Biomedical subjects

Hiroko Tada

Publications and source records attributed to Hiroko Tada.

At least 19 recordsLinked to original sources

PubMed2006

Anti-tumor effect in an in vivo model by human-derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties.

It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P < 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

Angiogenesis Inhibitors↗

PubMed2006

Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding.

The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli.

ADAM Proteins↗

PubMed2006

Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells.

Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

Base Sequence↗

PubMed2006

Endogenous erythropoietin system in non-hematopoietic lineage cells plays a protective role in myocardial ischemia/reperfusion.

OBJECTIVE: Recent studies suggested that erythropoietin (Epo) receptors (EpoR) are expressed not only in the hematopoietic lineage cells but also in the heart and that the administration of recombinant human Epo elicits protective effects in myocardial ischemia and reperfusion (I/R). We tested our hypothesis that endogenous Epo signals mediated by EpoR expressed in the non-hematopoietic lineage cells play a protective role against myocardial I/R injury. METHODS: Transgene-rescued EpoR null mutant mice (RES), which express EpoR exclusively in the hematopoietic lineage cells, were subjected to 30 min left coronary artery occlusion followed by reperfusion. RESULTS: Hematocrit, heart rate, blood pressure, heart weight, and echocardiographic parameters were comparable between wild-type mice (WT) and RES under the baseline condition. After 24 h of reperfusion, the infarct size in RES with I/R (RES/MI) was larger than that in WT/MI. Caspase-3 activity and number of TUNEL-positive cardiomyocytes in the ischemic area were increased in RES/MI compared with WT/MI. The extents of p38 and JNK phosphorylations in the ischemic area were significantly increased in WT/MI, but not in RES/MI as compared with corresponding sham-operated mice. Plasma Epo concentration in RES/MI did not differ from that in sham-operated RES, while that in WT/MI was peaked at 24 h post I/R. Additionally, left ventricular (LV) end-diastolic diameter was increased and LV fractional shortening tended to be reduced in the RES/MI compared with WT/MI at 21 days after I/R. CONCLUSIONS: These results suggest that the endogenous Epo-EpoR system in the non-hematopoietic lineage cells plays an important protective role against myocardial I/R injury.

Animals↗

PubMed2006

Reversible restricted diffusion of entire corpus callosum.

MR imaging of a 31-year-old man with bacterial meningo-encephalitis associated with infectious endocarditis demonstrated T2 prolongation and restricted diffusion involving the entire corpus callosum and symmetrically involving the cerebral hemispheric white matter. His neurological signs and symptoms were mild, and recovery was complete within a week. Follow-up MR imaging with a 9-day interval revealed complete resolution of these lesions. The clinical and radiological courses were identical to those previously reported with a reversible lesion isolated to the splenium, or to the splenium and peripheral fronto-parietal white matter. This suggests that such reversible lesions are not necessarily restricted to the splenium, but may involve the entire corpus callosum.

Adult↗

PubMed2006

Important role of endogenous erythropoietin system in recruitment of endothelial progenitor cells in hypoxia-induced pulmonary hypertension in mice.

BACKGROUND: Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow-derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine. METHODS AND RESULTS: A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR(-/-) rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR(-/-) rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR(-/-) rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR(-/-) rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR(-/-) rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR(-/-) rescued mice. CONCLUSIONS: These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.

Animals↗

PubMed2006

Engineered bio-nanocapsules, the selective vector for drug delivery system.

The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

Biomedical Engineering↗

PubMed2005

Identification of cell surface marker candidates on SV-T2 cells using DNA microarray on DLC-coated glass.

We analyzed gene expression profiles of normal mouse fibroblast BALB/c 3T3 cells and its SV40 transformant SV-T2 cells using our originally developed cell surface marker DNA microarray, which is prepared on a diamond-like carbon-coated glass. As a result, CD62L and IL-6 receptor alpha gene expressions were upregulated in SV-T2 and were thought to be candidates for call surface markers of the cells. The result of microarray analysis was validated by real-time quantitative PCR immunohistochemistry and biological assays. These data show that our cell surface marker DNA microarray should be useful in finding the candidates of cell type-specific surface markers.

Animals↗

PubMed2005

Differential regulation of diacylglycerol kinase isozymes in cardiac hypertrophy.

To examine the involvement of diacylglycerol kinase (DGK) and phosphatidic acid phosphatase (PAP) in pressure overloaded cardiac hypertrophy, rats were subjected to either ascending aortic banding for 3, 7, and 28 days or sham operation. In comparison with sham-operated rats, the left ventricular (LV) weight of the aortic-banded rats increased progressively. At 28 days after surgery, the expression of DGKepsilon mRNA but not DGKzeta or PAP2b mRNA in the LV myocardium significantly decreased in the aortic-banded rats compared with the sham-operated rats. DGKzeta protein in the LV myocardium translocated from the particulate to the cytosolic compartment in the aortic-banded rats. Furthermore, the myocardial content of 1,2-diacylglycerol and PKCdelta protein expression in the particulate fraction of the LV myocardium significantly increased in aortic-banded rats compared with sham-operated rats. These results suggest that DGKepsilon and DGKzeta play distinct roles in the development of pressure overloaded cardiac hypertrophy and that the two isozymes are differentially regulated.

Animals↗

PubMed2005

Anti-angiogenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1.

Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.

Angiogenesis Inhibitors↗

PubMed2005

High serum erythropoietin level is associated with smaller infarct size in patients with acute myocardial infarction who undergo successful primary percutaneous coronary intervention.

OBJECTIVES: We investigated whether a higher serum erythropoietin (EPO) level in patients with acute myocardial infarction (MI) subjected to successful primary percutaneous coronary intervention (PCI) can predict a smaller infarct size determined by creatine kinase (CK) release. BACKGROUND: Erythropoietin has been shown to protect cardiomyocytes from ischemia-reperfusion injury in rodents. METHODS: We prospectively studied 101 patients with first MI who received successful primary PCI within 12 h from the onset of MI. Blood samples were collected to examine the serum EPO level after the primary PCI and within 24 h from the onset of MI. RESULTS: The peak CK level and cumulative CK release were significantly lower in the above-median EPO group than in the below-median EPO group. Thrombolysis In Myocardial Infarction (TIMI) grades and collateral grades before PCI, infarct-related coronary arteries, time to the successful reperfusion from the onset of MI, and serum creatinine levels were similar in the two EPO groups. A stepwise multiple regression analysis revealed that the absolute serum EPO level (mU/ml) as well as TIMI grades after PCI and preinfarction angina was an independent predictor for the cumulative CK release. CONCLUSIONS: These data suggest that a high endogenous EPO level can predict a smaller infarct size in patients with acute MI subjected to successful primary PCI. This might be attributed to the potentially protective effect of endogenous EPO against ischemia-reperfusion injury in humans.

Aged↗

PubMed2005

Intracranial dural venous anomalies in familial cervical cystic hygroma.

This report describes a 3-year-old male with a familial cervical cystic hygroma and intracranial dural venous abnormalities consisting of a falcine sinus between the superior sagittal and the straight sinus with a rudimentary posterior part of the superior sagittal sinus; a prominent occipital sinus with narrowed transverse sinuses; and no intraparenchymal vascular abnormality. We hypothesize that a genetic factor that resulted in familial cervical cystic hygromas may also have caused the intracranial dural venous anomalies.

Cerebral Veins↗

PubMed2005

Magnetic resonance imaging confirms periventricular venous infarction in a term-born child with congenital hemiplegia.

Magnetic resonance imaging (MRI) of a 5-day-old female born at term with congenital leftsided hemiplegia revealed T2 shortening and T2* signal dropout lining the lateral wall of a focally enlarged right lateral ventricle, reflecting the deposition of haemosiderin or ferritin derived from periventricular venous infarction in utero. This observation strengthens the hypothesis that congenital hemiplegia in children born at term can result from a clinically silent periventricular venous infarction in utero. The loss of shortening of T2 in the right posterior limb of the internal capsule at birth was followed by prolongation of T2 at 1 year. It is important to evaluate the asymmetry of the posterior limb shown by MRI to predict future hemiplegia and enable early therapy.

Cerebral Infarction↗

PubMed2005

Protein transduction assisted by polyethylenimine-cationized carrier proteins.

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

Animals↗

PubMed2005

The specific delivery of proteins to human liver cells by engineered bio-nanocapsules.

A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

Amino Acid Sequence↗

PubMed2005

Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization.

In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

3T3 Cells↗

PubMed2005

Pituitary cysts in childhood evaluated by MR imaging.

BACKGROUND AND PURPOSE: Pituitary cysts are common findings on pathologic examination and imaging studies. They are generally considered to be rarer in children than in adults; however, no good data exist to substantiate this opinion. We reviewed MR imaging studies to evaluate the frequency and imaging features of pituitary cysts in children. METHODS: We retrospectively reviewed T1-weighted sagittal images in 341 patients <15 years of age to evaluate for pituitary cysts. Paramagnetic contrast was administered in 86 of the 341 patients. Sagittal or coronal fast spin-echo T2-weighted images were performed in 166 patients. For patients having pituitary cysts, pituitary function was examined by assessing blood levels of pituitary hormones. RESULTS: A cystic pituitary lesion was recognized in 4 patients (1.2%) aged 1-4 years. None of the 4 manifested endocrinologic signs or symptoms or were the results of their laboratory studies abnormal. All the lesions were sharply demarcated and situated just posterior to the anterior pituitary lobe. All were iso- or hypointense compared with the pons on T1-weighted images without contrast enhancement, suggesting a Rathke cleft cyst. MR imaging of a patient with probable low-grade gliomas in the left hypothalamic region and optic chiasma showed complete resolution of a pituitary cyst at a 1-year follow-up study. CONCLUSION: The frequency of pituitary cysts on MR imaging in childhood is almost equal to that of Rathke cleft cysts, as assessed in autopsy studies of subjects aged 10 to 29 years. These cysts are common in children and should be considered, in the absence of signs or symptoms of pituitary dysfunction, as incidental findings.

Brain Neoplasms↗

PubMed2004

Insertional-fusion of basic fibroblast growth factor endowed ribonuclease 1 with enhanced cytotoxicity by steric blockade of inhibitor interaction.

Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase.

Animals↗