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Biomedical subjects

H Ueno

Publications and source records attributed to H Ueno.

At least 55 records · Page 3Linked to original sources
PubMed2001

Development of high-performance stent: gelatinous photogel-coated stent that permits drug delivery and gene transfer.

Hydrogel-coated metallic stents may provide supplementary functions such as local drug delivery and gene transfer in addition to mechanical dilation function. To this end, we used a photoreactive material consisting of gelatin macromer (multiple styrene-derivatized gelatin) and carboxylated camphorquinone (photo-initiator). A few minutes of visible light irradiation of a stent after dip-coating of an aqueous solution of the photoreactive material resulted in the formation of a homogeneously crosslinked gelatinous layer on the entire exterior surface of the stent. As the metal stent, gold stents under development were used. Rhodamine-conjugated albumin as a model drug or adenoviral vector expressing bacterial beta-galactosidase (AdLacZ) as a model gene were photo-immobilized in the gelatinous gel layer. In vitro experiments using hybrid tubular tissue, which is a self-shrinkaged, vascular smooth muscle cell-incorporated type-I collagen gel, as a vascular model, showed that the immobilized dye-derivatized albumin was released on and permeated into tissues, as observed by confocal laser microscopy, and that the cells transfected with immobilized AdLacZ produced beta-galactosidase up to almost 3 weeks, as observed by x-gal staining. In preliminary in vivo experiments these drug- or adenovirus-immobilized stents were implanted in rabbit common carotid arteries. Within 3 weeks of implantation, drug permeation and gene expression in the vascular tissues were observed, indicating that the gelatinous photogel effectively serves as a matrix or coating for a bioactive stent,which permits drug release as well as gene transfer. This intraluminal approach has the potential to realize drug and gene therapy in atherosclerotic plaque.

Albumins↗
PubMed2001

Topical formulations and wound healing applications of chitosan.

Chitosan is being used as a wound-healing accelerator in veterinary medicine. To our knowledge, chitosan enhances the functions of inflammatory cells such as polymorphonuclear leukocytes (PMN) (phagocytosis, production of osteopontin and leukotriene B4), macrophages (phagocytosis, production of interleukin (IL)-1, transforming growth factor beta 1 and platelet derived growth factor), and fibroblasts (production of IL-8). As a result, chitosan promotes granulation and organization, therefore chitosan is beneficial for the large open wounds of animals. However, there are some reported complications of chitosan application. Firstly, chitosan causes lethal pneumonia in dogs which are given a high dose of chitosan. In spite of application of chitosan to various species, this finding is observed only in dogs. Secondly, intratumor injection of chitosan on mice bearing tumor increases the rate of metastasis and tumor growth. Therefore, it is important to consider these effects of chitosan, prior to drug delivery.

Administration, Topical↗
PubMed2001

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.

Alanine Racemase↗
PubMed2001

Contribution of enzymic alpha, gamma-elimination reaction in detoxification pathway of selenomethionine in mouse liver.

The objective of this study was to clarify the detoxification pathways of selenomethionine (SeMet) in mouse liver. It has been postulated that SeMet may be metabolized to selenocysteine (SeCyH) via a pathway similar to methionine (Met). CySeH may be decomposed to H(2)Se, which is consequently methylated to CH(3)SeH, (CH(3))(2)Se, and (CH(3))(3)Se(+). In this study, we estimated that the median lethal single oral dose (LD(50)) was 67.0 mg/kg. We also found that (CH(3))(3)Se(+) was quickly produced in mouse liver after single oral administration of SeMet. This result suggested the existence of a quick alpha,gamma-elimination pathway. We measured the amounts of alpha-ketobutyrate, NH(3), and CH(3)SeH produced by enzymic alpha,gamma-elimination reaction of SeMet in the liver of periodate-oxidized adenosine (PAD) or D,L-propargylglycine (PPG)-treated mice in order to verify the existence of alpha,gamma-elimination enzyme. PAD is an inhibitor of S-adenosylhomocysteinase (EC 3.3.1.1), which is necessary for conversion of SeMet to SeCyH. PPG is an effective inhibitor of the pyridoxal 5'-phosphate (PLP)-containing enzyme bacterial L-methionine gamma-lyase (EC 4.4.1.11) contributing to the alpha,gamma-elimination reaction of SeMet and cystathionine gamma-lyase (EC 4.4.1.1) relating to conversion of SeMet to SeCyH. When SeMet was incubated with the S9 fraction from liver of PAD-treated mice, the formation of alpha-ketobutyrate was much the same as that from nontreated mouse liver. However, the amount of alpha-ketobutyrate formed significantly decreased in the reaction of SeMet with S9 fraction from the liver of PPG-treated mice. In an in vivo experiment using mice treated with PAD before a toxic dosage of SeMet, the amount of SeMet in the liver decreased and the amount of acid-volatile Se derived from CH(3)SeH increased gradually. This phenomenon was not observed in the PPG-pretreated group. Furthermore, the protein fraction that had the alpha,gamma-elimination enzyme activity was found in mouse liver cytosol by gel chromatographic technique. The results of this study indicated that SeMet was directly metabolized to CH(3)SeH by an alpha,gamma-elimination enzyme analogous to bacterial L-methionine gamma-lyase, in addition to the generally acceptable pathway via SeCyH.

Ammonia↗
PubMed2001

Requirement of Ras for the activation of mitogen-activated protein kinase by calcium influx, cAMP, and neurotrophin in hippocampal neurons.

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1(m), or dominant negative Ras by means of adenovirus vectors. Gap1(m) expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

Adenoviridae↗
PubMed2001

Prognostic factors in patients with locally advanced pancreatic carcinoma receiving chemoradiotherapy.

BACKGROUND: The combination of radiation therapy and chemotherapy (chemoradiotherapy [CRT]) has been accepted as standard therapy for patients with locally advanced pancreatic carcinoma (PC). This study investigated prognostic factors in patients with locally advanced PC receiving CRT. METHODS: Fifty-five consecutive patients with locally advanced PC, who received concurrent radiotherapy (50.4 grays) and chemotherapy using 5-fluorouracil or cisplatin, were analyzed retrospectively to investigate prognostic factors. RESULTS: Median survival time and overall survival rates at 1 and 2 years were 301 days, 35.1% and 2.4%, respectively. By multivariate analysis using the Cox proportional hazards model, performance status of 0-1 (P < 0.01), absence of regional lymph node swelling (P < 0.01), and serum CA 19-9 level of less than 1000 (P = 0.02) were independent favorable prognostic factors. A prognostic index based on the coefficients of those prognostic factors was used to classify patients into three groups with good, intermediate, and poor prognoses. The median survival times for these three groups were 410, 239, and 143 days, respectively (P < 0.01). CONCLUSIONS: The results may be helpful in predicting life expectancy, determining treatment strategies, and designing future clinical trials.

Adult↗
PubMed2001

Molecular basis for paradoxical carriers of adenosine deaminase (ADA) deficiency that show extremely low levels of ADA activity in peripheral blood cells without immunodeficiency.

Adenosine deaminase (ADA) deficiency causes an autosomal recessive form of severe combined immunodeficiency and also less severe phenotypes, depending to a large degree on genotype. In general, ADA activity in cells of carriers is approximately half-normal. Unexpectedly, healthy first-degree relatives of two unrelated ADA-deficient severe combined immunodeficient patients (mother and brother in family I; mother in family II) had only 1-2% of normal ADA activity in PBMC, lower than has previously been found in PBMC of healthy individuals with so-called "partial ADA deficiency." The level of deoxyadenosine nucleotides in erythrocytes of these paradoxical carriers was slightly elevated, but much lower than levels found in immunodeficient patients with ADA deficiency. ADA activity in EBV-lymphoblastoid cell lines (LCL) and T cell lines established from these carriers was 10-20% of normal. Each of these carriers possessed two mutated ADA alleles. Expression of cloned mutant ADA cDNAs in an ADA-deletion strain of Escherichia coli indicated that the novel mutations G239S and M310T were responsible for the residual ADA activity. ADA activity in EBV-LCL extracts of the paradoxical carriers was much more labile than ADA from normal EBV-LCL. Immunoblotting suggested that this lability was due to denaturation rather than to degradation of the mutant protein. These results further define the threshold level of ADA activity necessary for sustaining immune function.

Adenosine Deaminase↗
PubMed2001

Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect. METHODS AND RESULTS: We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial ss-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3+/-8.7% (mean+/-SD, n=14) in saline-treated arteries and 33.3+/-4.2% in AdLacZ-infected arteries (P:=NS versus saline). However, it was reduced to 25.5+/-8.5% in rTFPI-irrigated arteries (P:<0.01 versus saline) and to 20.7+/-5.3% in AdTFPI-infected arteries (P:<0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6+/-4.7% (P:<0.01 versus rTFPI, P:<0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals. CONCLUSIONS: A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans.

Adenoviridae↗
PubMed2001

Heterogeneity of the signal transduction pathways for VEGF-induced MAPKs activation in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) activates ERK and p38 MAPK in endothelial cells (ECs). The present study was aimed to compare its intracellular signal transduction pathways between three primary cultures of human ECs including human aortic ECs (HAECs), human umbilical vein ECs (HUVECs), and human microvascular ECs (HMVECs). VEGF activated ERK and p38 MAPK in all of three ECs. Isoforms of p38 MAPK that were activated by VEGF in HUVECs were p38-alpha and p38-delta. GF109203X, a specific inhibitor of PKC, markedly inhibited VEGF-induced activation of ERK and p38 MAPK in HAECs and HUVECs, whereas it exhibited little effect in HMVECs. In contrast, dominant negative mutant of Ha-Ras almost completely abrogated VEGF-induced activation of ERK and p38 MAPK in HMVECs. Although dominant negative mutant of Ha-Ras substantially inhibited the basal activities of ERK and p38 MAPK, it exhibited marginal effect on VEGF-induced activation of ERK and p38 MAPK in HUVECs and HAECs. The activation of Ras by VEGF appeared to be most prominent in HMVECs. These results indicate that intracellular signal transduction pathways for VEGF-induced activation of MAPKs are heterogeneous and vary depending on the origin of ECs.

Aorta↗
PubMed2001

Characterization of the gene EPAC2: structure, chromosomal localization, tissue expression, and identification of the liver-specific isoform.

The liver-specific protein cAMP-GEFII (also known as Epac2) belongs to a family of cyclic adenosine monophosphate (cAMP) binding proteins having guanine nucleotide exchange factor (GEF) activity (the cAMP-GEF family). Here we clone the gene EPAC2, encoding cAMP-GEFII, from a human liver cDNA library. Human EPAC2 has at least 31 exons and is mapped to human chromosome 2q31. Analyses by primer extension, reverse transcriptase-polymerase chain reaction, and in situ hybridization revealed the presence of three transcription start sites of liver-specific Epac2: two major sites located in exon 10 and a minor site in intron 9. The same translation start site is used in all three transcripts. Liver-specific cAMP-GEFII protein, which lacks the first cAMP-binding domain and the Dishevelled/Egl-10/Pleckstrin domain, was detected at 79 kDa by immunoblot analysis, confirming the presence of the short form of cAMP-GEFII in the liver. Liver-specific cAMP-GEFII also has GEF activity toward Rap1. These results demonstrate the presence of liver-specific cAMP-GEFII. Together with the previous finding that cAMP-GEFII is responsible for cAMP-dependent exocytosis in secretory cells, our study suggests that cAMP-GEFII may have a distinct role in liver.

Amino Acid Sequence↗
PubMed2001

Endoscopic carpal tunnel release and nerve conduction studies.

We investigated the outcome of endoscopic carpal tunnel release (ECTR) for patients with carpal tunnel syndrome (CTS) in comparison with the results of preoperative nerve conduction studies. The compound muscle action potential (CMAP) of the abductor pollicis brevis muscle (APB) and the second lumbrical muscle (L2) was recorded following median nerve stimulation at the wrist. A total of 38 hands in 35 patients were classified into four categories. Hands with a similarly prolonged distal motor latency for the APB and L2 were classified as type I (n=25), while those with a more prolonged distal motor latency for the APB than for the L2 (>0.7 ms) were classified as type 2 (n=10). Hands with a CMAP for the APB, but not L2, were classified as type 3 (n=1), and hands with no CMAP for either the APB or L2 were classified as type 4 (n=2). After ECTR, all of the type 1 and 2 hands were improved. Patients with type 3 and type 4 hands did not show satisfactory improvement, which may have been due to anatomical variation of the recurrent motor branch of the median nerve.

Action Potentials↗
PubMed2001

Nitric oxide inhibits the release of acetylcholine in the isolated retina.

BACKGROUND: Recent studies have revealed that administration of nitric oxide (NO) donors increases the release of neurotransmitters in various brain regions. In the retina, NO synthetase (NOS) is found in retinal amacrine and ganglion cells, and it is evident that NO is involved in encoding visual information. In the present study, therefore, NO donors were used to study the effect of exogenous NO on the high K(+)-evoked release of endogenous acetylcholine (ACh) in the rat retina. METHODS: Isolated rat retinal preparations were superfused with modified Krebs-Ringer bicarbonate buffer solution. In each experiment, stimulation for 10 min with 30 mM KCl was done twice. The amounts of ACh released by the first or second KCl stimulation were termed S1 and S2 respectively. Test agents were applied just before the second KCl stimulation. The effects of test agents were evaluated as S2 divided by S1. ACh was converted to hydrogen peroxide and electrochemically assayed by high-performance liquid chromatography. RESULTS: S-Nitro-N-acetyl-DL-penicillamine (SNAP), an NO donor, dose-dependently inhibited the high K(+)-evoked release of endogenous ACh. Such inhibition by NO was confirmed also by another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxy imino]-5-nitro-3-hexenamide (NOR3). The inhibitory effect of SNAP was abolished by both carboxy-PTIO, an NO scavenger, and bicuculline, an antagonist of GABAA receptors. CONCLUSIONS: The NO-induced decrease of ACh release is probably due to an NO-induced increase of GABAergic system inhibition.

Acetylcholine↗
PubMed2001

In vivo modulation of FGF biological activity alters cranial suture fate.

Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.

Adenoviridae↗
PubMed2001

Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes.

Chitosan is a copolymer of beta(1 --> 4) glucosamine and N-acetyl-D-glucosamine, which accelerates the infiltration of polymorphonuclear leukocytes (PMN) in the early phase of wound healing. In the granulation tissue treated with chitosan in canine experimental wound, osteopontin (OPN) was strongly positive in PMN immunohistochemically. OPN is a glycosylated phosphoprotein and promotes the attachment or spread of a variety of cell types. In addition, OPN may play a role in granulomatous inflammation. Production of OPN in PMN was therefore investigated in vitro using human PMN in this study. PMN stimulated with granulocyte-colony stimulating factor (G-CSF) and chitosan accumulated OPN mRNA, and released OPN into their culture supernatants. These findings suggest that OPN is synthesized by migrating PMN which plays the novel role of regulating the evolution of wound healing with chitosan treatment at the early phase of healing.

Animals↗
PubMed2001

Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages.

Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors.

Animals↗
PubMed2001

Criteria for extramural perineural invasion as a prognostic factor in rectal cancer.

BACKGROUND: An abundant extramural autonomic nerve network is an anatomical feature of the rectum. Extramural perineural invasion (PNI) may influence the prognosis after resection of rectal cancer, however, few assessment criteria exist. METHODS: PNI was investigated in 364 patients who underwent curative surgery for rectal cancer penetrating the muscular layer. A grading system was established based on the 'intensity' (number of PNI foci in a 20-power field) and 'depth' (distance from the muscularis propria) of PNI. PNI-0 was defined as without PNI, PNI-1 as 'intensity' of less than five foci and 'depth' less than 10 mm, and PNI-2 as five or more foci or 10 mm or greater 'depth' of invasion. RESULTS: PNI was observed in 52 patients (14 per cent) and strongly correlated with pathological tumour node metastasis (pTNM) stage. Five-year survival was related to PNI grade (74 per cent in PNI-0, 50 per cent in PNI-1 and 22 per cent in PNI-2). The rate of local recurrence was also related to PNI stage: 43 per cent in PNI-2 and 9 per cent in both PNI-0 and PNI-1. Multivariate analyses showed that graded PNI was associated both with local recurrence and long-term survival, independent of tumour depth (pTNM T) and nodal involvement (pTNM N). CONCLUSION: The PNI grading system may be useful in prognosis and allow case selection for intensive postoperative adjuvant therapy.

Adult↗