Biomedical subjects

Osamu Nakajima

Publications and source records attributed to Osamu Nakajima.

At least 19 recordsLinked to original sources

PubMed2006

ELISA method for monitoring human serum IgE specific for Cry1Ab introduced into genetically modified corn.

Enzyme-linked immunosorbent assay (ELISA) is the most convenient method of monitoring the occurrence of IgE antibodies specific for novel proteins in genetically modified (GM) foods. The levels of IgE specific for a recombinant protein, Cry1Ab, were determined using an ELISA method. A soluble form of the Cry1Ab protein purified from pCold1 vector-transformed Escherichia coli pTf16/BL21 was used as the ELISA coating antigen, and 1M NaCl was used as the washing buffer to remove IgE non-specifically bound to the coated antigen. Sera from 44 patients allergic to major food allergens were obtained, diluted 20-fold, tested, and found no identifiable IgE above background levels. We also tested sera from patients with corn allergy against whole extracts of non-GM and GM-corn (MON 810) using immunoblotting. The staining patterns were similar for the two types of corn. These results indicate that significant levels of IgE antibodies specific to Cry1Ab were not found in the sera of Japanese patients with food allergies.

Bacillus thuringiensis Toxins↗

PubMed2006

In vivo oxygen imaging using green fluorescent protein.

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520-550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.

Animals↗

PubMed2006

Transgenic rescue of erythroid 5-aminolevulinate synthase-deficient mice results in the formation of ring sideroblasts and siderocytes.

Molecular defects in erythroid 5-aminolevulinate synthase (ALAS-E), the first enzyme in the heme biosynthetic pathway, cause X-linked sideroblastic anemia (XLSA). However, ring sideroblasts, the hallmark of XLSA, were not found in ALAS-E-deficient mouse embryos, indicating that simple ALAS-E-deficiency is not sufficient for ring sideroblast formation. To investigate the developmental stage-specific pathogenesis caused by heme-depletion, we attempted a complementation rescue of ALAS-E-deficiency. We exploited transgenic mouse lines expressing human ALAS-E at approximately half that of wild-type levels. In these hypomorphic embryos, most of the primitive erythroid cells were transformed into ring sideroblasts. The majority of the circulating definitive erythroid cells became siderocytes, enucleated erythrocytes containing iron deposits, and definitive ring sideroblasts were also observed. These iron-overloaded cells suffered from an alpha/beta globin chain imbalance. Despite the iron overload, transferrin receptors were highly expressed in the erythroid cells, suggesting they contribute to the formation of ring sideroblasts and siderocytes. These results indicate that a partially depleted heme supply provokes ring sideroblast formation. The experimental generation of ring sideroblasts in animals would contribute to our understanding of the iron metabolism and its disorder in erythroid cells.

5-Aminolevulinate Synthetase↗

PubMed2005

Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice.

BACKGROUND: Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. METHODS AND RESULTS: To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. CONCLUSIONS: These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Angiotensin II↗

PubMed2005

Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods.

For monitoring the occurrence of IgE antibody specific for novel proteins in genetically modified (GM) foods, ELISA is the most convenient method. The levels of IgE specific for recombinant proteins, phosphinothricin-N-acetyltransferase (PAT), CP4-EPSPS, and Cry9C were determined by ELISA using the sera from patients allergic to known allergens. Ovalbumin (OVA) and OVA-positive patient sera were used as positive control. In the ELISA, 20-fold-diluted sera tested were mostly negative for the specific IgE. However, the PAT-specific, but not CP4-EPSPS- or Cry9C-specific IgE in some patients was apparently higher than that of the healthy volunteers. To clarify the binding specificity of the antibody, we pre-incubated the sera with soluble PAT, but the inhibition was marginal, suggesting that the binding was non-specific. Therefore, we used 1M NaCl as a washing buffer to remove IgE non-specifically bound to the coated PAT. This washing step efficiently decreased non-specific binding. In contrast, OVA-specific IgE binding to OVA-coated plate was not affected by the washing. Finally, in this pilot study significant levels of IgE antibodies specific for the three proteins were not detected in the sera of Japanese food-allergy patients.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2005

Differential gene expression profiling between wild-type and ALAS2-null erythroblasts: identification of novel heme-regulated genes.

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific delta-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.

5-Aminolevulinate Synthetase↗

PubMed2005

Genetic oxygen sensor: GFP as an indicator of intracellular oxygenation.

We report in this article a new method for in vivo oxygen measurement using green fluorescence protein (GFP). COS7 cells were transiently transfected with an expression vector, pCMX-GFP, using a polyethylenimine reagent and cultured for 48 hrs. After exposure of the cell to anoxic gas (O2 < .001%), a 1 min illumination of the cell to strong 470-490 nm light evoked a significant red fluorescence (excitation 520-550 nm, emission > 580 nm) that had been negligible before the photoactivation. This red shift of (green) GFP fluorescence was never observed in normoxia. We then examined the validity of this method in transgenic mice in which GFP is stably expressed (green mice). All the ventricular myocytes isolated from the green mice showed significant green fluorescence, although the intensity was approximately 1/200 of the transiently GFP-expressing COS7 cells. The photoactivation in anoxia increased the red fluorescence in these cells, but the magnitude was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia. In GFP-expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.

Animals↗

PubMed2005

Performance of a semi-quantitative whole blood test for human heart-type fatty acid-binding protein (H-FABP).

OBJECTIVES: We evaluated the accuracy of visually reading the whole blood Rapicheck H-FABP panel test using the quantitative plasma H-FABP concentration as the reference. SUBJECTS AND METHODS: Consecutive patients with chest pain (n = 237) who were suspected of having acute myocardial infarction were recruited. The appearance of an evident test line within 5 min was given a grade of +3 (strongly positive), appearance within 15 min +2 (moderately positive) and the appearance of a weak test line within 15 min +1 (weakly positive). RESULTS AND DISCUSSION: The concordance rates were 91.8% for positive, 70.1% for negative and 80.2% for overall. Plasma H-FABP concentrations were above the cut-off value for 9.2% of negative (0) results. Fifty percent of weakly positive (+1) and 25.0% of moderately positive (+2) results had H-FABP concentrations lower than the cut-off value. All of the strongly positive (+3) were above the cut-off value. These results suggested that the false-positive and false-negative results of Rapicheck H-FABP were caused by over or underestimation in visual reading when the plasma H-FABP concentration was near the cut-off concentration. CONCLUSIONS: Low accuracy of visual reading of Rapicheck H-FABP was due to poor estimation by manual reading around the cut-off value.

Aged↗

PubMed2005

The distal sequence element of the selenocysteine tRNA gene is a tissue-dependent enhancer essential for mouse embryogenesis.

Appropriate expression of the selenocysteine tRNA (tRNA(Sec)) gene is necessary for the production of an entire family of selenoprotein enzymes. This study investigates the consequence of disrupting an upstream enhancer region of the mouse tRNA(Sec) gene (Trsp) known as the distal sequence element (DSE) by use of a conditional repair gene targeting strategy, in which a 3.2-kb insertion was introduced into the promoter of the gene. In the absence of DSE activity, homozygous mice failed to develop in utero beyond embryonic day 7.5 and had severely decreased levels of selenoprotein transcript. Cre-mediated removal of the selection cassette recovered DSE regulation of Trsp, restoring wild-type levels of tRNA(Sec) expression and allowing the generation of viable rescued mice. Further analysis of targeted heterozygous adult mice revealed that the enhancer activity of the DSE is tissue dependent since, in contrast to liver, heart does not require the DSE for normal expression of Trsp. Similarly, in mouse cell lines we showed that the DSE functions as a cell-line-specific inducible element of tRNA(Sec). Together, our data demonstrate that the DSE is a tissue-dependent regulatory element of tRNA(Sec) expression and that its activity is vital for sufficient tRNA(Sec) production during mouse embryogenesis.

Animals↗

PubMed2004

The transcriptional programme of antibody class switching involves the repressor Bach2.

Activated B cells differentiate to plasma cells to secrete IgM or, after undergoing class switch recombination (CSR), to secrete other classes of immunoglobulins. Diversification of antibody function by CSR is important for humoral immunity. However, it remains unclear how the decision for the bifurcation is made. Bach2 is a B-cell-specific transcription repressor interacting with the small Maf proteins whose expression is high only before the plasma cell stage. Here we show that Bach2 is critical for CSR and somatic hypermutation (SHM) of immunoglobulin genes. Genetic ablation of Bach2 in mice revealed that Bach2 was required for both T-cell-independent and T-cell-dependent IgG responses and SHM. When stimulated in vitro, Bach2-deficient B cells produced IgM, as did wild-type cells, and abundantly expressed Blimp-1 (refs 9, 10) and XBP-1 (ref. 11), critical regulators of the plasmacytic differentiation, indicating that Bach2 was not required for the plasmacytic differentiation itself. However, they failed to undergo efficient CSR. These findings define Bach2 as a key regulator of antibody response and provide an insight into the orchestration of CSR and SHM during plasma cell differentiation.

Animals↗

PubMed2004

Effect of two nitrogenous diphenyl ether pesticides on mast cell activation.

We examined the effect of two nitrogenous diphenyl ether pesticides, nitrofen (NIP) and chlornitrofen (CNP), on mast cell activation. RBL-2H3 (rat basophilic leukemia) cells were exposed to NIP or CNP for 30 min to investigate their effect on degranulation, and for 3 h to investigate their effect on cytokine production and gene expression. NIP and CNP increased IgE receptor-mediated beta-hexosaminidase release, MCP-1 release, and TNF-alpha release in a dose-dependent manner. The increasing effect of CNP on their release was greater than that of NIP. In the gene expression experiment, 30 microg/ml CNP significantly upregulated Egr-1, MCP-1 and GADD45a gene expression. These results suggest that at higher concentrations (more than 30 microg/ml) the nitrogenous diphenyl ether pesticides had both a degranulation-enhancing effect and proinflammatory cytokine-production enhancing effect through the expression of some transcription factors in RBL-2H3 cells.

Animals↗

PubMed2003

Distinct response to dioxin in an arylhydrocarbon receptor (AHR)-humanized mouse.

There are large inter- and intraspecies differences in susceptibility to dioxin-induced toxicities. A critical question in risk assessment of dioxin and related compounds is whether humans are sensitive or resistant to their toxicities. The diverse responses of mammals to dioxin are strongly influenced by functional polymorphisms of the arylhydrocarbon receptor (AHR). To characterize responses mediated by the human AHR (hAHR), we generated a mouse possessing hAHR instead of mouse AHR. Responses of these mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene were compared with the responses of naturally sensitive (C57BL6J) and resistant (DBA2) mice. Mice homozygous for hAHR exhibited weaker induction of AHR target genes such as cyp1a1 and cyp1a2 than did C57BL6J (Ahr(b-1/b-1)) mice. DBA2 (Ahr(d/d)) mice were less responsive to induction of cyp genes than C57BL6J mice. hAHR and DBA2 AHR exhibit similar ligand-binding affinities and homozygous hAHR and Ahr(d/d) mice displayed comparable induction of AHR target genes by 3-methylcholanthrene. However, when TCDD was administered, a greatly diminished response was observed in homozygous hAHR mice compared with Ahr(d/d) mice, indicating that hAHR expressed in mice is functionally less responsive to TCDD than DBA2 AHR. After maternal exposure to TCDD, homozygous hAHR fetuses developed embryonic hydronephrosis, but not cleft palate, whereas fetuses possessing Ahr(b-1) or Ahr(d) developed both anomalies. These results suggest that hAHR may define the specificity of the responses to various AHR ligands. Thus, the hAHR knock-in mouse is a humanized model mouse that may better predict the biological effects of bioaccumulative environmental toxicants like TCDD in humans.

Animals↗

PubMed2003

HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin.

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Animals↗

PubMed2003

GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene in transgenic mouse.

BACKGROUND: The erythroid transcription factor GATA-1 is also expressed in Sertoli cells of the testis. The testicular expression of GATA-1 is regulated in a developmental and spermatogenic stage-specific manner. To further clarify the regulatory mechanisms of testicular GATA-1 gene expression, we carried out transgenic reporter gene expression analyses. RESULTS: We found that GATA-1 expression in Sertoli cells is markedly decreased concomitant with the emergence of elongated spermatids in the seminiferous tubules. Transgenic reporter mouse analyses revealed that a 15 kb GATA-1 genomic region is sufficient to recapitulate the gene expression profile in Sertoli cells. While the GATA-1 haematopoietic enhancer and the proximal first exon are included within the 15 kb genomic region, these regulatory elements are not essential for GATA-1 expression in Sertoli cells. Further analyses using deletion constructs revealed that a 1.5 kb region 5' to the GATA-1 haematopoietic enhancer is essential for gene expression in Sertoli cells and this region is referred to as the GATA-1 testis activation region. CONCLUSION: These results thus demonstrated that the GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene. The 15 kb genomic region is applicable and useful for the expression vector system specific for adult Sertoli cells in stage VII to IX.

Animals↗

PubMed2003

Functional analysis of basic transcription element binding protein by gene targeting technology.

Basic transcription element binding protein (BTEB) is a transcription factor with a characteristic zinc finger motif and is most remarkably enhanced by thyroid hormone T(3) treatment (R. J. Denver et al., J. Biol. Chem. 272:8179-8188, 1997). To investigate the function of BTEB per se and to touch on the effects of T(3) (3,5,3'-triiodothyronine) on mouse development, we generated BTEB-deficient mice by gene knockout technology. Homologous BTEB(-/-) mutant mice were bred according to apparently normal Mendelian genetics, matured normally, and were fertile. Mutant mice could survive for at least 2 years without evident pathological defects. From the expression of lacZ, which was inserted into the reading frame of the BTEB gene, BTEB showed a characteristic tissue-specific expression profile during the developmental process of brain and bone. Dramatically increased expression of BTEB was observed in Purkinje cells of the cerebellum and pyramidal cell layers of the hippocampus at P7 when synapses start to form in the brain. Although general behavioral activities such as locomotion, rearing, and speed of movement were not so much affected in the BTEB(-/-) mutant mice, they showed clearly reduced activity levels in rotorod and contextual fear-conditioning tests; this finding was probably due to defective functions of the cerebellum, hippocampus, and amygdala.

Amygdala↗

PubMed2003

[Study on a method for delivering scFv recombinant antibody into cultured cells].

We try to develop a method for delivering antibody from blood circulation through blood brain barrier to brain. In order to achieve this goal, antibody has to cross cellular membrane of brain capillary endothelial cells twice. As a first step of our study, we examined the ability for scFv antibody to cross cellular membrane of RBL-2H3 cells once and be delivered into the inside of the cultured cells with the help of TAT peptide. TAT peptide was originally found in Tat protein from the HIV-1 virus and known as one of protein transduction domains. First, oligonucleotide encoding TAT peptide was linked to 5' terminal of gene fragment of scFv antibody by PCR technology. TAT-linked scFv gene fragment was subcloned into pET-23b vector and successfully expressed in E. coli as inclusion body. After solubilization and purification, TAT-linked scFv recombinant protein was added to the culture of RBL-2H3 cells. TAT-linked scFv delivered into RBL-2H3 cells was detected by means of immunocytochemistry using fluorescence microscopy. TAT-linked scFv crossed cellular membrane more efficiently than scFv without TAT peptide.

Animals↗

PubMed2002

Aberrant iron accumulation and oxidized status of erythroid-specific delta-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts.

Alas2 encodes the erythroid-specific delta-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with the Alas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking the Alas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype of Alas2-null definitive erythroblasts was examined. Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes in Alas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate that Alas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron, Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.

5-Aminolevulinate Synthetase↗

PubMed2002

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene.

Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho-1. Here we show that a heme-binding factor, Bach1, is a critical physiological repressor of ho-1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho-1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho-1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho-1. Thus, regulation of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor-activator antagonism.

3T3 Cells↗