CD5, CD8, CD20, CD30, terminal deoxynucleotidyl transferase, and T-cell receptor β. An in situ hybridization study showed positive signals for Epstein-Barr virus encoding small ribonucleic acids on tumor cells. Despite chemotherapy, the patient died of respiratory failure due to disease progression 2 months after admission. No autopsy was done.Conclusion: Based on the above data, we definitively diagnosed this case as extranodal NK/T-cell lymphoma of nasal type. It appeared to be unique in clinical manifestation, primarily affecting the pericardium.

Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)-derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)-expressing BM cells. The distribution and characteristics of EGFP-positive (EGFP+) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP+ cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP-negative (EGFP−) liver resident cells, expressed matrix metalloproteinase (MMP)-13 and MMP-9. Whereas MMP-13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP-9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony-stimulating factor (G-CSF) treatment significantly enhanced migration of BM-derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G-CSF and HGF synergistically increased MMP-9 expression along the fibrous septa. Conclusion: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis. (HEPATOLOGY; 2007:213–222.)

Global statistics estimate that 15% of all cases of lung cancer in men and 53% in women are not attributable to smoking, and these data indicate that worldwide, approximately 25% of patients with lung cancer are never smokers. The etiology of lung cancer is disputed. The present study reviews the genes associated with susceptibility to lung cancer among never smokers and suggests possibilities for the involvement of metabolic syndrome. The environment appears to have changed the genes susceptible to lung cancer. Classical genes associated with lung cancer are decreasing and novel emerging genes may reflect changes in lifestyle. We provide evidence that the genes associated with susceptibility to lung cancer in never smokers are very similar to those reported in patients with metabolic syndrome, and that simply quitting smoking is not sufficient as the primary means of preventing lung cancer.

Background: Alcohol intake can have hypoglycemic or hyperglycemic effects in patients with type 2 diabetes mellitus. The present study was designed to investigate the glycemic control of male patients with diabetes mellitus from the aspect of the genetic status of alcohol metabolism. Methods: One hundred sixty-three men with type 2 diabetes mellitus were enrolled in the present study. They were all outpatients at the Diabetes Center of Saiseikai Central Hospital. The genotype of the aldehyde dehydrogenase 2 (ALDH2) gene of each patient was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the patients were divided into those with active or inactive ALDH2 phenotype. We compared the amount of habitual alcohol intake and clinical data that included physical findings and blood chemistry of the patients in the active and inactive ALDH2 groups. The glycemic control of each patient was evaluated by the serum level of HbAlc. Results: Of the 163 patients with type 2 diabetes mellitus, 90 patients had the active ALDH2 phenotype and 73 patients had the inactive ALDH2 phenotype. The mean HbAlc level of the active ALDH2 group was nearly the same as that of the inactive ALDH2 group. However, the HbA1c level of the light-to-moderate drinkers (1-400 g/week) in the inactive ALDH2 group was highest and was significantly higher than the HbAlc level of the light-to-moderate drinkers of the active ALDH2 group. The HbAlc of the patients with diabetic complications was higher than the HbAlc of those without diabetic complications in both the active and inactive ALDH2 groups. However, the HbAlc level of the light-to-moderate drinkers without diabetic complications in the inactive ALDH2 group was significantly higher and the incidence of 24 hr urinary C-peptide was higher than the respective level of the light-to-moderate drinkers without diabetic complications in the active ALDH2 group. Conclusions: Habitual light-to-moderate alcohol intake worsens glycemic control in diabetic patients who have the inactive ALDH2 phenotype. The data on 24 hr urinary C-peptide level suggested that increased acetaldehyde after light-to-moderate drinking by inactive ALDH2 diabetic patients may increase the HbAlc value by the insulin-resistant condition that resulted in hyperinsulinemia.

An all-electron calculation of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata was carried out using the density functional method based on Gaussian-type orbitals with workstations. Our computer program ProteinDF coded by the object-oriented language C++, was originally designed for all-electron calculations of proteins and controlled in parallel with high efficiency. The numbers of residues, atoms, orbitals and auxiliary functions of the calculated protein were 43, 629, 3615 and 6545, respectively. This Letter reports timing data of the computation and some calculated electronic properties. Our program will open the door for advanced studies of larger proteins.

The ionization potentials of one-electron oxidized states of cytochrome c 3 were evaluated by numerical calculations. In the ionization processes from cytochrome c 3 0+ to c 3 1+ , four ionization potentials were observed with respect to four hemes involved in cytochrome c 3 . Ab initio SCF MO calculations were carried out for the four heme complexes to obtain their ionization potentials under vacuum (IP vacuum s). The electrostatic potentials (EPs) which the apo-protein and the aqueous solvent produce at the heme irons were calculated by the particle–particle/particle–mesh (PPPM) algorithm based on the self-consistent boundary (SCB) method. Final theoretical ionization potentials of cytochrome c 3 (IP protein s) were evaluated as the sum of IP vacuum s and EPs. The calculated IP protein s successfully reproduced the order of corresponding experimental values. The maximum difference among IP protein s was 130 mV which was approximately twice as large as that of experimental ones. The small differences among IP protein s were given by the delicate cancellations of IP vacuum s and EPs. We also discuss the detailed electronic structures of hemes in cytochrome c 3 .

Background and Objectives The mechanism of the desmoplastic response in gastric carcinoma tissues is largely unknown. The objective of this study is to determine the localization of prolyl 4-hydroxylase (PH), an enzyme that plays a crucial role in collagen biosynthesis. Methods Freshly prepared gastric carcinoma tissues from 51 cases, including 13 of the scirrhous type (diffusely infiltrative type), were immunostained by using monoclonal antibodies to human placental PH. Results Although cytoplasmic staining for PH was observed in both fibroblasts and carcinoma cells, there was increased expression of the α-subunit in fibroblasts and no difference in expression between the scirrhous and non-scirrhous type gastric carcinomas. In scirrhous type samples, there was increased PH expression in fibroblasts located in the tumor periphery when compared with fibroblasts in the tumor center. These findings suggested that maintenance of a balance between production and degradation of collagen in gastric carcinoma tissues might be important for stroma formation. Conclusions It is speculated that activated fibroblasts participate in collagen biosynthesis at the tumor periphery rather than in the tumor center and that increased collagen biosynthesis at the tumor periphery in scirrhous gastric carcinoma may assist further invasion of tumor cells. J. Surg. Oncol. 1999;70:239–246. © 1999 Wiley-Liss, Inc.

A restricted open shell Kohn–Sham (ROKS) equation is introduced in this article. This ROKS equation was constructed analogously to the restricted open shell Hartree–Fock (ROHF) equation. We derived a set of simultaneous eigenequations by the variation of orbitals and combined them into a unified ROKS equation. A program module to solve the ROKS equation was coded by the object-oriented language C++ to realize the software reliability and an efficient use of the limited memory. The module was implemented into a density functional theory (DFT) program we had developed. We solved a cationic state of bis(imidazole)porphinato-iron by our program with the ROKS method. The complex is a model for the reaction centers embedded in cytochrome c 3 . For the comparison of the electron distribution, the restricted Kohn–Sham (RKS) and the unrestricted Kohn–Sham (UKS) calculations were also carried out for the neutral state and the cationic state of the model complex, respectively.

In general, most of execution time for large scale forging simulations by rigid plastic finite element method is spent to solve linear equations called stiffness equation. For speedup of large scale forging simulations, it is necessary to solve stiffness equation at high speed. In this paper, we present the result of accelerated computation for stiffness equation by the parallelized conjugated gradient method on the distributed multiprocessor system called cluster of workstations. As a result, the cluster of workstations, which are inter connected hierarchically by high speed network, spend less communication time than the ordinary non-hierarchical cluster of workstations. By using high speed network TPDDI (Twisted Pair Distributed Data Interface) and three-class model of hierarchical cluster of workstations which has two layers of slave CPUs, we achieved 50% reduction of communication time and about fivefold speedup of execution time of the parallelized conjugated gradient method by sixteen slave CPUs.

The histological observation that well-differentiated cancer cells in early hepatocellular carcinoma (HCC) invade portal tracts and/or fibrous bands and that these fibrous tissues then disappear suggests the participation of matrix metalloproteinase-1 (MMP-1) in the degradation of fibrous tissue. To confirm this hypothesis, the authors investigated the localization of both the MMP-1 protein and its messenger RNA (mRNA) in early HCC immunohistochemically and by in situ hybridization using complementary DNA (cDNA) and synthetic antisense probe of MMP-1; they then compared the results with those in advanced HCC. MMP-1 gene transcripts and protein were observed in well-differentiated cancer cells of early HCC but not in moderately or poorly differentiated cancer cells. Thus, cancer cells producing MMP-1 in early HCC may destroy the portal tract tissue adjacent to the cancer lesion and/or the fibrous band of cirrhosis. These results seem to have demonstrated a difference in the mechanism of cancer growth and invasion between early and advanced HCCs.

In this article, we propose a new molecular orbital program for all-electron calculation of proteins which is based on density functional theory. To carry it out in a fully analytical way, we adopted the (pure-) analytical Xα method and modified it for saving a lot of memories for large-scale calculations. The recent software technology sophisticated in information science is inevitably applied to achieve calculations of large molecular systems. The program is coded by the object-oriented language C + +, its output is shown graphically, and the most of the procedures in this program are controlled through an efficient graphical user interface developed by ourselves. Such technology supports the safe construction of the huge software, the tidy representation of enormous data, and the ready control of complex calculations. Test calculations with various sizes of glycine polypeptides indicate that the computation time is proportional to the 1.7 powers of the number of residues. This result suggests that the all-electron calculations of proteins consisting of over 1000 atoms could be performed with distributed and/or massively parallel computers. © 1997 John Wiley & Sons, Inc. Int J Quant Chem 63: 245–256, 1997

The expression of matrix metalloproteinase-1 (MMP-1) gene and the presence of MMP-1 protein in gastric cancer were examined by in situ hybridization and immunohistochemistry. Expression of the interstitial collagenase (MMP-1) gene was detected within the stroma of the neoplastic glands, and infiltration of eosinophils was observed to be associated with regions of MMP-1 gene expression. The degree of eosinophilic infiltration correlated with the level of MMP-1 mRNA expression. Immunostaining showed localization of MMP-1 protein in the stromal cells, and additionally in the neoplastic glands. These findings indicate that the stromal cells may play an important role in the expression of MMP-1, and suggest a pathophysiological role for MMP-1 in the invasion and metastasis of gastric cancer.

Using data compiled from 216,707 births from the population-based Kanagawa Birth Defects Monitoring Program (KAMP), we conducted a case-control study to evaluate the effect of maternal smoking and/or drinking during pregnancy on the risk of infants' anal atresia in 1989-1994. The frequency of maternal smoking (including passive smoking) and/or maternal drinking during pregnancy among 84 infants with anal atresia was compared with 174 matched controls. The 84 anal atresias include 49 cases of isolated anal atresia and 35 cases of syndromal anal atresia. Our findings suggest that maternal drinking during early pregnancy is associated with an increased risk of isolated anal atresia (OR = 4.8, 95% CI 1.2 to 19.1, p0.05). A slightly increased trend was also observed in the association of maternal smoking during pregnancy with both in the pooled groups of anal atresia (OR = 1.4, 95% CI 0.5 to 3.6).

Metabolic changes of basement membrane proteins, collagenous and non-collagenous, will be followed by morphological changes and develop into diabetic microangiopathy. In this study, serum concentrations of laminin and type IV collagen were measured in 87 cases of non-insulin-dependent diabetes mellitus (NIDDM) and examined with the development of diabetic nephropathy and retinopathy. Serum type IV collagen was measured by two methods, assays of the 7S portion (7S-RIA) and of both the 7S and central triple helical domain (IV-EIA). IV-EIA showed more significant elevation with development of diabetic microangiopathy than 7S-RIA and laminin. After measurement of basement membrane proteins, 66 cases were followed clinically for 3 or 4 years. In 45 cases without worsening of diabetic nephropathy, IV-EIA and laminin concentrations in serum were significantly lower than those in 21 cases who showed worsening of diabetic nephropathy in the same period. Multivariate analysis revealed a good model including three dependent variables of serum values of laminin, IV-EIA and mean blood pressure for discriminating between the worsened group and the non-worsened group. High values of IV-EIA and laminin in serum may be useful in predicting the worsening of diabetic nephropathy.

To evaluate the relation between the working conditions and the workers' health, particularly the prevalences of obesity, liver disorder and hyperlipidemia, we analyzed physiological examination data and the questionnaire survey about life behaviors and working conditions during the terms of car manufacturing work and car sales work among 61 male subjects. In the physiological examination data, compared with the term of car manufacturing work, the values of body weight, body mass index (BMI), GOT, GPT, gamma-GTP, TG and T-CHO elevated and the prevalences of obesity and liver disorder increased during the term of car sales work. During the term of car sales work, the prevalences of alcohol drinkers and cigarette smokers increased and the changes of food intake behaviors were noted. It was estimated that the changes of food intake behaviors associated with the differences of working conditions contributed increasing number of obesity and liver disorder that was based on fatty liver caused by hyperlipidemia. These results of this study suggested that working conditions associated with the prevalences of obesity, liver disorder and hyperlipidemia were important to conduct the effective health education in the present occupational health administration.

Objective: to observe the influence of aging on urinary secretion of 3-hydroxyproline (3-Hyp) in normal subjects and in cancer patients and study the effects of aging on the accuracy of urinary 3-Hyp as a general-purpose cancer screening test.Design: We reanalyzed, from the aspect of gerontology, the amounts of 3-Hyp in the urine samples of the 211 healthy persons and 94 patients with cancer reported earlier. The two groups were divided in 3 age subgroups: adult, 30 to 44 years, middle-aged, 45 to 64 years, and aged, 65 years and older. The cancer stage was separated into 3 categories: stage 1, the early stage without any invasion, stage 2, locally invasive with no distant metastasis, and stage 3, advanced cancer. The correlation of age and urinary 3-Hyp was analyzed in normal subjects and cancer patients. Sensitivity and specificity were calculated for different cutoff points in the diverse groups to construct the receiver operating characteristic (ROC) curves.Main Results: We observed that aging decreases the urinary secretion of 3-Hyp in normal subjects, with a correlation coefficient of -0.250 (p

Hepatocellular carcinoma (HCC) is a cancer with extremely poor prognosis. This review discusses the pathological characteristics of multi-step hepatocarcinogenesis, tumor growth, invasion and metastasis, the expression of matrix metalloproteinases (MMPs) and their inhibitors via signal transduction in relation to dedifferentiation of hepatoma cells. It introduces the reports on anti-cancer agents in the field of MMP science, and finally describes novel strategies for the early stages of HCC in relation to cancer stem cells.

Objective: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. Methods: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl4) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl4 injection. The role of MMP-13 in stably expressing cell lines was also analyzed. Results: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. Conclusion: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.

The polymorphism of CYP1A1*2A or CYP1A1*2B, and the linkage of CYP1A1*2A, CYP1A1*2B, GSTM1 and GSTT1 polymorphisms have been established as susceptible genes or gene-gene interactions of tobacco-related lung cancer. New candidate genes susceptible for lung cancer such as NQO1 (NAD(P)H:quinine oxidoreductase), NAT2 (N-acetyltransferase 2), and several others have been reported. In the present review we focus on new candidate genes susceptible for lung cancer, then examine all Japanese references by meta-analysis on susceptible genes over the past 20 years, and discuss whether new candidates and changing trend in Japan could be caused by environmental change.

We reported that endogenous urinary 3-hydroxyproline (3-Hyp) is useful for cancer screening because cancer invasion involves the destruction of basement membrane. A simple and sensitive assay is desired.An ELISA method using a specific antibody against a synthetic peptide of 10 amino acids including 3-Hyp corresponding to the amino acid sequences of collagen type IV alpha chain was applied to urine samples from 180 healthy controls and 22 cancer patients.The values in controls were 2.44+/-1.90 (SD) mg peptide/g creatinine for 52 men and 2.87+/-2.01 for 128 women, while the values in 22 cancer patients were very low at 0.110+/-0.137 (p0.001).The discrepancy in the data between our previous and present studies is based on the difference of targets measured. 3-Hyp-containing peptides in cancer patients might be destroyed by the elevated peptidase levels found in these patients.This ELISA assay may be useful for cancer screening.

The Ψ and Φ torsion angles around glycosidic bonds in a glycoside chain are the most important determinants of the conformation of a glycoside chain. We determined force-field parameters for Ψ and Φ torsion angles around a glycosidic bond bridged by a sulfur atom, as well as a bond bridged by an oxygen atom as a preparation for the next study, i.e., molecular dynamics free energy calculations for protein-sugar and protein-inhibitor complexes. First, we extracted the Ψ or Φ torsion energy component from a quantum mechanics (QM) total energy by subtracting all the molecular mechanics (MM) force-field components except for the Ψ or Φ torsion angle. The Ψ and Φ energy components extracted (hereafter called “the remaining energy components”) were calculated for simple sugar models and plotted as functions of the Ψ and Φ angles. The remaining energy component curves of Ψ and Φ were well represented by the torsion force-field functions consisting of four and three cosine functions, respectively. To confirm the reliability of the force-field parameters and to confirm its compatibility with other force-fields, we calculated adiabatic potential curves as functions of Ψ and Φ for the model glycosides by adopting the Ψ and Φ force-field parameters obtained and by energetically optimizing other degrees of freedom. The MM potential energy curves obtained for Ψ and Φ well represented the QM adiabatic curves and also these curves' differences with regard to the glycosidic oxygen and sulfur atoms. Our Ψ and Φ force-fields of glycosidic oxygen gave MM potential energy curves that more closely represented the respective QM curves than did those of the recently developed GLYCAM force-field. © 2009 Wiley Periodicals, Inc., J Comput Chem, 2009

Aims Glycyrrhizin has been widely used for the treatment of chronic hepatitis C. It decreases the serum levels of aminotransferases, and suppresses progression of liver fibrosis as well as subsequent occurrence of hepatocellular carcinoma. Although previous studies have shown that glycyrrhizin and its metabolite inhibit collagen gene expression, its underlying mechanisms are virtually unknown. This study was aimed to explore molecular mechanisms responsible for the inhibitory effect of glycyrrhizin on type I collagen gene transcription. Main methods Effects of glycyrrhizin and its metabolite, glycyrrhetinic acid, on collagen promoter activity were examined by using transgenic reporter mice harboring α2(I) collagen gene (COL1A2) promoter. Their effects on the TGF-β/Smad signaling pathway were studied by cell transfection assays and immunofluorescence studies using cultured hepatic stellate cells. Key findings Administration of glycyrrhizin or its metabolite, glycyrrhetinic acid, significantly suppressed COL1A2 promoter activation and progression of liver fibrosis induced by repeated carbon tetrachloride injections. In cultured hepatic stellate cells, glycyrrhetinic acid, but not glycyrrhizin, inhibited type I collagen synthesis mostly at the level of gene transcription. This inhibitory effect of glycyrrhetinic acid was abolished by a mutation introduced into a Smad3-binding region within the COL1A2 promoter. Glycyrrhetinic acid did not affect gene expression of TGF-β receptors or Smad proteins, but inhibited nuclear accumulation of Smad3 in activated hepatic stellate cells. In addition to those direct inhibitory effects on COL1A2 transcription, glycyrrhetinic acid also suppressed activation of quiescent hepatic stellate cells in primary culture. Significance The results provide a molecular basis for the anti-fibrotic effect of glycyrrhizin treatment.

Background & Aims: Hepatocyte growth factor (HGF) and transforming growth factor-β (TGF-β) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-β is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-β-stimulated profibrogenic signal transduction. Methods: Effects of HGF on TGF-β-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring α2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. Results: Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-β-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-β-stimulated expression of those target genes. Conclusions: These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

A questionnaire survey was conducted to examine a relationship between indefinite complaints and life styles in junior high school students and to apply the results to life guidance.Twenty nine public junior high schools were selected by random sampling of 13 cities and 3 towns in Kanagawa Prefecture. A self-reported questionnaire was supplied to all subjects, containing 100 life-style related items, including a simplified CMI (Cornel Medical Index) questionnaire.The number of students who were evaluated as "point medical examination" based on physical symptoms of CMI and "disorder" based on the mental and physical symptom increased with the grade for both males and females. The students who reported "the life rhythm was always irregular" tended to be in the group evaluated as "disorder" or "point medical examination". The life rhythm was related to bedtime, sleeping hours and eating habits. It was observed that the students whose daily habits were always irregular had late bedtime, short sleeping hours and an irregular eating habit. As a result of the quantification method for the second type of analysis, it was found that sleeping time was the most related factor to the life rhythm. Furthermore 90% and more of students reported they did not consider nutritional balance with regard to their eating habits.It was suggested that those students who had irregular life rhythm needed sufficient sleep and nutrition guidance to maintain their health.