Biomedical subjects

E Harada

Publications and source records attributed to E Harada.

At least 19 recordsLinked to original sources

PubMed2006

Mechanisms involved in the enhancement of osteoclast formation by enamel matrix derivative.

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. MATERIAL AND METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. RESULTS: Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.

Animals↗

PubMed2006

Binding characteristics and distribution of lactoferrin receptors in the gut and choroid plexus in newborn calves.

Lactoferrin (Lf), an iron-binding multifunctional glycoprotein, abundantly present in colostrum and milk of different species such as humans, bovines, and mice has been shown that bovine colostral Lf is transported into the CSF via plasma in newborn calves. Specific Lf-receptors (Lf-R) are present in different cells of different species. In the present study, we report for the first time, the presence and distribution of Lf-R in the intestine and choroid plexus in newborn calves. Brush-border membrane vesicles (BBMV) were prepared from the mucosa of duodenum, jejunum, ileum, colon, epithelium overlying Peyer's patches (EOPP) in jejunum (EOPPJ) and ileum (EOPPI), and choroid plexus membranes. Receptor binding assays were carried out using 125I labeled bovine Lf. Specific and saturable Lf-R were found in BBMV of all the intestinal segments and choroid plexus examined. Nonlinear regression and Scatchard plot analyses clearly revealed that EOPP had the highest binding maximal (Bmax), and lowest in colon. The maximum dissociation constant (Kd) 0.7 microM was in colon while, Bmax and Kd in choroid plexus membrane were 16.87 nmol/mg protein and 0.34 microM, respectively. All these findings together strongly suggested that Lf was transported into CSF via plasma through receptor mediated transcytosis.

Animals↗

PubMed2003

Characteristics of lactoferrin receptor in bovine intestine: higher binding activity to the epithelium overlying Peyer's patches.

Several lines of evidence have recently demonstrated the occurrence of specific lactoferrin (Lf) receptors in different cells. We report here, for the first time, the characteristics of binding, and distribution of Lf receptors in the bovine intestinal tract with special emphasis on the epithelium overlying Peyer's patches (EOPP). Brush-border membrane vesicles (BBMV) were prepared from the mucosa of duodenum, jejunum, ileum, colon, EOPP in jejunum and EOPP in ileum. Receptor binding assays were carried out using 125I-labelled bovine Lf. Specific and saturable Lf receptors were found in BBMV of all the intestinal segments examined. Non-linear regression and Scatchard plot analyses clearly revealed that EOPP had the highest binding maximal (Bmax), and lowest in colon. The maximum dissociation constant (Kd) 3.74 microm was in the ileum. We found that bovine transferrin competed with Lf for the same binding site of receptors. In contrast, no binding of bovine serum albumin occurred. It was concluded that Lf receptors in the mucosal lining are attributable to mediate multifunctional activities of Lf in the gut, especially in the EOPP.

Animals↗

PubMed2003

Impaired pancreatic endocrine and exocrine responses in growth-retarded piglets.

The alteration of pancreatic endocrine and exocrine secretory responses induced by secretagogues and neural input was investigated in post-weaning growth-retarded (GR) piglets. Blood and pancreatic juice were collected from these animals (6-8-weeks old). Plasma insulin and pancreatic digestive enzymes induced by nutrients, drugs and vagal stimulation were measured biochemically. The pancreas was inspected by immunohistochemical analysis. In GR piglets, the plasma glucose and insulin concentrations at the resting state were very low, and the secretory response was also markedly reduced, with maximum inhibition of 90% by glucose administration and 83% by arginine administration. The insulin secretion was not increased by 2-deoxy-D-glucose administration in GR piglets. The pancreatic juice secretions induced by vagal stimulation and secretagogues in GR piglets were not different from those induced in the control piglets. However, amylase activity in the pancreatic juice and in the pancreas was significantly decreased in GR piglets, although trypsin and chymotrypsin activities were not different. In the immunohistochemical analysis, the numbers of islets and the staining degree for insulin antibody also declined in the pancreases of GR piglets. These results indicated the reduction of insulin and amylase secretions from the pancreas in GR piglets, suggesting that a dysfunction of pancreatic endocrine and exocrine secretion during growth after weaning may be an important factor in the induction of growth retardation in piglets.

Amylases↗

PubMed2002

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs.

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.

Animals↗

PubMed2002

Transport of colostral macromolecules into the cerebrospinal fluid via plasma in newborn calves.

The objective of this study was to investigate the transfer of bovine colostral macromolecules especially the lactoferrin (Lf), transferrin (Tf), immunoglobulin G (IgG), and epidermal growth factor (EGF) from the gastrointestinal tract to the cerebrospinal fluid (CSF) via systemic circulation in newborn calves. Cannulae were placed into the jugular vein and cisterna magna to collect blood and CSF, respectively at various time points. The colostrum, plasma, and CSF were analyzed by ELISA, SDS-PAGE, two-dimensional PAGE, and Western blotting. The concentration of total protein, Lf, Tf, and IgG in plasma averaged 47 mg, 204 ng, 101 microg and 15 microg/ml before colostrum feeding and increased to the peak values of 64 mg, 2413 ng, 820 microg, and 4608 microg/ml 8 h after feeding, respectively. Before colostral feeding CSF, total protein, Lf, Tf, and IgG averaged 0.44 mg, 10.3 ng, 0.31 microg, and 0.11 microg/ml, but peak values after feeding averaged 2.0 mg, 173 ng, 71 microg and 72 microg/ml after 10 h, respectively. Immunologically, six EGF-positive protein bands were detected in colostrum as well as in three bands higher density in plasma and CSF after colostral feeding. This study revealed that the colostral macromolecules were not only absorbed into the systemic circulation, but also some of them including Lf, Tf, IgG, and EGF-like proteins were transported into the CSF in a time-dependent manner through blood-CSF or blood-brain barrier of the newborn calves.

Animals↗

PubMed2001

Aldosterone induces angiotensin-converting-enzyme gene expression in cultured neonatal rat cardiocytes.

BACKGROUND: The cardiac renin-angiotensin-aldosterone system is activated in failing hearts in proportion to the severity of the disease. We hypothesized that a positive feedback mechanism might exist within this system and contribute to the progression of the heart failure. Methods and Results-- To test this hypothesis, we examined whether angiotensin II or aldosterone induces the expression of angiotensin-converting-enzyme (ACE) mRNA in cultured neonatal rat ventricular cardiocytes. Expression of ACE mRNA was detected and quantified using real-time reverse transcription-polymerase chain reaction. Exposure to angiotensin II (10(-5) mol/L) for 24 hours had no significant effect on the expression of ACE mRNA (0.7+/-0.5-fold versus control, P=NS), but similar treatment with aldosterone (10(-5) mol/L) induced a 23.3+/-7.9-fold increase (P<0.01) in ACE mRNA expression. The effect of aldosterone was both time- (maximal effect, 24 hours) and dose-dependent (EC(50), 4x10(-7) mol/L), and it was significantly (P<0.01) inhibited by spironolactone, a specific mineralocorticoid receptor antagonist. CONCLUSIONS: Aldosterone upregulates ACE mRNA expression, which is blocked by spironolactone in neonatal rat cardiocytes. Thus, spironolactone may suppress the progression of heart failure by blocking the effects of aldosterone and angiotensin II.

Aldosterone↗

PubMed2001

Aldosterone production is activated in failing ventricle in humans.

BACKGROUND: Recent reports have indicated that aldosterone is produced in extra-adrenal tissues in animals. The present study was designed to examine whether aldosterone is produced in human heart. METHODS AND RESULTS: Plasma levels of aldosterone, BNP, and angiotensin-converting enzyme were measured in anterior interventricular vein (AIV), coronary sinus (CS), and aortic root (Ao), respectively, in 20 patients with left ventricular systolic dysfunction (LVSD), 25 patients with LV diastolic dysfunction (LVDD), and 23 control subjects. Aldosterone levels were significantly higher in AIV and CS than Ao in LVSD (98+/-10 versus 72+/-9 pg/mL, P:<0.001, and 97+/-11 versus 72+/-9 pg/mL, P:<0.001, respectively) and LVDD (87+/-10 versus 71+/-9 pg/mL, P:<0.01, and 84+/-10 versus 71+/-9 pg/mL, P:<0.01, respectively) groups, but no differences were observed in levels for these sites in the control group. Levels of ACE activity and BNP also were higher in AIV than Ao in both LV dysfunction groups. The difference in aldosterone levels between AIV and Ao and those in BNP and angiotensin-converting enzyme had a significant positive correlation with LVEDP and a significant negative correlation with LV ejection fraction in the LVSD group. CONCLUSIONS: Production of aldosterone, angiotensin-converting enzyme, and BNP are activated in failing human ventricle in proportion to severity.

Adult↗

PubMed2001

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes.

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 microm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes.

Biotransformation↗

PubMed2001

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Amino Acid Sequence↗

PubMed2001

Differential toxicity expression of gentamicine in five-sixths nephrectomized rats assigned to three progressive stages of renal dysfunction--establishment of a new screening approach.

Progressive renal dysfunction in 5/6 nephrectomized (NX) rats can be physiologically divided into three stages, coinciding with morphological stages, after definition of physiological parameters for identification of stage. Now, for the establishment of a toxicity screening approach using 5/6 NX rats, our concept, "Differential toxicity synchronized with renal dysfunction process could be identified using 5/6 NX rats" was examined by dosing gentamicin. Firstly, electrophoretic fractional changes of urinary proteins during gentamicin treatment were clarified with determination of amino acid sequences and the three differential features were proven, revealing the unpredictable depression of urinary albumin with progression of the stages in NX rats. Secondly, marked elevation of urinary lactate dehydrogenase (LDH) and glucose (GLU) was evident, indicating the intensified hypoxic conditions and glycolysis in tubular cells synchronized with increased tubular damage. Thirdly, these transit metabolic changes were proven as intensive cause for the advancement of renal dysfunction by the reduction of FRelectrolytes and water at the end of each dosing period. These results indicate that toxicity studies of newly developed drugs using 5/6 NX rats have potentiality prior to clinical dosing to the patients.

Albumins↗

PubMed2000

Plasma levels of A- and B-type natriuretic peptides in patients with hypertrophic cardiomyopathy or idiopathic dilated cardiomyopathy.

We investigated the relation between left ventricular structure and the secretion patterns of A- and B-type natriuretic peptides (ANP and BNP) by comparing their plasma levels in patients with hypertrophic cardiomyopathy (HC) and patients with idiopathic dilated cardiomyopathy (IDC). The secretion of ANP and BNP was much higher in patients with HC than in those with IDC; this shows that left ventricular cavity size is a key factor that regulates the secretion of ANP and BNP.

Aged↗

PubMed2000

T(-786)--> C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with myocardial infarction, especially without coronary organic stenosis.

Recently, we discovered a T(-786)-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene that is associated with coronary spasm. The precise mechanism(s) of myocardial infarction (MI), especially without coronary organic stenosis, has not been elucidated, but it seems possible that coronary spasm plays a key role in the mechanism. In this study, we examined the frequency with which the T(-786)-->C mutation occurred in 359 patients with MI who were compared with 195 controls. In the MI group, the frequency of C/C, C/T, and T/T genotypes was 1%, 22%, and 77%, respectively. In the control group, the frequency of C/C, C/T, and T/T genotypes were 0%, 8%, and 92%, respectively. The frequency of the C allele was significantly higher in the MI group than in the control group (p < 0.001). In the MI group, 30 of 359 patients (8%) with MI had no stenosed vessels angiographically, 158 (44%) had 1 stenosed vessel, 80 (22%) had 2 stenosed vessels, and 91 (25%) exhibited 3 stenosed vessels. Total and low-density lipoprotein cholesterol levels and the incidence of diabetes mellitus increased as the number of stenosed vessels increased (p < 0.01, respectively). The frequency of the T(-786)-->C mutation was significantly higher in MI patients with no stenosed vessels (50%) than in those with stenosed vessels (p < 0.003). In conclusion, the T(-786)-->C mutation was strongly associated with MI, especially without coronary arterial stenosis, in Japanese patients. The association may be due to the impaired effects of nitric oxide in the cardiovascular system.

Adult↗

PubMed2000

A T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene and coronary arterial vasomotility.

In the endothelium, synthesis of nitric oxide (NO) from the amino acid L-arginine is catalyzed by the endothelial NO synthase (eNOS), and the continuously generated NO serves to maintain basal vascular tone. Recently, we discovered a T-786-->C mutation in the 5'-flanking region of the eNOS gene; this mutation reduced the promoter activity of the eNOS gene and was associated with coronary spasm. We examined the vasomotility of the epicardial coronary artery in subjects with and without T-736-->C mutation. We examined vasomotility in 32 consecutive subjects who were heterozygotes for the T-786-->C mutation and in 68 subjects without the T-786-->C mutation who had equivalent age, sex, and smoking status at the proximal and distal segments of the left descending coronary artery by performing quantitative coronary angiography. In subjects with the mutant allele (-786C allele), basal diameters of proximal and distal segments before intracoronary injection of acetylcholine (ACh) were less than diameters in subjects without the mutant allele (p <0.05), although there was no difference between subjects with and without the mutant allele in the diameters of coronary arteries after isosorbide dinitrate (ISDN) administration. When we compared the changes in diameters, both ACh-induced vasoconstriction and ISDN-induced vasodilatation in subjects with the mutant allele were significantly increased in the proximal (p <0.01, p <0.001, respectively) and distal segments (p <0.03, p <0.01, respectively). Taken together, these findings strongly suggest that the T-786-->C mutation increases the basal tone of the coronary artery, and enhances the response to the constrictor effects of ACh and the dilator effect of ISDN because of reducing the endothelial NO synthesis.

5' Flanking Region↗

PubMed2000

Interaction on metabolic clearance between A-type and B-type natriuretic peptides in patients with heart failure.

A-type and B-type natriuretic peptides (ANP and BNP) are secreted into the systemic circulation via the coronary sinus. Plasma levels of ANP and BNP at the coronary sinus should directly determine the systemic circulating levels. However, the metabolic clearance of these hormones are dependent on similar systems, natriuretic peptide clearance receptor (NPR-C) and neutral endopeptidase 24.11 (NEP), suggesting a possible interaction between ANP and BNP on metabolic clearance. In this study, we examined the interaction on metabolic clearance in patients with heart failure. We obtained blood samples from the coronary sinus and aortic root in 100 patients with heart failure and 28 control subjects. The difference in ANP and BNP levels between the coronary sinus and the aortic root is reflected partly by the metabolic clearance in the pulmonary circulation. In this study, we examined the possible interaction on metabolic clearance between ANP and BNP using a statistical procedure. The ratio of the level of BNP to ANP (BNP/ANP) was significantly higher in the aortic root than in the coronary sinus at any stage of heart failure. We performed multiple regression analysis using ANP and BNP levels at the coronary sinus as independent variables (X1 and X2, respectively) and the ANP level at the aortic root as a dependent variable (Y). The analysis showed that both X1 and X2 were significant variables in the equation. On the other hand, we performed the same analysis using the BNP level at the aortic root as a dependent variable (Y). The analysis showed that only X2 was a significant variable in the equation. This study suggests that (1) the metabolic clearance in the pulmonary circulation is higher for ANP versus BNP and (2) the amount of ANP cleared in the pulmonary circulation depends on the amount of both ANP and BNP secreted from the heart, whereas the amount of BNP cleared in the pulmonary circulation is dependent solely on the amount of BNP secreted from the heart.

Aorta↗

PubMed1999

Effect of interleukin-1 beta on cardiac hypertrophy and production of natriuretic peptides in rat cardiocyte culture.

This study was designed to examine the effects of interleukin-1 beta (IL-1 beta) on myocyte (MC) hypertrophy and the production of A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in rat ventricular cardiocyte culture, and to investigate the role of nonmyocyte (NMC) in this process. We examined the effects of IL-1 beta on the production of ANP and BNP in comparison with the effects of endothelin-1 (ET-1) by using two types of neonatal rat cardiocyte culture; MC-enriched culture and MC-NMC coculture. In the MC-enriched culture, the increase in secretion of ANP and BNP was small in treatment with IL-1 beta (1000 pg/ml), while ET-1 (10 nM) markedly augmented the secretion of ANP and BNP. In the MC-NMC coculture, IL-1 beta and ET-1 each significantly augmented the secretion of ANP and BNP. The degree of the increase of ANP and BNP was equivalent between IL-1 beta and ET-1. As for the morphological changes of MCs, IL-1 beta induced the star-shaped MC hypertrophy characterized by elongation and pointed edges only in the MC-NMC coculture, while ET-1 induced the MC hypertrophy characterized by shapes of squares, triangles or circles in both cultures. This study shows that IL-1 beta induces unique cardiac hypertrophy and the marked secretion of ANP and BNP, and that NMC is indispensable when treated with IL-1 beta.

Animals↗

PubMed1999

Developmental changes in the regional Na+/glucose transporter mRNA along the small intestine of suckling rats.

We investigated the postnatal development of Na(+)-dependent glucose transporter (SGLT1) as a change in the level of the gene expression of the transporter during the suckling period in rats. We measured the changes in the expression of SGLT1 mRNA in various regions of the intestine during the development of rat pups, using a Northern blot analysis. We found a pronounced gradient in SGLT1 mRNA, with a high level in the duodenum declining gradually, but significantly, to a relatively low level near the ileocecal junction. The level of SGLT1 mRNA in the jejunum region increased in proportion to postnatal age. SGLT1 mRNA was not found at the colon. These data indicate that the glucose uptake in the small intestine of rat pups changes according to regional and age-related transporter activity.

Actins↗

PubMed1999

Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets.

Lactoferrin is a major iron-binding protein in milk from several species, such as humans, monkeys, mice and sows. Using neonatal and weaner piglets, the characteristic transfer of lactoferrin from intestinal lumen into bile via the circulation was investigated. Bovine lactoferrin (1 or 3 g/kg body weight) was infused into the stomach through a polyethylene tube or into the duodenum through a duodenal catheter over 5 min. Peripheral blood and bile samples were collected after the infusion. Lactoferrin absorbed into plasma and bile were assayed quantitatively by double-antibody enzyme-linked immunosorbent assay, and homogeneity of bovine lactoferrin in plasma and bile was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting methods. Morphological investigation was carried out according to the peroxidase anti-peroxidase method. Following oral administration in neonatal pigs, bovine lactoferrin appeared in the blood circulation and reached a peak level after 2 h. It was confirmed immunohistochemically that lactoferrin was transported by endocytosis via the epithelial cells. Lactoferrin absorbed into the blood was also detected in the bile and reached a peak value 12 h after oral administration. Transportation of lactoferrin from the intestinal lumen into the bile via the bloodstream was also observed in weaner piglets. Lactoferrin transported into plasma and bile was confirmed to be the same substance as administrated lactoferrin by electrophoresis and immunoblotting methods. Lactoferrin transported into bile was re-absorbed into the blood in neonatal pigs. These results demonstrate that lactoferrin contained in milk is transported into the circulation from the intestinal lumen and excreted into the bile, suggesting the possibility of entero-hepatic circulation of lactoferrin in neonatal pigs.

Administration, Oral↗