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Bovine ceruloplasmin underwent fragmentation following non-enzymatic glycosylation. Western blot and ELISA analyses indicated that a polyclonal rabbit antiserum to hexitolysine reacted with bovine ceruloplasmin after incubation with 0.1 M glucose. The same fragmentation was seen upon exposure of the protein to a hydrogen peroxide bolus. Both catalase and EDTA blocked peroxide-dependent fragmentation. Incubation with glucose resulted in a time-dependent release of Cu2+. The released Cu2+ appeared to participate in a Fenton-type reaction to produce hydroxyl radicals, which effected the fragmentation. Hydroxyl radical scavengers such as thiourea, mannitol, methionine, and formate inhibited this cleavage. ESR spectral studies also supported participation of hydroxyl radicals. Inhibition by EDTA of the fragmentation induced by an H2O2 bolus also supports a role for copper in a Fenton-type reaction. Taken together these results suggest that reactive oxygen species, such as superoxide anion and H2O2, were formed by the Maillard reaction which led to hydroxyl radicals being produced by a copper-dependent Fenton-type reaction. Both processes are likely to be involved in the fragmentation of ceruloplasmin.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently characterized member of the EGF family of peptide signaling factors that acts as an early response gene to growth stimuli in vascular smooth muscle cells, as well as being a potent mitogen for these cells. As many of these growth stimuli also induce a hypertrophic response in heart muscle, we examined the regulation of HB-EGF mRNA abundance and function in primary cultures of neonatal rat ventricular myocytes and adult rat ventricular myocytes (ARVM). HB-EGF mRNA levels increased 40- and 6-fold in neonatal rat ventricular myocytes and ARVM, respectively, following a 2-4-h exposure to the alpha-adrenergic agonist phenylephrine, a known hypertrophic stimulus for these cells. Phenylephrine had no effect on HB-EGF mRNA stability, and induction of HB-EGF could be blocked completely by actinomycin D. HB-EGF mRNA abundance was also increased 15-fold in ARVM maintained in defined medium that had been induced to contract at 3 Hz by continual uniform electric field stimulation, a mechanical stimulus that we have shown preserves contractile function and induces cell growth in vitro. To determine whether cardiac myocytes would respond to exogenous HB-EGF, quiescent ARVM were exposed to defined medium conditioned by transfected COS MT cells overexpressing HB-EGF. These myocytes exhibited nearly a 2-fold increase in protein content at 24 h compared with unstimulated control ARVM exposed to medium conditioned by COS cells transfected with the plasmid vector alone. Thus, neonatal and adult cardiac muscle cells respond to both neurohumoral and mechanical growth stimuli with a marked increase in HB-EGF mRNA, which may act as an early response gene to facilitate hypertrophic growth in these cells.

Small wireless nodes in sensor networks are increasing being deployed in a wide variety of applications. In this paper, we present ActiveBeans, a generic and secure software platform in a small wireless node employing the Java technologies for data management and sensor/actuator control applications inside a building. Small wireless nodes have been deployed everywhere, and have been used to manage data for applications, perform operations using the data or control sensor/actuator attached to the node. To deal with management data items and controlled devices in a node in a uniform manner, we introduce a node description in the XML format. ActiveBeans provides a Java software platform to access the node description securely. ActiveBeans has the following features: a small Java virtual machine with small footprint management for various input and output device using the generic framework, the data management, and the fine-grained access control. To decrease the Java program size, a generator customizes and optimizes the access control module in a host machine, which is downloaded into the node with a lightweight wireless management protocol.

By immobilizing fructose-modified dendrimers on a polystyrene culture plate, the number of initially adhered hepatocytes on it was increased. Moreover, increasing the number of generations of fructose-modified dendrimer (fructose-dendrimer) increased the number. Urea synthesis per unit area also was increased, corresponding to the increase in the number of initially adhered hepatocytes. This result suggests that the fructose-dendrimers do not cause a decline in cell function. On the other hand, apoptosis of hepatocytes occurs during cultivation, and results in a decrease in the number of adhered cells and a decline in cell function. Fructose-dendrimers were found to suppress apoptosis of hepatocytes. This characteristic is considered to be responsible for the increase in the number of initially adhered hepatocytes without a decline in cell function. Fructose-dendrimers are shown to be very suitable scaffolds for use in a high-performance bioartificial liver support system.

Recent studies have demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in carcinogenesis and carcinoma progression. In this study we investigated the expression of HB-EGF in human colonic non-neoplastic and neoplastic tissues.We performed immunohistochemistry using a polyclonal antibody against HB-EGF for normal colon hyperplastic polyps, adenomas and adenocarcinomas.Normal human colon and hyperplastic polyps did not express HB-EGF. In adenomas with moderate or severe dysplasia, HB-EGF was positive in 92% of the cases, whereas only 14.6% of those with mild dysplasia, expressed HB-EGF (p0.0001). HB-EGF expression was observed in 75% of carcinoma-in-adenoma cases. In adenocarcinomas, the incidence of HB-EGF expression significantly decreased as compared to adenomas with moderate or severe dysplasia (p0.0001) and CIA (p = 0.0005), with only 27.5% of the cases being classified as positive. In adenocarcinomas, HB-EGF expression was inversely linked to carcinoma differentiation (p = 0.0003) and lymph node metastasis (p = 0.0358).Our results demonstrated the bimodal expression of HB-EGF in colonic neoplasms and suggested that HB-EGF may play a role in colonic carcinogenesis and at an early phase of the progression of colonic adenocarcinoma.

Angiotensin II (Ang II)-mediated signals are transmitted via heparin binding epidermal growth factor (EGF)-like growth factor (HB-EGF) release followed by transactivation of EGF receptor (EGFR). Although Ang II and HB-EGF induce angiogenesis, their link to the angiopoietin (Ang)-Tie2 system remains undefined. We tested the effects of Ang II on Ang1, Ang2, or Tie2 expression in cardiac microvascular endothelial cells expressing the Ang II receptors AT(1) and AT(2). Ang II significantly induced Ang2 mRNA accumulations without affecting Ang1 or Tie2 expression, which was inhibited by protein kinase C inhibitors and by intracellular Ca(2+) chelating agents. Ang II transactivated EGFR via AT(1), and inhibition of EGFR abolished the induction of Ang2. Ang II caused processing of pro-HB-EGF in a metalloproteinase-dependent manner to stimulate maturation and release of HB-EGF. Neutralizing anti-HB-EGF antibody blocked EGFR phosphorylation by Ang II. Ang II also upregulated vascular endothelial growth factor (VEGF) expression in an HB-EGF/EGFR-dependent manner. AT(2) inhibited AT(1)-mediated Ang2 expression and phosphorylation of EGFR. In an in vivo corneal assay, AT(1) induced angiogenesis in an HB-EGF-dependent manner and enhanced the angiogenic activity of VEGF. Although neither Ang2 nor Ang1 alone induced angiogenesis, soluble Tie2-Fc that binds to angiopoietins attenuated AT(1)-mediated angiogenesis. These findings suggested that (1) Ang II induces Ang2 and VEGF expression without affecting Ang1 or Tie2 and (2) AT(1) stimulates processing of pro-HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of Ang2 and VEGF, whereas AT(2) attenuates them by blocking EGFR phosphorylation. Thus, Ang II is involved in the VEGF-Ang-Tie2 system via HB-EGF-mediated EGFR transactivation, and this link should be considerable in pathological conditions in which collateral blood flow is required.

A human histiocytic lymphoma cell line, U-937 cells, secretes several vascular endothelial cell growth inhibitors including leukemia inhibitory factor, oncostatin M, tumor necrosis factor-alpha, and transforming growth factor-beta1. Characterization of partially purified fractions from the conditioned media of phorbol ester-treated U-937 cells suggested the existence of unknown endothelial growth inhibitors. Using a combination of copper affinity, heparin affinity, cation exchange, and reversed phase liquid chromatographies, a growth inhibitor for endothelial cells was purified to homogeneity from conditioned media. The purified growth inhibitor migrated as a 65 kDa band on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Microsequencing analyses of the tryptic fragments of the growth inhibitor and a BLAST search analysis revealed a unique sequence showing no homology to known proteins. The purified protein inhibited endothelial cell growth in a dose-dependent manner, but had no effect on smooth muscle cell growth. The factor also blocked endothelial cell growth induced by both fibroblast growth factor-2 and vascular endothelial growth factor, and was additively effective in inhibiting the growth of endothelial cells by U-937 cell-derived endothelial cell growth inhibitors. Thus, this factor appears to be a novel inhibitor with specificity for vascular endothelial cells, and is tentatively called endothelial cell inhibitor (ECI) in this study.

A novel member of the epidermal growth factor (EGF) family, the neural and thymus-derived activator for ErbB kinase (NTAK) has been cloned from the cDNA library of a rat pheochromocytoma cell line, PC12 cells and human neuroblastoma cell line, SK-N-SH cells. Four alternative spliced isoforms from rat cDNA have been detected by the methods of RT-PCR. The rat NTAK alpha 2a isoform exhibits 94% identity in its sequence with the human NTAK alpha isoform. Three characteristic Ig-like, EGF-like and hydrophobic domains have been identified in rat and human NTAK molecules. Recombinant NTAK, the soluble 46 kDa form, binds directly to ErbB3 and ErbB4, but not ErbB1 and B2. NTAK, however, transactivates with heterodimer such as ErbB1/B3, B1/B4, B2/B3, B2/B4, and B3/B4. NTAK stimulates the differentiation of MDA-MB-453 cells, derived from blast carcinoma. NTAK competitively inhibits the binding of [125I] NRG-1 to these cells. Thus, NTAK is a new member of the EGF family displaying NRG-1 properties.

The development of the pancreas appears to be regulated by various growth factors. We report here the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in the developing pancreas. Immunostaining of fetal and neonatal rat pancreata, in which endocrine cells are visible as cell clusters often associated with primitive ducts or ductular cells, revealed that most of the cluster-forming cells and primitive ducts or ductular cells express HB-EGF protein. In contrast, the exocrine pancreas lacked HB-EGF expression. Based on findings that the expression pattern was similar to that of the homeodomain-containing transcription factor PDX-1 (IDX-1/STF-1/IPF1) and that the regulatory region of the HB-EGF gene contained sequences similar to the PDX-1-binding A element, we examined whether PDX-1 could be a potential activator of HB-EGF gene expression. The results of reporter gene analyses suggested that the HB-EGF gene promoter is PDX-1-responsive and that the activity of the promoter in pancreatic beta cell-derived betaTC1 cells depends on the PDX-1 binding site-like sequences. Gel-mobility shift analyses using an anti-PDX-1 antibody indicated that PDX-1 is a specific and dominant binding factor for an A element-like sequence in the HB-EGF gene. These observations suggest the possible involvement of HB-EGF in pancreas development. While PDX-1 is essential for pancreas development, HB-EGF may function as a mediator of PDX-1 and thus be involved in the development of the endocrine pancreas.

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2',7'-dichlorofluorescin diacetate. N-Acetyl-L-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC. DL-Buthionine-(S, R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

Heparin-binding EGF-like growth factor (HB-EGF) is a potent chemoattractant and mitogen for smooth muscle cells (SMC) in culture. To elucidate whether HB-EGF is implicated in the pathogenesis of human atherosclerosis, we examined immunohistochemical localization of HB-EGF in human aortic walls and atherosclerotic plaques. The medial SMC of the aorta in babies and children synthesized HB-EGF protein, while the number of SMC producing HB-EGF was dramatically decreased in young and middle-aged adults. In atherosclerotic plaques, however, marked production of HB-EGF protein was detected in SMC and macrophages of the plaques. Furthermore, EGF receptors, to which HB-EGF is known to bind, were detected in plaque SMC. These data suggest that HB-EGF may be implicated in the migration and proliferation of SMC that occurs in the normal development of arterial walls, and in the formation of atherosclerotic plaques.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently characterized member of the EGF family of peptide signaling factors that acts as an early response gene to growth stimuli in vascular smooth muscle cells, as well as being a potent mitogen for these cells. As many of these growth stimuli also induce a hypertrophic response in heart muscle, we examined the regulation of HB-EGF mRNA abundance and function in primary cultures of neonatal rat ventricular myocytes and adult rat ventricular myocytes (ARVM). HB-EGF mRNA levels increased 40- and 6-fold in neonatal rat ventricular myocytes and ARVM, respectively, following a 2-4-h exposure to the alpha-adrenergic agonist phenylephrine, a known hypertrophic stimulus for these cells. Phenylephrine had no effect on HB-EGF mRNA stability, and induction of HB-EGF could be blocked completely by actinomycin D. HB-EGF mRNA abundance was also increased 15-fold in ARVM maintained in defined medium that had been induced to contract at 3 Hz by continual uniform electric field stimulation, a mechanical stimulus that we have shown preserves contractile function and induces cell growth in vitro. To determine whether cardiac myocytes would respond to exogenous HB-EGF, quiescent ARVM were exposed to defined medium conditioned by transfected COS MT cells overexpressing HB-EGF. These myocytes exhibited nearly a 2-fold increase in protein content at 24 h compared with unstimulated control ARVM exposed to medium conditioned by COS cells transfected with the plasmid vector alone. Thus, neonatal and adult cardiac muscle cells respond to both neurohumoral and mechanical growth stimuli with a marked increase in HB-EGF mRNA, which may act as an early response gene to facilitate hypertrophic growth in these cells.

Heparin-binding (HB) epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF protein family, is a potent mitogen for fibroblasts, smooth muscle cells, and keratinocytes that was initially identified as a secreted product of macrophage-like cells. HB-EGF and EGF appear to act on target cells utilizing the same receptor, but HB-EGF is distinguishable from EGF by its strong affinity for heparin. To facilitate studies of structure-function relationships in HB-EGF, a bacterial recombinant expression system was established that produced biologically active HB-EGF with the expected disulfide bonding pattern. Mutagenesis and protease digestion studies of the recombinant HB-EGF, coupled with heparin-binding analyses of synthetic peptides, indicated that the sequences within HB-EGF mediating its interaction with heparin are located primarily in a stretch of 21 amino acids characterized by a high content of lysine and arginine residues. Most of this heparin-binding domain lies in an amino-terminal region of HB-EGF that has no counterpart in EGF, but a portion of the 21-residue sequence extends into the EGF-like region of HB-EGF. In addition, the mutagenesis and synthetic peptide studies indicated that sequences in HB-EGF lying outside of the 21-residue stretch can also influence the interaction with heparin. Finally, a synthetic peptide derived from the 21-residue stretch was found to compete with HB-EGF for binding to Chinese hamster ovary cells, suggesting that the heparin-binding sequences in HB-EGF may also mediate the interaction of this factor with cell surface heparan sulfate proteoglycan.

Proliferation of smooth muscle cells (SMCs) in atherosclerosis may be modulated by several growth regulatory molecules. At least two mitogens for SMCs, platelet-derived growth factor (PDGF) A-chain and heparin-binding epidermal growth factor-like growth factor (HB-EGF), can be produced by SMCs themselves and may stimulate smooth muscle proliferation in an autocrine or paracrine fashion. We examined the effects of thrombin, which may be generated at the site of vascular injury during atherogenesis, and the potent anti-inflammatory glucocorticoid, dexamethasone (DEX), on the expression of the genes encoding these two growth factors. Since both PDGF A-chain and HB-EGF have affinity for heparin, we also examined the effect of thrombin and DEX on the release of heparin binding mitogenic activity from SMCs. Treatment of SMCs with thrombin resulted in increases both in the level of the PDGF-A and HB-EGF transcripts in the cells, as well as in released heparin-binding growth factor activity. DEX inhibits the thrombin-stimulated release of mitogenic activity in a dose-dependent manner. An enzyme-linked immunoadsorbent assay showed that DEX inhibits both constitutive and thrombin-stimulated release of PDGF-AA. DEX also decreases both constitutive and thrombin-stimulated mRNA levels for PDGF A-chain and HB-EGF and destabilizes the transcripts for both growth factors. A nuclear run-on assay revealed that DEX acts, in addition, to inhibit constitutive and thrombin-stimulated transcription of the PDGF A-chain and HB-EGF genes. Thus, these findings indicate that expression of PDGF A-chain and HB-EGF may be regulated by thrombin and glucocorticoid at the transcription level. Our results are consistent with the involvement of thrombin-induced growth factor expression in neointimal SMC proliferation and suggest the possibility that intimal proliferation may be attenuated by glucocorticoids.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a newly described member of the epidermal growth factor (EGF) family that is mitogenic for BALB/c 3T3 cells, inhibits the binding of 125I-EGF to its receptor, and triggers autophosphorylation of the EGF receptor. HB-EGF was purified from the conditioned medium of U-937 cells using cation exchange, copper affinity, heparin affinity, and two rounds of C4 reversed phase liquid chromatography. The elution profile of the first round of C4 column chromatography contained four growth factor activity peaks with similar specific biological activities. N-terminal and tryptic fragment microsequencing demonstrated that these peaks contained different structural forms of the HB-EGF protein. Some of the differences in the various forms of HB-EGF were found to be due to N-terminal heterogeneity. Microsequencing of tryptic fragments indicated that the mature HB-EGF polypeptide can contain at least 86 of the 208 amino acids predicted by nucleotide sequence to be the HB-EGF precursor molecule. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the various forms of HB-EGF have apparent molecular masses of 19-23 kDa. Further analysis of the most predominant form of HB-EGF found in U-937 cell conditioned medium indicated that it has a pI of 7.2-7.8 and is O-glycosylated.

A combined prednisolone-clomiphene regimen is effective for patients with clomiphene failure1–3. In this study, the effect of prednisolone on the positive feedback function was investigated.

To investigate the actions of nitroglycerin on left ventricular (LV) systolic and diastolic functions in coronary artery disease, high-fidelity LV pressures and LV biplane cineventriculograms were simultaneously made for 16 patients with old myocardial infarction before and 5-10 minutes after the sublingual administration of 0.6 mg of nitroglycerin. Before and after the nitroglycerin administration, heart rates were maintained constant at an average of 73 beats/min by right atrial pacing. LV volume and segmental wall shortening were derived from frame by frame (20 msec) analysis of LV biplane angiograms. The results were as follows: There were significant decreases in LV end-diastolic pressures (-44%) and volumes (-10%), mean arterial pressures (-12%) and LV systolic pressures (-16%), suggesting preload and afterload reductions. Global ejection fraction was significantly increased from 0.41 to 0.45, while cardiac output decreased significantly (-8%) because of a decrease in preload. Augmented ejection fraction was associated with increased shortening of the normal and hypokinetic segments. The dyskinetic segment remained unchanged. Thus, the degree of nitroglycerin-induced potentiation of LV regional contraction seemed related to the degree of regional myocardial ischemia and fibrosis. The time constant (T) of the LV pressure decay was slightly but significantly decreased, which may imply the possibility of improving LV relaxation using nitroglycerin. LV early filling significantly decreased as evidence by a fall in the peak filling rate (PFR) and the normalized PFR. The decrease of the PFR, however, may be related mainly to preload reduction by nitroglycerin. In 11 of 16 patients, there was a displacement of LV diastolic pressure-volume curves downward and to the left. The shift of the curves may be caused by the effects of ventricular interaction and/or the pericardium.

To assess left ventricular (LV) diastolic function in hypertrophic non-obstructive cardiomyopathy (HCM), biplane angiograms and pressures (catheter-tip manometer) were analyzed in 23 patients with HCM and 10 normal subjects (N). The effects of calcium antagonist on LV diastolic function was also evaluated by sublingual administration of 20 mg of nifedipine in 17 patients with HCM. Frame-by-frame (20 msec) analysis of angiograms was performed. LV relaxation was assessed by the time constant (T) of isovolumic LV pressure decline. 1. As compared with N, HCM showed greater increase in LV end-diastolic pressure and time constant (T). There was a significant prolongation of time from end-systole to the peak filling rate (PFR) during rapid filling period in HCM, while PFR did not differ between N and HCM. Left and upward shifts of LV diastolic pressure-volume relations were noted in HCM. Thus, abnormal LV diastolic function in HCM was characterized by impaired relaxation, delayed early diastolic filling and decreased LV compliance. 2. Clinical symptom of chest pain in HCM was associated with a decreased normalized PFR (PFR/SV and PFR/EDV). This finding suggested that an abnormal LV diastolic filling may be one of the contributing factors of chest pain in HCM. 3. Sublingual nifedipine (20 mg) did not always improve impaired relaxation and diminished LV compliance in HCM. Further study will be needed in the treatment of abnormal LV diastolic function in HCM with calcium blocking agents.

Effects of short and long term administrations of an oral contraceptive preparation on the pituitary responses to the synthetic LH-RH were compared in the present experiment. Five normal cycling women were used as controls in the late proliferative phase. Five healty volunteers taking an oral contraceptive agent (1.0 mg norethindrone with 0.05 mg mestranol) for less than 10 cycles were served as short term group and other 4 women taking the same preparation for more than 40 cycles were studied as long term group. Volunteers in each group were kept fasting overnight and 5 ml of blood was drawn at 9 to 10 am on the day of experiment. Ten minutes after drawing blood, 200 mug synthetic LH-RH was injected subcutaneously in the late proliferative phase in the normal cycling women. According to the same procedure, women taking pills were administered LH-RH on the 7th to 12th day after the beginning of taking the first tablet in each cyle. Three ml of blood was taken at 15, 30, 60, 120 minutes and 24 hours, and serum LH and Fsh were determined by the double antibody radioimmunoassay. The 2nd IRP-HMG was used as the standard materials and expressed as mIU/ml of serum. Mean baseline serum LH and FSH concentrations were not suppressed in a short term administration of norethindrone-mestranol combination. Fifteen minutes after the subcutaneous injection of LH-RH, mean LH level was significantly elevated, and thereafter the level was not significantly changed as compared with that seen in the late proliferative phase of the cycle. Concerning the FSH response to LH-RH, a short term administration did not induce a significant rise. ...