Association between underweight, serum albumin levels, and height loss in the Japanese male population: a retrospective study.

BACKGROUND: Previous study has shown that height loss (defined as the highest quartile of height loss per year) was inversely associated with serum albumin levels. Furthermore, comparatively healthy hyponutrition has been linked with being underweight; as such, underweight might be inversely associated with serum albumin levels and positively associated with height loss. METHODS: To clarify the associations between serum albumin level, underweight status, and height loss, we conducted a retrospective study of 8,096 men over 4.0 years (median). RESULTS: Serum albumin level at baseline was inversely associated with being underweight (body mass index [BMI]: < 18.5 kg/m2) at baseline and height loss. The known cardiovascular risk factor adjusted odds ratio (OR) and 95% confidence interval (CI) of underweight at baseline and of height loss for 1 standard deviation increment of serum albumin (0.28 g/dL) was 0.79 (0.70, 0.90) and 0.84 (0.80, 0.88). Underweight was also shown to be positively associated with height loss: with the reference of normal-low weight (BMI: 18.5-22.9 kg/m2), the adjusted OR (95% CI) was 1.60 (1.21, 2.10). CONCLUSION: Comparative healthy hyponutrition, which is related to low serum albumin levels and being underweight, is a significant risk factor for height loss among Japanese men. These results help to clarify the mechanisms underlying height loss.

Medical and exercise consultation use for low back and knee pain among cardiovascular mass screening population: A cross-sectional study.

Low back and knee pain, as major symptoms and early signs of osteoarthritis, have restricted healthy life expectancy, and numerous guidelines have recommended therapeutic exercise as the first-line treatment for chronic pain. Proportions of medical and exercise consultation use for those pain have been unclear, and these may change in the future. We performed a cross-sectional study of 2,954 persons aged over 30 years in 2017 as a part of the Circulatory Risk in Communities Study. A generalized linear model with logit link and 11-year age-group moving averages were used to estimate sex- and age-specific average proportions of lifetime pain, chronic pain, and dysfunctional chronic pain of the low back and knee, and history of medical and exercise consultation use. The medical consultation use increased in the order of lifetime pain, chronic pain, and dysfunctional chronic pain, reaching 69.1 % [65.2, 72.8] in women and 74.9 % [70.3, 79.0] in men for chronic low back pain, and 70.3 % [66.1, 74.2] in women and 55.6 % [49.3, 61.7] in men for chronic knee pain. On the other hand, the exercise consultation use accounted for 36.5 % [32.6, 40.6] in women and 28.8 % [24.4, 33.5] in men for chronic low back pain, and 40.8 % [36.5, 45.2] in women and 20.6 % [16.0, 26.0] in men for chronic knee pain. This survey revealed the differences in the multilayer proportions of medical and exercise consultation use for low back and knee pain in the cardiovascular mass screening, suggesting exercise consultation was less often provided compared to medical consultation.

Association between smoking and height loss in Japanese workers: A retrospective study.

Height loss is reported to be an independent risk factor for all-cause and cardiovascular mortality. Smoking, which is responsible for a considerable proportion of deaths due to any cause, is also associated with lumbar disc degeneration, a major risk factor for height loss. Therefore, smoking could be an independent risk factor for height loss. To clarify the association between smoking status and height loss, a retrospective study with 8,984 (5,518 men and 3,466 women) Japanese workers was conducted. The present study population comprised 9,681 workers aged 40-74 years who participated in annual medical examinations between 2011 and 2017 (baseline). Subjects without a height measurement during 2012-2018 (endpoint) were excluded from the analysis (n = 697). Height loss was defined as being in the highest quartile of annul height decrease (1.48 mm/year for men and 1.79 mm/year for women). Independent of known cardiovascular risk factors, smoking was positively associated with height loss among men but not among women. With never smokers as the referent group, the adjusted odds ratio (95% confidence interval) was 1.15 (0.98, 1.35) for former smokers and 1.24 (1.05, 1.46) for current smokers among men, respectively. Among women, the corresponding values were 0.98 (0.79, 1.21) and 0.90 (0.71, 1.16), respectively. Since height loss and smoking are independent risk factors for all-cause and cardiovascular mortality, these results help clarify the mechanisms underlying the association between height loss and mortality risk.

HbA1c and height loss among Japanese workers: A retrospective study.

Evaluating the risk of height loss could be an efficient way to evaluate endothelial health, which might be associated with all-cause and cardiovascular mortality. Diabetes is an established risk factor both for intervertebral disk degeneration and osteoporosis-related fractures, which are major risk factors for height loss among adults. Therefore, hemoglobin A1c (HbA1c), as an indicator of the presence of diabetes, could be positively associated with height loss. A retrospective study of 10,333 workers aged 40 to 74 years was conducted. Height loss was defined as being in the highest quintile of height decrease per year. HbA1c in the normal range was positively associated with height loss. The known cardiovascular risk factors-adjusted odds ratio (OR) and 95% confidence interval (CI) for height loss with a 1-standard deviation (SD) increase in HbA1c (0.38% for both men and women) was 1.06 (1.02, 1.10) for men and 1.15 (1.07, 1.23) for women, respectively. When limit those analysis among those without diabetes, the magnitude was slightly higher; the fully adjusted OR and 95% CI for height loss with a 1-SD increase in HbA1c was 1.19 (1.11, 1.28) for men and 1.32 (1.20, 1.44) for women, respectively. Even when HbA1c is within the normal range, higher HbA1c is a significant risk factor for height loss among workers.

Association between serum albumin levels and height loss in Japanese workers: a retrospective study.

BACKGROUND: Height loss starting in middle age was previously shown to be associated with high cardiovascular mortality in later life. However, the factors associated with height loss remain unknown. Since low serum albumin levels are reported to be associated with high mortality caused by cardiovascular disease, they may also contribute to height loss. METHODS: To clarify the association between serum albumin and height loss, we conducted a retrospective study of 7637 Japanese workers who participated in general health check-ups from 2008 to 2019. Height loss was defined as the highest quartile of height loss per year. RESULTS: Individual with high serum concentration of albumin possess beneficial influence on preventing incidence of height loss. In both men and women, serum albumin level was significantly inversely associated with height loss. After adjustment for known cardiovascular risk factors, the adjusted odd ratio (OR) and 95% confidence interval (CI) for height loss per 1 standard deviation of albumin (0.2 g/dL for both men and women) were 0.92 (0.86, 0.98) in men and 0.86 (0.79, 0.95) in women. Even when the analysis was limited to participants without hypoalbuminemia, essentially same association was observed, with fully adjusted corresponding ORs (95%CI) of 0.92 (0.86, 0.98) in men and 0.86 (0.78, 0.94) in women. CONCLUSION: Independent of known cardiovascular risk factors, higher serum albumin levels may prevent height loss among Japanese workers. While several different diseases cause hypoalbuminemia, they may not be the main reasons for the association between serum albumin and height loss. Though further research is necessary, this finding may help clarify the mechanisms underlying the association between height loss and higher mortality in later life.

Eating speed and height loss in relation to overweight: A retrospective study.

Fast eating is an independent risk factor for weight gain. Our previous study involving Japanese workers revealed that overweight (body mass index ≥ 25.0 kg/m2) is an independent risk factor for height loss. However, no studies have clarified the association between eating speed and height loss in relation to overweight status. A retrospective study of 8,982 Japanese workers was conducted. Height loss was defined as being in the highest quintile of height decrease per year. Compared with slow eating, fast eating was revealed to be positively associated with overweight; the fully adjusted odds ratio (OR) and 95% confidence interval (CI) was 2.92 (2.29, 3.72). Among non-overweight participants, fast eaters had higher odds of height loss than slow eaters. Among overweight participants, fast eaters had lower odds of height loss; the fully adjusted OR (95% CI) was 1.34 (1.05, 1.71) for non-overweight individuals and 0.52 (0.33, 0.82) for overweight individuals. Since overweight was significantly positively associated with height loss [1.17(1.03, 1.32)], fast eating is not favorable for reducing the risk of height loss among overweight individuals. Those associations indicate that weight gain is not the main cause of height loss among Japanese workers who eat fast.

Effects of Low-Dose Therapist-Led Self-Exercise Education on the Management of Chronic Low Back Pain: Protocol for a Community-Based, Randomized, 6-Month Parallel-Group Study.

INTRODUCTION: Chronic low back pain (CLBP), defined as low back pain persisting for at least 3 months, leads to limitations in the activities of daily living and decreased quality of life. Individualized self-exercise education could be a preferable treatment option, especially in community-dwelling people with CLBP. Previous studies, however, did not directly compare the effects of therapist-led self-exercise education and material-only education, and there are only a few studies investigating the effects of low-dose (comprising a few sessions) self-exercise education on CLBP. We present a protocol of community-based, randomized study to evaluate the effects of low-dose (comprising a few sessions), therapist-led self-exercise education on CLBP. METHODS: Forty-eight participants with CLBP (men and women, aged 40-74 years) will be allocated to therapeutic self-exercise education programs, either a therapist-led group (2-week therapist's consultation and material use) or material-only group (material use only), in a randomized controlled trial. Pain intensity (NRS, numeric rating scale), pain disability (RDQ, Roland-Morris disability questionnaire), pain self-efficacy (PSEQ, pain self-efficacy questionnaire), and quality of life score (EQ-5D, European quality of life-5 dimensions) will be measured at baseline and at 4, 12, and 24 weeks. We will apply a repeated-measures design with mixed-effect models to estimate group differences from the baseline. Ethics/Trial registration number: The protocol was approved by the Ethics Committees of the Osaka Center for Cancer and Cardiovascular Disease Prevention and Osaka University. The trial registration number is registered on the University Hospital Medical Information Network (UMIN000024537).

Myofibroblasts: Biochemical and proteomic approaches to fibrosis.

Fibrosis is a state, in which excess amounts of extracellular matrix are deposited in the tissue. Fibrosis can occur in various organs, including the liver, lung, kidney and heart. The progression of fibrosis involves interstitial hypercellularity, accumulation of extracellular matrix, and atrophy of epithelial structures, resulting in a loss of normal function. Myofibroblasts play a crucial role in the development and progress of fibrosis. When stimulated, myofibroblasts actively synthesize connective tissue components and cause organ fibrosis. As a result, the process and the mechanism of myofibroblast activation represent a target for antifibrotic treatment. As yet, however, an effective treatment has not been developed, and new treatment modalities are expected. Because activation of myofibroblasts is a key event during fibrosis development, there is great interest in identifying and characterizing proteins whose expression is changed after this activation. In this review, fibrosis is outlined and the role of myofibroblasts in this disorder is described. Furthermore, the search for candidate proteins to target for treatment and the prospects of antifibrotic therapy are discussed.

Conditioned media from lung cancer cell line A549 and PC9 inactivate pulmonary fibroblasts by regulating protein phosphorylation.

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-ß (TGF-ß) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease α-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-ß-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process.

Transforming growth factor-beta upregulates the expression of integrin and related proteins in MRC-5 human myofibroblasts.

Myofibroblasts are defined as fibroblasts that express certain features of smooth muscle differentiation. Activation of myofibroblasts plays a central role in fibrosis. Transforming growth factor-beta (TGF-beta) is a potent activator of myofibroblasts; namely, TGF-beta causes changes in myofibroblast phenotypes including morphological alterations and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Because it has been well known that humoral factors, especially, TGF-beta, and extracellular matrix components cause myofibroblast activation, we examined the expression of integrin and related proteins during activation of MRC-5 human myofibroblasts with TGF-beta. Western blot analysis revealed that TGF-beta treatment for 3 days increased the expression of alpha-SMA, which was also immunocytochemically observed as actin stress fibers. In the early phase of TGF-beta treatment, fibronectin expression was greatly increased, followed by the increased expression of integrin alphav and alpha11 and integrin beta1 and beta3. Co-immunoprecipitation assays revealed that the integrin alphav subunit was co-precipitated with integrin beta1 and beta3, and that integrin beta1 was co-precipitated with alpha11, alphav, alpha2, and alpha5. The expression of focal adhesion kinase and integrin-linked kinase proteins was also upregulated by treatment with TGF-beta. In addition, the expression of type I collagen mRNA was increased by TGF-beta, but not type III collagen mRNA, as judged by real-time PCR analysis. These results suggest the possibility that TGF-beta induces fibronectin expression in MRC-5 cells, which subsequently induces the expression of integrin receptors, alphavbeta3, alphavbeta1, and alpha11beta1. This report also shows that expression of integrin alpha11 is upregulated during the TGF-beta-mediated activation of myofibroblasts.

Decreased expression of catechol-O-methyltransferase in the renal cortex of malignant spontaneously hypertensive rats.

Essential hypertension is a disease of unknown pathogenesis, although renal function has been implicated as an important factor in its cause. Stroke-prone spontaneously hypertensive (SHRSP) rats provide an animal model of essential hypertension. To understand the cause of hypertension, identifying proteins that are differentially expressed between hypertensive and normotensive rats may provide a key. Here, proteins in the renal cortex from SHRSP rats, malignant stroke-prone spontaneously hypertensive (M-SHRSP) rats, and Wistar Kyoto (WKY) rats as a normotensive control were subjected to two-dimensional difference gel electrophoresis (2D-DIGE). After in-gel digestion by trypsin, proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Several proteins showed differential expression patterns between hypertensive and normotensive rats. Among them, we focused on catechol-O-methyltransferase (COMT) because this enzyme inactivates catecholamines, possibly affecting blood pressure. To confirm the differential expression of COMT in each animal, we conducted Western blot analysis, which revealed that the expression of COMT is lower in M-SHRSP rats than in control and SHRSP rats, indicating that blood pressure and expression of COMT are related. In fact, the blood pressure of M-SHRSP rats was significantly higher than that of SHRSP rats at age of 10 weeks. Immunohistochemical and immunofluorescence studies showed that COMT in renal cortex is localized in tubular epithelial cells. The expression of COMT is lower in the renal cortex tubular epithelium of M-SHRSP rats than in normotensive rats. These results suggest that the decreased expression of COMT may be an important factor leading to the development of hypertension.

Hypocretin-1/orexin-A activates subfornical organ neurons of rats.

Central injection of hypocretins/orexins in rats induces water intake. As the subfornical organ (SFO) plays an important role in drinking behavior, hypocretins may excite SFO neurons. In this study, effects of hypocretins on SFO neurons were investigated electrophysiologically in slice preparations. In extracellular recordings, hypocretin-1 excited SFO neurons, but hypocretin-2 did not or it was little. After the block of synaptic inputs, the excitatory responses to hypocretin-1 remained, but some disappeared. In whole-cell patch-clamp recordings, hypocretin-1 reduced the frequencies of miniature inhibitory presynaptic currents with inward currents occasionally in SFO neurons, but hypocretin-2 did not. These results suggest that hypocretin-1 excites SFO neurons via the activation of hcrtR1 on premembranes and postmembranes.

Intraperitoneal injection of pilocarpine activates neurons in the circumventricular organs and hypothalamus in rats.

It has been suggested that while the sialogogue pilocarpine elicits salivary secretion by acting directly on acinar cells of the salivary glands, it induces drinking behavior by acting on muscarinic receptors in the central nervous system. To study which brain regions are affected by the peripherally injected pilocarpine, we investigated changes in the numbers of c-Fos immunoreactive cells. The injections increased the numbers of c-Fos immunoreactive cells in the subfornical organ, median nucleus of preoptic area, organum vasculosum of lamina terminalis, paraventricular nucleus and supraoptic nucleus. Intracerebroventricular injection of pilocarpine produced similar changes in the expression of c-Fos immunoreactivity. The increases in immunoreactive expression induced by both the intraperitoneally and intracerebroventricularly injected pilocarpine were suppressed by previous intracerebroventricular injection of the muscarinic receptor antagonist atropine. Electrophysiological experiments using slice preparations and whole cell recordings showed that pilocarpine depolarized the membrane of neurons in the subfornical organ and suppressed the inhibitory GABAergic synaptic currents by a presynaptic action. The results suggest that peripherally applied pilocarpine does not act only on the salivary glands as a sialogogue, but also evokes thirst sensation by acting on the center controlling body fluid balance in the central nervous system.

Effects of decorin on the expression of alpha-smooth muscle actin in a human myofibroblast cell line.

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing alpha-smooth muscle actin (alpha-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of alpha-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell-cell contact or serum starvation, and examined the relationship between decorin and alpha-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell-cell contact. In contrast, the expression of alpha-SMA in cells made quiescent by cell-cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of alpha-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of alpha-SMA.

Clathrin light chain b is capable of affecting potently a major protein phosphatase from microtubules (MT-PP1).

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.

Activation of subfornical organ neurons in rats through pre- and postsynaptic alpha-adrenoceptors.

The effects of noradrenaline (NA) and its analogs on subfornical organ (SFO) neurons in rat slice preparations were investigated by using whole cell patch-clamp recording. In the current-clamp mode, the application of NA at 10-100 microM produced membrane depolarization (63%, 17 responsive neurons/27 neurons tested) and hyperpolarization (22%, 6/27 neurons). In the voltage-clamp mode, NA application at 1-100 microM produced inward currents (69%, 42/61 neurons) and outward currents (23%, 14/61 neurons). These currents remained in the presence of TTX or both glutamate and GABA receptor antagonists. In most of the neurons (25/31 neurons) showing inward currents in the presence of NA, the membrane conductance was not changed by voltage ramps or hyperpolarizing pulse stimulation. Similar responses were obtained by the application of the alpha1-agonist phenylephrine. The phenylephrine-induced inward currents were inhibited by the alpha1-antagonist prazosin. The alpha2-agonist clonidine decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (4/10 neurons). In addition, RT-PCR assay and immunohistochemical staining showed the existence of alpha1-adrenoceptors in the SFO. The results suggest that SFO neurons in rats are activated postsynaptically through alpha1-adrenoceptors and that the activation is enhanced by suppressing GABAergic inhibitory synaptic inputs through presynaptic alpha2-adrenoceptors.

Transient outward K+ currents in rat dissociated subfornical organ neurones and angiotensin II effects.

Although angiotensin II inhibits transient outward K+ currents (I(A)s) in subfornical organ neurones, there is no evidence concerning which Kv channels are involved. We investigated I(A)-generating Kv channels in dissociated rat subfornical organ neurones, using molecular, electrophysiological and pharmacological techniques, and studied the effects of angiotensin II. Conventional RT-PCR showed the presence of mRNAs for channels of the Kv3.4, Kv1.4 and Kv4 families, which are capable of generating I(A)s. Tetraethylammonium at 1 mm, which blocks Kv3 channel-derived currents, and blood-depressing substance-I, a Kv3.4-specific blocker, at 2 microm suppressed the I(A)-like component of whole-cell outward currents in some neurones. 4-Aminopyridine at 5 mm inhibited I(A)s in the presence of tetraethylammonium at 1 mm. Cd2+ at 300 microm shifted the activation and inactivation curves of the 4-aminopyridine-sensitive and tetraethylammonium-resistant I(A)s positively. The tetraethylammonium-resistant I(A)s showed fast and slow components during the process of recovery from inactivation, but the slow component was not seen in all neurones. The time constant of the fast recovery component was less than 200 ms, while that of the slow recovery component was around 1 s. Using single-cell RT-PCR, mRNAs for Kv4.2 and Kv4.3L were detected frequently, but those for Kv1.4 and Kv3.4 were seen only rarely. Angiotensin II at 30 nm inhibited the fast recovery component of tetraethylammonium-resistant I(A)s in many neurones. These results suggest that the fast recovery component of the tetraethylammonium-resistant I(A) in subfornical organ neurones depends upon Kv4, and that it can be modulated by angiotensin II.

Central nicotinic stimulation reduces vascular conductance in the gingiva in anesthetized rats.

OBJECTIVE: The central effects of nicotine on gingival blood flow and vascular conductance were investigated. BACKGROUND: Nicotine is absorbed in cigarette smoking, which is a risk factor for periodontal disease. Although studies have shown peripheral effects of nicotine on gingival blood flow and gingival vascular conductance, little is known about its central effects. METHODS: We used laser Doppler flow measurements to investigate the changes of gingival blood flow produced by 5 min intracerebroventricular (i.c.v.) injection of (-)-nicotine ditartrate in rats anesthetized by urethane and alpha-chloralose, along with occasional measurements of the blood pressure from the femoral artery. RESULTS: The i.c.v. injection of nicotine at 15, 50, and 150 microg reduced the gingival blood flow significantly, compared with saline. The maximal reduction was dose-dependent. Next, we measured the blood pressure and gingival blood flow in the i.c.v. injection of nicotine at 50 microg, to calculate the gingival vascular conductance. The blood pressure was reduced, along with the change of gingival blood flow immediately after the injection, whereas the gingival vascular conductance fell with a time-lag. CONCLUSION: These results suggest that the reduction of gingival blood flow by central nicotinic stimulation is accompanied in part by a change of vascular tonus in the gingiva.

Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

Purification and characterization of decorin from the culture media of MRC-5 cells.

Myofibroblasts play an important role in fibrogenesis. Myofibroblasts secrete several components of the extracellular matrix, including decorin. To clarify the properties of decorin synthesized by myofibroblasts, we have purified and characterized decorin secreted into culture medium by the myofibroblast cell line MRC-5. Decorin was purified by successive chromatography steps using Hitrap Q and Superdex 200. Purified decorin showed a broad band on SDS-polyacrylamide gel electrophoresis, which was resolved into two smaller molecular weight bands after digestion with chondroitinase ABC. Further digestion with N-glycanase resolved these two bands into a single band, indicating that the N-glycation pattern of decorin is heterogeneous. The N-terminal amino acid sequence analysis of the purified protein and its reactivity towards an antibody raised against a C-terminal peptide of decorin indicate that MRC-5 cells secrete full-length decorin into the culture medium. To characterize the glycosaminoglycan chains attached to decorin, glycosaminoglycans from the purified protein were treated with chondroitinase ACI, chondroitinase ACII, chondroitinase ABC and chondroitinase B. The resulting disaccharides were analyzed by chromatography, which indicated that decorin secreted by MRC-5 cells is a dermatan sulfate proteoglycan. In conclusion, the decorin secreted by MRC-5 cells has similar characteristics to the decorin expressed in several tissues. Thus, culturing MRC-5 cells may be highly useful for studying the role of decorin and myofibroblasts in fibrosis.