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N Koizumi

Publications and source records attributed to N Koizumi.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of capsid-modified adenovirus vectors containing heterologous peptides in the fiber knob, protein IX, or hexon.

Adenovirus (Ad) vectors are widely used in gene therapy and in vitro/in vivo gene transfer because of their high transduction efficiency. However, Ad vector application in the gene therapy field is limited by poor transduction into cells not expressing the primary receptor, coxsackievirus and adenovirus receptor. To overcome this problem, several types of capsid-modified Ad vectors have been developed. The HI loop or C-terminus of the fiber knob, the C-terminus of the protein IX (pIX) and the hypervariable region 5 of the hexon are promising candidate locations for displaying foreign peptide sequences. In the present study, we constructed Ad vectors in which each of the above region was modified by a simple in vitro ligation-based method, and examined the characterization of each Ad vector containing the FLAG tag (DYKDDDDK) or RGD (CDCRGDCFC) peptide. Enzyme-linked immunosorbent assay examining the surface expression of foreign peptides on the virus suggested that foreign peptides are exposed on virion surfaces in all types vectors and that the hexon was the most efficiently reacted, reflecting the copy number of the modification. However, in the case of the transduction efficiency of Ad vectors containing the RGD peptides, the modification of pIX and the hexon showed no effect. The modification of the HI loop of the fiber knob was the most efficient, followed by the modification of the C-terminus region of the fiber knob. These comparative analyses, together with a simple construction method for each modified Ad vector, could provide basic information for the generation of capsid-modified Ad vectors.

Adenoviridae↗
PubMed2006

Adenovirus serotype 35 vector-mediated transduction into human CD46-transgenic mice.

We previously demonstrated that systemic administration of adenovirus serotype 35 (Ad35) vectors to mice does not mediate efficient transduction in organs, probably because expression of the mouse analog of the subgroup B Ad receptor, human CD46 (membrane cofactor protein), is limited to the testis. Here, we describe the in vitro and in vivo transduction characteristics of Ad35 vectors by using homozygous and hemizygous human CD46-transgenic (CD46TG) mice, which ubiquitously express human CD46. An Ad35 vector more efficiently transduced the primary dendritic cells and macrophages prepared from CD46TG mice than those from wild-type mice. In vivo transduction experiments demonstrated that CD46TG mice are more susceptible to Ad35 vector-mediated in vivo transduction than are wild-type mice. In particular, homozygous CD46TG mice, which express higher levels of CD46 in the organs than hemizygous CD46TG mice, tend to exhibit higher transduction efficiencies after intraperitoneal administration than hemizygous CD46TG mice. Intraperitoneal administration of Ad35 vectors resulted in efficient transduction into the mesothelial cells of the peritoneal organs in homozygous CD46TG mice. These results indicate that an Ad35 vector recognizes human CD46 as a cellular receptor in CD46TG mice. However, the in vivo transduction efficiencies of Ad35 vectors in CD46TG mice are much lower than those of conventional Ad5 vectors in wild-type mice.

Adenoviridae↗
PubMed2004

Comparison of transgene expression mediated by several Fiber-modified adenovirus vectors in trophoblast cells.

The transfer of genes of interest is a useful method for studying placental biology. Recombinant adenovirus (Ad) vector is an efficient vector for transgene expression. An interaction between the fiber of Ad and the coxsackievirus and adenovirus receptor on the cell membrane is the first step in infection. We previously developed fiber-modified Ad vectors and showed that they improved transgene activity in several cell lines when compared to wild-type vector. In the present study, we examined the ability of three fiber-modified Ad vectors to transduce human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and rat trophoblast cell lines (Rcho-1, TR-TBT 18d-1 and TR-TBT 18d-2). We compared the transgene efficacy of wild-type Ad-L2 vector, Ad-RGD(HI)-L2 vector containing an Arg-Gly-Asp motif, Ad-K7(C)-L2 vector containing a 7-tandem lysine motif, and Ad-RGD(HI)K7(C)-L2 vector containing both motifs in the fiber. We used the luciferase gene as a reporter gene. In the human and rodent trophoblast cell lines, Ad-RGD(HI)-L2 had the greatest infectious potential, followed by Ad-RGD(HI)K7(C)-L2, Ad-K7(C)-L2 and Ad-L2. Compared to the amount of luciferase produced by wild-type vector, Ad-RGD(HI)-L2 mediated 8.1-fold the amount of luciferase in JEG-3 cells, 13.5-fold in JAR cells, 76.8-fold in BeWo cells, 5.0-fold in Rcho-1, 19.4-fold in TR-TBT 18d-1 and 15.0-fold in TR-TBT 18d-2. These results indicate that Ad-RGD(HI) is a potential recombinant Ad vector for transgene expression in some trophoblast cell lines.

Adenoviridae↗
PubMed2004

[Evaluation of spinal cord ischemia in endovascular stent graft repair and surgical operation of descending thoracic or thoracoabdominal aortic aneurysms].

Between October 1996 and June 2003, endovascular stent graft repair was performed in 87 patients with descending thoracic aortic aneurysms, graft replacement was performed in 24 patients with thoracoabdominal aortic aneurysms, and endovascular stent graft repair with concomitant surgical bypass of abdominal visceral arteries was performed in 3 patients with thoracoabdominal aortic aneurysms. The retrievable stent graft was inserted and evoked spinal cord potential were monitored in order to predict spinal cord ischemia for stent graft repair. There was no paraplegia or hospital death, although 3 patients had paraparesis in stent graft repair. Two of the 3 patients with paraparesis made a full neurologic recovery. There were no cases of paraplegia or paraparesis in surgical operations with thoracoabdominal aortic aneurysm. The concomitant surgical procedure was a good technique for patients in whom cardiopulmonary bypass could not be used. Our results of stent graft repair and surgical operation for descending thoracic or thoracoabdominal aortic aneurysms were acceptable. The retrievable stent graft was useful for prediction of spinal cord ischemia before endovascular stent graft repair of descending thoracic or thoracoabdominal aortic aneurysm.

Adult↗
PubMed2002

Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants.

In order to identify genes that are involved in the defense reaction against pathogen attack, we screened for examples that are regulated during the hypersensitive response (HR) to infection by tobacco mosaic virus (TMV) of tobacco ( Nicotiana tabacum cv. Xanthi nc) carrying the N gene, which confers resistance to TMV. Among seven genes initially identified by fluorescent differential display, one clone was further characterized because its transcripts accumulated rapidly and transiently after the onset of HR. Its full-length cDNA of 1346 bp encoded a polypeptide consisting of 258 amino acids. The deduced protein contained a single WRKY domain, a Cys(2)His(2) zinc-finger motif and a leucine-zipper motif, showing high similarity to WIZZ, a member of the family of WRKY transcription factors in tobacco. The gene was thus designated TIZZ. A GFP-TIZZ fusion protein was found to localize to the nucleus upon introduction into epidermal cells of onion. Bacterially expressed TIZZ was able to bind to the W-box (TTGAC) element that is recognized by other WRKY proteins, but transactivation assays showed it to be unable to activate reporter gene expression by itself. TIZZ transcripts were induced in TMV-infected nahG transgenic tobacco plants, in which salicylic acid fails to accumulate. Neither exogenously applied salicylic acid nor mechanical wounding induced TIZZ transcript accumulation. These results indicate the presence of salicylic acid-independent pathways for HR signal transduction, in which a novel type of WRKY protein(s) may play a critical role for the activation of defense.

Amino Acid Sequence↗
PubMed2002

CAR- or alphav integrin-binding ablated adenovirus vectors, but not fiber-modified vectors containing RGD peptide, do not change the systemic gene transfer properties in mice.

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and alphav integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and alphav integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while alphav integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as alphav integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with alphav integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with alphav integrin is likely to be crucial to the development of targeted Ad vectors.

Adenoviridae↗
PubMed2002

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Amnion↗
PubMed2002

Diminished inibitory action of ethanol on the contraction of gallbladder isolated from chronically ethanol-fed Guinea pigs.

Ethanol is known to decrease the gallbladder contractility. The purpose of this study was to clarify the mechanism of tolerance to the inhibitory action of ethanol on the gallbladder contractility. Male guinea pigs were fed ethanol (3%) or calorie-matched sucrose in the drinking water for 4 weeks. Then, the gallbladder was isolated, and its isometric tension was measured. The contractile responses to KCl, BAY K8644, histamine, and phorbol 12,13-dibutyrate in the normal medium were not different between the gallbladder strips from ethanol-fed and control guinea pigs. Ethanol at 25 mmol/l in vitro did not affect the contractile responses to KCl and BAY K8644 in the gallbladder strips from both ethanol-fed and control guinea pigs. On the other hand, ethanol at 25 mmol/l in vitro significantly inhibited the contractile responses to histamine and phorbol 12,13-dibutyrate in the gallbladder strips from the control guinea pigs, but it did not affect the contractile response to histamine and significantly augmented that to phorbol 12,13-dibutyrate in the strips from the ethanol-fed guinea pigs. Diphenhydramine, a selective H(1) receptor antagonist, abolished the histamine contraction in gallbladder strips from both control and ethanol-fed guinea pigs, while cimetidine, a selective H(2) receptor antagonist, did not affect histamine contraction, implying that histamine-induced contraction of guinea pig gallbladders is mediated only by H(1) receptors. Verapamil (1 micromol/l) completely inhibited the phorbol 12,13-dibutyrate-induced contraction of the strips from both ethanol-fed and control guinea pigs. The histamine-induced contraction was partly inhibited in the absence of Ca(2+) in the medium. In the gallbladder strips from both ethanol-fed and control guinea pigs, ethanol at 25 mmol/ in vitro did not affect the histamine-induced contraction in the absence of extracellular Ca(2+). Tolerance to the inhibitory action of ethanol developed selectively on contractile responses to histamine and phorbol 12,13-dibutyrate. Chronic ethanol administration produces tolerance to in vitro gallbladder contractility mediated by the Ca(2+) entry through L-type Ca(2+) channels linked with protein kinase C activation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide.

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

Adenoviridae↗
PubMed2001

PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by Newcastle disease virus.

Virus infection triggers innate responses to host cells including production of type I interferon (IFN). Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-beta gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-beta gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-beta enhancer, increased activity of NF-kappaB and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-beta gene expression.

Amino Acid Sequence↗
PubMed2001

An expression profile of active genes in human conjunctival epithelium.

The purpose of this study was to describe the quantitatively and qualitatively genes expressed in in vivo human conjunctival epithelium. A cDNA library was created from human conjunctival epithelial cells obtained from 38 normal eyes by brush cytology. Poly A(+)RNA isolated from these cells was used as a template for cDNA synthesis by the vector-priming method. A 3'-directed cDNA library consisting of the cDNA moiety covering the poly A(+)RNA tail through the nearest Mbo I site was constructed and transformed into Escherichia coli. Inserts in 933 randomly chosen clones were sequenced. The resulting sequences were compared to determine frequency of appearance in the library, and to establish an expression profile of genes in the conjunctival epithelium. The sequences were sent to GenBank for gene identification. The result was an expression profile of active genes reflecting their relative abundance in the conjunctival epithelial mRNA population. The expression profile of human conjunctival epithelium showed that the most abundant gene transcript in human conjunctival epithelium was that for cytokeratin 13. Altogether 102 genes were found to be very active, including beta-2 microglobulin, lipocortin I and insulin-like growth factor binding protein-3. The gene expression profile of the conjunctival epithelium reflects the unique properties and functions of this tissue. Comparison of this expression profile with that obtained from corneal epithelium discloses clear differences and helps us better understand the physiology and pathophysiology of the ocular surface in humans.

Adult↗
PubMed2001

Two novel genes encoding SNF-1 related protein kinases from Arabidopsis thaliana: differential accumulation of AtSR1 and AtSR2 transcripts in response to cytokinins and sugars, and phosphorylation of sucrose synthase by AtSR2.

We searched for genes encoding members of the group-3 SNF1-related protein kinase (SnRK3) family in the Arabidopsis thaliana database, and seven independent sequences were identified. Transcripts of two of them were found to accumulate differentially upon treatment with light, cytokinins and sugars. Full-length cDNAs were isolated and designated as AtSR1 and AtSR2; they encode polypeptides of 442 and 429 amino acids with relative molecular masses of 50.3 kDa and 48.2 kDa, respectively. In etiolated seedlings, no transcripts of either gene were observed. However, upon exposure to light or cytokinins, transcripts of AtSR1 but not AtSR2 began to accumulate. The induction with light was greatly reduced in the presence of a cytokinin antagonist, suggesting that cytokinins are involved in light-signaling pathways. In contrast, transcription of AtSR2, but not of AtSR1, was greatly increased by sucrose, as well as glucose and fructose. AtSR2 expressed in E. coli efficiently phosphorylated sucrose synthase in the presence of manganese ions. These results suggest that, although SnRK3 proteins may generally be involved in sugar metabolism, expression of AtSR1 and AtSR2 is differentially and distinctly regulated by various external signals, and AtSR2 may function in the regulation of sucrose synthase by specific phosphorylation.

Amino Acid Sequence↗
PubMed2001

Early results of a prospective study of limited resection for bronchioloalveolar adenocarcinoma of the lung.

BACKGROUND: We reported that bronchioloalveolar adenocarcinoma (BAC) without active fibroblastic proliferation of the lung had no lymph node and pulmonary metastasis and had a favorable prognosis. However, there has been no prospective trial regarding limited pulmonary resection for this type of BAC. The purpose of this study is to confirm the effectiveness of limited resection for histologically confirmed BAC without active fibroblastic proliferation. METHODS: From 1996 through 1999, 42 patients who had small peripheral lung tumors (< or = 20 mm), suspected of being BAC, were enrolled in this trial. The patient population consisted of 24 men and 18 women with a mean age of 58.4 years. Limited resection was completed when BAC, without both active fibroblastic proliferation and lymph node metastasis, was confirmed histologically by intraoperative pathologic examination. RESULTS: Limited resection was completed in 36 patients, wedge resection in 34, and segmentectomy in 2 patients. In 6 patients, the procedure was converted into lobectomy because of pathologic invasive sign in 3, active fibroblastic proliferation in 1, and for other reasons in 2 patients. All patients have been followed for a median follow-up period of 30 months and are alive without sign of recurrence. CONCLUSIONS: Our early results indicate that limited resection may be an acceptable alternative to lobectomy for histologically confirmed BAC without active fibroblastic proliferation.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2001

Cultivated corneal epithelial stem cell transplantation in ocular surface disorders.

PURPOSE: To investigate the outcome of cultivated corneal epithelial transplantation for severe stem cell deficiencies using denuded amniotic membrane (AM) as a carrier. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Thirteen eyes of 11 patients were studied. These consisted of five eyes with acute Stevens-Johnson syndrome (SJS), two with chronic SJS, one with an acute chemical injury, two with chronic chemical injuries, two with ocular cicatricial pemphigoid, and one with drug-induced pseudopemphigoid. All of these eyes had total stem cell deficiencies. MAIN OUTCOME MEASURES: Adaptation of the cultivated corneal epithelium onto the host corneal surface was confirmed 48 hours after surgery. The reconstruction of the ocular surface and visual acuity were measured. METHODS: Corneal limbal epithelium from donor corneas was cultivated for 4 weeks on a denuded AM carrier, with 3T3 fibroblast coculture and air lifting. The cultivated corneal epithelium showed four to five layers of stratification and was well differentiated. After conjunctival tissue removal from the cornea up to 3 mm outside the limbus and subconjunctival tissue treatment with 0.04% mitomycin C, cultivated allocorneal epithelium, including the AM carrier, was transplanted onto the corneal surface up to the limbus. Lamellar keratoplasty, using preserved donor graft without epithelium, was performed simultaneously for five chronic-phase patients showing corneal stromal scarring. Systemic immunosuppression was used to prevent allograft rejection. RESULTS: In all 13 eyes, the entire corneal surface, on which cultivated allocorneal epithelium had been placed, was free from epithelial defects 48 hours after surgery, indicating complete survival of the transplanted corneal epithelium. Visual acuity improved in all eyes after surgery, and 10 of the 13 eyes were restored to good vision (postoperative visual acuity improved two or more lines) 6 months after the operation. During the follow-up period (mean +/- standard deviation, 11.2 +/- 1.3 months), the corneal surfaces were clear, although three eyes experienced epithelial rejection. CONCLUSIONS: Cultivated corneal epithelial transplantation using denuded AM as a carrier can be used for severe stem cell deficiencies.

Adult↗
PubMed2001

A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

Adenoviridae↗
PubMed2001

An Arabidopsis SNF1-related protein kinase, AtSR1, interacts with a calcium-binding protein, AtCBL2, of which transcripts respond to light.

AtSR1 is a protein kinase of Arabidopsis thaliana, which belongs to the SNF1-related protein kinase subfamily 3. We previously showed accumulation of its transcripts to be responsive to light. In this study, we examined the interaction between AtSR1 and six calcineurin B like proteins of Arabidopsis and found that AtSR1 prominently interacts with one of them, AtCBL2, by yeast two-hybrid assay. Interaction between AtSR1 and AtCBL2 could also be directly confirmed in vitro by pull down assay. RNA blot and reverse transcription-polymerase chain reaction analyses showed that transcripts of AtCBL2, and also of AtCBL1, another CBL, increased upon illumination of leaves. The physiological meaning of the interaction of AtSR1and AtCBL2 is not clear, but they presumably function in signal transduction of light.

Amino Acid Sequence↗