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Biomedical subjects

T Ueno

Publications and source records attributed to T Ueno.

At least 19 recordsLinked to original sources
PubMed2001

Intracoronary beta-radiation exacerbates long-term neointima formation in balloon-injured pig coronary arteries.

BACKGROUND: Long-term biological effects of ionizing radiation on coronary arteries remain poorly defined. We examined late arterial responses 6 months after balloon angioplasty and beta-radiation in normal pig coronary arteries. METHODS AND RESULTS: Coronary arteries of 25 adult pigs were randomized to receive 20 Gy (n=8) or 30 Gy (n=9) of (186)Re beta-radiation or sham radiation (n=8) immediately after balloon angioplasty. Aspirin was given daily during follow-up. The study vessels were analyzed histopathologically at 6 months. beta-Radiation decreased lumen area (20 Gy, 1.55+/-0.99 mm(2); 30 Gy, 1.03+/-0.82 mm(2); and 0 Gy, 2.05+/-0.80 mm(2); P<0.05) but not overall vessel area. The neointimal area was significantly larger within the injured segment with beta-radiation (20 Gy, 1.92+/-1.23 mm(2); 30 Gy, 1.51+/-0.97 mm(2); and 0 Gy, 0.89+/-0.31 mm(2); 0 Gy versus 20 Gy, P<0.05), and a significant increase of edge stenosis was observed with beta-radiation. Irradiated vessels also had larger thrombus areas within the neointima (30 Gy, 0.24+/-0.61 mm(2); 20 Gy, 0.98+/-1.57 mm2; and 0 Gy, 0.00+/-0.01 mm(2); P<0.05) and larger adventitial areas (20 Gy, 2.25+/-0.75 mm(2); 30 Gy, 2.38+/-0.98 mm(2); and 0 Gy, 1.23+/-0.29 mm(2); 0 Gy versus 20 or 30 Gy, P<0.05) that showed substantial collagen accumulation. CONCLUSIONS: Intracoronary beta-radiation did not inhibit neointima formation in balloon-injured normal pig coronary arteries 6 months after the interventional procedure. Unresorbed thrombus contributed to, but was not the sole component of, augmented neointima formation. Irradiated vessels demonstrated more adventitial thickening and fibrosis. These observations may have relevance for long-term clinical outcomes after intracoronary beta-radiation.

Angioplasty, Balloon, Coronary↗
PubMed2001

Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

Animals↗
PubMed2001

Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant.

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.

Catalysis↗
PubMed2001

Analysis of dominant-negative effects of mutant Env proteins of human immunodeficiency virus type 1.

The Env protein of human immunodeficiency virus type 1 is assembled into a stable trimer, and oligomerization is required for maintenance of viral infectivity. This property of Env suggests that Env mutants may have a dominant-negative effect on virus infectivity. To investigate this possibility, we established a packaging cell line in which both wild-type and mutant Env proteins could be expressed simultaneously in a single cell. We analyzed the effects of two types of Env mutants: cytoplasmic tail-truncated TM mutants and a mutant defective in gp120/gp41 cleavage. The cytoplasmic tail-truncated proteins were found to be incorporated into virions by forming an oligomer with wild-type TM, but could not inhibit the wild-type function. In contrast, phenotypic mixing of cleavage-defective Env with the wild-type protein caused dramatic inhibition of infectivity, indicating that this mutant has a strong dominant-negative phenotype.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis.

Akt is a common mediator of cell survival in a variety of circumstances. Although some candidate Akt targets have been described, the function of Akt is not fully understood, particularly because of the cell type- and context-dependent apoptosis regulation. In this study, we demonstrate that one of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77, a transcription factor implicated in T-cell receptor-mediated apoptosis. It has been suggested that Akt phosphorylates Nur77 directly, but whether Akt suppresses biological functions of Nur77 remains unknown. We found that Akt inhibited the DNA binding activity of Nur77 and stimulated its association with 14-3-3 in a phosphorylation site-dependent manner. Moreover, we found that expression of Akt suppressed Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas. The inhibition of Nur77 by Akt suggests a mechanism that explains how T-cell receptor activation can promote survival in some instances even when Nur77 is induced. Collectively, these results may suggest that Akt is a negative regulator of Nur77 in T-cell apoptosis.

14-3-3 Proteins↗
PubMed2001

Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.

BACKGROUND: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood (PB) and contribute to neovascularization. However, little is known regarding whether EPCs and their putative precursor, CD34-positive mononuclear cells (MNC(CD34+)), are mobilized into PB in acute ischemic events in humans. METHODS AND RESULTS: Flow cytometry revealed that circulating MNC(CD34+) counts significantly increased in patients with acute myocardial infarction (n=16), peaking on day 7 after onset, whereas they were unchanged in control subjects (n=8) who had no evidence of cardiac ischemia. During culture, PB-MNCs formed multiple cell clusters, and EPC-like attaching cells with endothelial cell lineage markers (CD31, vascular endothelial cadherin, and kinase insert domain receptor) sprouted from clusters. In patients with acute myocardial infarction, more cell clusters and EPCs developed from cultured PB-MNCs obtained on day 7 than those on day 1. Plasma levels of vascular endothelial growth factor significantly increased, peaking on day 7, and they positively correlated with circulating MNC(CD34+) counts (r=0.35, P=0.01). CONCLUSIONS: This is the first clinical demonstration showing that lineage-committed EPCs and MNC(CD34+), their putative precursors, are mobilized during an acute ischemic event in humans.

Aged↗
PubMed2001

Inhibition of the hepatitis C virus NS3 protease activity by Fv fragment of antibody 8D4.

An antibody variable domain fragment (Fv) is a candidate for a specific inhibitor of the hepatitis C virus (HCV) NS3 protease. Here we report the functional characterization of the Fv of antibody 8D4, which is specific for the active site of the HCV NS3 protease domain. The variable fragments of 8D4 in the forms of Fv and scFv (VH-(G(4)S)(3)-VL) were expressed as insoluble fractions in the periplasm of Escherichia coli, and were subsequently solubilized, purified under denaturing conditions, and refolded. The Fv had an inhibition profile almost identical to that of the parent IgG, with an IC(50) of 71.3 nM, whereas the scFv had a greatly decreased affinity to NS3 and was the same as the isolated VH fragment. To date, this is the first report of an antibody Fv fragment specific for the HCV NS3 protease domain, aimed at designing potent protease inhibitors and antiviral drugs.

Amino Acid Sequence↗
PubMed2001

Augmentation of postnatal neovascularization with autologous bone marrow transplantation.

BACKGROUND: Endothelial progenitor cells (EPCs) have been identified in adult human peripheral blood. Because circulating EPCs should originate from bone marrow (BM), we examined whether BM mononuclear cells (BM-MNCs) can give rise to functional EPCs and whether transplantation of autologous BM-MNCs might augment angiogenesis and collateral vessel formation in a rabbit model of hindlimb ischemia. METHODS AND RESULTS: Rabbit BM-MNCs were isolated by centrifugation through a Histopaque density gradient and cultured on fibronectin. EPCs developed from BM-MNCs in vitro, as assessed by acetylated LDL incorporation, nitric oxide (NO) release, and expression of von Willebrand factor and lectin binding. Unilateral hindlimb ischemia was surgically induced in rabbits (n=8), and fluorescence-labeled autologous BM-MNCs were transplanted into the ischemic tissues. Two weeks after transplantation, fluorescence microscopy revealed that transplanted cells were incorporated into the capillary network among preserved skeletal myocytes. In contrast, transplanted autologous BM-fibroblasts did not participate in EC capillary network formation (n=5). Then, in an additional 27 rabbits, saline (control; n=8), autologous BM-MNCs (n=13; 6.9+/-2.2x10(6) cells/animal), or BM-fibroblasts (n=6; 6.5+/-1.5x10(6) cells/animal) were injected into the ischemic tissues at postoperative day 7. Four weeks after transplantation, the BM-MNC-transplanted group had more angiographically detectable collateral vessels (angiographic score: 1.5+/-0.34 versus 0.94+/-0.26 and 1.1+/-0.14; P:<0.05), a higher capillary density (23+/-5.8 versus 10+/-1.9 and 11+/-0.8 per field; P:<0.001), and a greater laser Doppler blood perfusion index (505+/-155 versus 361+/-35 and 358+/-22 U; P:<0.05) than the control and BM-fibroblast-transplanted groups. CONCLUSIONS: Direct local transplantation of autologous BM-MNCs seems to be a useful strategy for therapeutic neovascularization in ischemic tissues in adults, consistent with "therapeutic vasculogenesis."

Angiography↗
PubMed2001

The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation.

Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.

Amino Acid Sequence↗
PubMed2001

A recurrent large Alu-mediated deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene associated with Lesch-Nyhan syndrome.

We identified the identical large genomic deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene in two Japanese patients with Lesch-Nyhan (LN) syndrome. This deletion spanned from an Alu sequence in the promoter region to another Alu-sequence in intron 1, a length of 2,969 base pairs including exon 1. In order to ask whether this deletion was a recurrent mutation, we developed a simple alternative method to determine the separate origin of the HPRT1 mutation of the patients as assessed with an apparent mtDNA polymorphism. Considering that an LN syndrome-causing mutation is not transmitted from patient to offspring as LN syndrome is a fatal disease in childhood and that mtDNA is maternally inherited, HPRT1 mutations and mtDNA would be co-transmitted from carrier mother to offspring since both appeared in females. Two bases were different in the hypervariable region I of the mtDNA between the two patients, indicating the separate origin of their mtDNA over at least several thousand years as calculated based on the molecular evolution rate in this region. We thus conclude that the identical deletion found in HPRT1 of the two patients was derived from recurrent events of genomic recombination. Given that the same Alu-mediated deletion of HPRT1 has not been reported among somatic mutations at the same locus, this region of the HPRT1 gene flanked by Alu-sequences is likely a mutational hot spot in the germline but not in somatic cells. In addition, we also report novel LN-syndrome-conferring mutations in intron 6 (IVS6+1G --> C) and intron 8 (IVS7-9T --> G) that resulted in exclusions of exon 6 and exon 8, respectively.

Alternative Splicing↗
PubMed2001

Modulation of H reflex of pretibial and soleus muscles during mastication in humans.

A previous study in our laboratory demonstrated that the soleus H reflex was facilitated during mastication in humans. In the present study, we investigated whether there was any modulation of the magnitude of the pretibial H reflex during mastication in five healthy adult volunteers. The pretibial H reflex was significantly facilitated during mastication, and there was no significant difference in the facilitation between jaw-closing and jaw-opening phases; that is, the gain of the H reflex was modulated tonically but not in a phase-dependent manner during mastication. Furthermore, in the same subjects, we confirmed that the soleus H reflex was facilitated during mastication. Based on our findings, we conclude that the H reflexes in both the pretibial and soleus muscles undergo a nonreciprocal facilitation during mastication. It is suggested that mastication contributes to stabilization of postural stance in humans.

Adult↗
PubMed2001

Insecticidal and binding activities of N3-substituted imidacloprid derivatives against the housefly Musca domestica and the alpha-bungarotoxin binding sites of nicotinic acetylcholine receptors.

N3-substituted imidacloprid congeners containing C1-C6 alkyl groups or various analogous groups, and their corresponding nitromethylene analogues, were used in this study. Their insecticidal activity against the housefly, Musca domestica, and their binding activity toward the nicotinic acetylcholine receptor were determined. The insecticidal test was conducted using the synergists piperonyl butoxide and propargyl propyl phenylphosphonate. The binding assay was performed with housefly head membrane preparations using radio-labelled alpha-bungarotoxin. Both insecticidal and binding activities were drastically lowered by the introduction of alkyl/allyl groups at the imidazolidine NH sites of both nitroimino and nitromethylene compounds. The binding activity of N3-substituted nitromethylene analogues was much higher than that of the corresponding nitroimino analogues. However, the insecticidal activity of both series of compounds with a given substituent was nearly identical. The insecticidal activity correlated positively with the binding activity after taking into account the structural difference of the nitroimino and nitromethylene moieties and a structural feature of the N3-substituents.

Animals↗
PubMed2001

Laparascope-assisted versus conventional restorative proctocolectomy with rectal mucosectomy.

To assess the advantages of a laparoscope-assisted proctocolectomy with ileal J-pouch anal anastomosis compared with conventional procedures, we retrospectively analyzed the results of the two procedures as follows: Eleven patients including five patients with familial adenomatous polyposis (FAP) and six with ulcerative colitis (UC) underwent a laparoscope-assisted proctocolectomy and hand-sewn ileal J-pouch anal anastomosis at our department from June 1997 to November 1999. This laparoscope-assisted colectomy (LAC) group was then compared with a group of 13 patients who had undergone conventional ileal pouch anal anastomosis using a standard laparotomy from 1986 to 1997. The median operative time of the LAC group was 8h 23min, which was 81 min longer than that of the standard colectomy (SC) group. The number of days during which eating was prohibited were similar in the two groups but the median postoperative hospital stay was significantly shorter in the LAC group (24.1 days). In the LAC group, the small incisions showed better cosmetic results and there was also a remarkable reduction in the degree of postoperative pain. In conclusion, a laparoscope-assisted proctocolectomy with ileal J-pouch anal anastomosis can be employed widely in patients with FAP and also in selected patients with UC.

Adenomatous Polyposis Coli↗
PubMed2001

Penetrating atherosclerotic ulcer.

The pathogenesis of penetrating atherosclerotic ulcer (PAU) in aortic disease remains controversial. Between January 1995 and April 1999, five patients underwent treatment for a PAU in our hospital. All were men, ranging in age from 46 to 74 years, with a mean age of 66.2 years. The PAU was located on the thoracic descending aorta in three patients and on the abdominal aorta in two. Preoperative diagnosis was established by contrast-enhanced computed tomographic scan and aortogram. Surgery was performed in four patients, as graft replacement in three, and patch plasty in one. The remaining patient is being carefully observed on antihypertensive therapy. No connective tissue disorder, trauma, dissection, or infection was seen in any of the patients who underwent surgery, all of whom had uneventful postoperative courses with no perioperative complications or enlargement of the aorta. We conclude that surgical treatment should be performed for patients with PAU to prevent an aortic catastrophe caused by rapid development of the lesion. This report might provide further evidence of the importance of PAU.

Aged↗
PubMed2001

Modification of virus infectivity by cytoplasmic tail of HIV-1 TM protein.

Envelope glycoprotein incorporation is an essential process in formation of infectious particles of human immunodeficiency virus. Accumulated data have indicated that the cytoplasmic tail of Env gp41 is required for efficient incorporation. By analyzing mutant viruses with truncated cytoplasmic tails, we found that the domain was required in a cell-type-dependent manner for maintaining virus infectivity. Although the viruses with truncated cytoplasmic tails produced from HeLa, A3.01 and SupT1 cells showed a greatly reduced infectivity, those from SW480 and MT-4 cells retained a significant infectivity. To clarify the different effect of the cytoplasmic tail mutation on virus infectivity, we performed biochemical studies on the virions produced from HeLa and SW480 cells. Although the truncation of cytoplasmic tail appeared to reduce the Env incorporation in both cell lines, it caused a significant incorporation of Env precursor with HeLa cells. The results suggested that the cytoplasmic tail regulated selective incorporation of processed Env into virions in a cell-type-dependent manner.

Cell Line↗
PubMed2001

Laminin deposition to type IV collagen enhances haptotaxis, chemokinesis, and adhesion of hepatoma cells through beta1-integrins.

BACKGROUND/AIMS: In hepatocellular carcinoma, laminin deposition to type IV collagen along the sinusoids is observed with the development of arterial network, coinciding with intrahepatic metastasis. We investigated the influence of laminin deposition to type IV collagen on hepatoma cell adhesion, motility and secretion of matrix metalloproteinases (MMPs), which are indispensable behaviors for tumor metastasis. METHODS: Hepatoma cell lines (KYN-1, -2 and -3) were used. The expression of integrin subunit mRNAs in hepatoma cells was confirmed by RT-PCR. The influence of laminin addition to type IV collagen on the adhesion, chemokinesis, and migration of KYN-1, -2 and -3 was evaluated by the haptotactic migration, phagokinetic track motility, and cell adhesion assays. The effects of integrin subunits on these activities were evaluated using the function-blocking antibodies for integrins. Phosphorylation of MEK1/2 and secretion of MMPs were investigated by Western blotting and gelatin zymography. RESULTS: Integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunit mRNAs were detected. The combination of type IV collagen and laminin enhanced the migration, chemokinesis, and adhesion of hepatoma cells compared to that of type IV collagen when used alone. The enhanced activity was significantly suppressed by function-blocking antibodies for integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunits. Hepatoma cells cultured on the combination of type IV collagen and laminin showed phosphorylation of MEK1/2 and increased secretion of MMPs. CONCLUSIONS: The addition of laminin to type IV collagen enhances hepatoma cell adhesion and motility through beta1-integrins.

Carcinoma, Hepatocellular↗