Biomedical subjects

A Takashima

Publications and source records attributed to A Takashima.

At least 37 records · Page 2Linked to original sources

PubMed2001

Regulation by two CatR proteins that differ in binding affinity to catB promoters expressing two cat gene clusters.

We isolated the two LysR-type regulatory proteins CatR1 and CatR2, which regulate the expression of cat1 and cat2 gene clusters, respectively, required for catechol degradation in the bacterium Frateuria sp. ANA-18. In a gel mobility shift assay using CatR1 and the DNA fragment containing the catB1 promoter region, the formation of two complexes, complex 1-1 (C1-1) and complex 1-2 (C1-2), was observed in the presence of cis,cis-muconate. On the other hand, CatR2 and the DNA fragment containing the catB2 promoter region formed only complex 2-2 (C2-2) at a lower concentration of cis,cis-muconate than that at which C1-1 and C1-2 were formed. As the concentration of cis,cis-muconate decreased, the production of the muconate cycloisomerase isozyme MC II encoded by catB2 decreased as well as that of MC I encoded by catB1. However, the amount of MC II synthesized was larger than that of MC I at low concentrations. On the basis of these results, we concluded that the catB2 promoter was activated at low concentrations of cis,cis-muconate.

Bacterial Proteins↗

PubMed2001

[Analysis of mouse model exhibiting neurofibrillary changes].

Dysfunction and filamentous microtubule-binding tau protein are key markers of neurodegenerative pathologies, including the pathology and neural degeneration associated with Alzheimer's disease (AD). Immunocytochemical studies of NFT-bearing neurons showed that NFTs are composed of ubiquitin and phosphorylation-dependent tau. Congo-red birefringency and thioflavin-S reactivity in NFT-bearing neurons also demonstrated that the tau aggregation forms a beta-sheet structure. Discovery of the molecular mechanisms of NFT formation may lead to more insight about events occurring during neurodegeneration. In frontotemporal dementia parkinsonism 17 (FTDP17), genetic studies indicated that tau is a causative gene, and mutation is found in exons and introns of tau gene. A patient who possesses this mutation exhibits pathologically NFT and clinically personality change and cognitive dysfunction. Then, we produced the Tg mice expressing human longest tau with missense mutation V337M. In the present study, neurons of hippocampus and cerebral cortex in our Tg mice showed phosphorylated and ubiquitinated tau aggregations with a beta-sheet structure. This was demonstrated by Congo-red and thioflavin-S positive staining, a histological criterion used to identify NFTs observed in neurodegenerative disorders. The mice also displayed altered behaviors that were associated with NFT formation. Thus, V337M mice provide a first animal model exhibiting altered behavior due to NFTs.

Animals↗

PubMed2000

Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking.

Hyaluronan (HA), a high molecular weight glycosaminoglycan, is expressed abundantly in the extracellular matrix and on cell surfaces. Although HA is known to bind many adhesion molecules, little information has been available with respect to its direct physiological role. In this study, we developed a novel 12-mer (GAHWQFNALTVR) peptide inhibitor of HA, termed "Pep-1," by using phage display technology. Pep-1 showed specific binding to soluble, immobilized, and cell-associated forms of HA, and it inhibited leukocyte adhesion to HA substrates almost completely. Systemic, local, or topical administration of Pep-1 inhibited the expression of contact hypersensitivity responses in mice by blocking skin-directed homing of inflammatory leukocytes. Pep-1 also inhibited the sensitization phase by blocking hapten-triggered migration of Langerhans cells from the epidermis. These observations document that HA plays an essential role in "two-way" trafficking of leukocytes to and from an inflamed tissue, and thus provide technical and conceptual bases for testing the potential efficacy of HA inhibitors (e.g., Pep-1) for inflammatory disorders.

Amino Acid Sequence↗

PubMed2000

Induction of Th2-directed immune responses by IL-4-transduced dendritic cells in mice.

Dendritic cell (DC)-based vaccines have been used to generate Th1-mediated, protective immunity against cancers and infectious microorganisms. As an attempt to develop a new vaccine protocol for the induction of Th2-directed responses, we introduced an IL-4 plasmid vector into the XS106 DC line (derived from A/J mice). Although relatively small fractions of XS106 cells exhibited apparent intracellular deposition of IL-4, they secreted biologically relevant amounts of the cytokine. IL-4-transduced XS106 DC and control XS106 DC transfected with vector alone were pulsed with KLH and injected s.c. into A/J mice. The overall magnitude of KLH-specific cellular and humoral responses was comparable between the two animal groups. However, they differed in the isotype profile albeit only transiently, with the IL-4-transduced DC group showing higher IgE and lower IgG2a responses, and in the cytokine profile, with spleen cells isolated from the IL-4-transduced DC group producing higher IL-13 and lower IL-12. Thus, delivery of IL-4 gene to relatively small numbers of DC is sufficient to modify the immunological outcome of DC-based vaccines.

Animals↗

PubMed2000

Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

Amino Acid Sequence↗

PubMed2000

Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms.

Using a subtractive cDNA cloning strategy, we isolated previously five novel genes that were expressed abundantly by the murine dendritic cell (DC) line XS52, but not by the J774 macrophage line. One of these genes encoded a unique, DC-associated C-type lectin, termed "dectin-1." Here we report the characterization of a second novel gene that was also expressed in a DC-specific manner. Clone 1B12 encoded a type II membrane-integrated polypeptide of 209 amino acids containing a single carbohydrate recognition domain motif in the COOH terminus. The expression pattern of this molecule, termed "dectin-2," was almost indistinguishable from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptase-polymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 (alpha isoform) is encoded by 6 exons, whereas truncated isoforms (beta and gamma) are produced by alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins.

Alternative Splicing↗

PubMed2000

Proteolytic processing and degradation of human presenilin-1 expressed in yeast.

Numerous mutations causing early-onset familial Alzheimer's disease have been identified in the presenilin-1 gene. Presenilin-1 protein is produced as a 47 kDa holoprotein and proteolytically processed to an N-terminal 28 kDa and a C-terminal 19 kDa fragments by unidentified presenilinase in mammalian cells. We have demonstrated that this proteolytic processing also occurs in yeast. We also show that degradation of C-terminal fragment of presenilin-1 is dependent of proteasomal function. This yeast system will be a good tool to identify presenilinase and to study the role of presenilin-1 in amyloid precursor protein processing.

Blotting, Western↗

PubMed2000

Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope.

A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimer's disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.

Alzheimer Disease↗

PubMed2000

T cells reactive to keratinocyte antigens are generated during induction of contact hypersensitivity in mice. A model for autoeczematization in humans?

BACKGROUND: The role of keratinocytes (KC) in contact hypersensitivity (CH) has been examined more with respect to cytokine secretion and tolerance induction and less as a source of antigenic proteins to which chemical haptens can conjugate. OBJECTIVE: To determine whether KC-derived proteins can serve as antigenic carriers for haptens such as dinitrofluorobenzene (DNFB). METHODS: We examined the capacity of draining lymph node cells from BALB/c mice sensitized to DNFB to proliferate in response to hapten or to hapten-conjugated protein extracts derived from a KC line (DNP-Pam KC extract). Using limiting dilution microculture of these lymph node cells, we established DNP-specific T cell clones as well as DNP-Pam KC extract-reactive T cell clones. We also examined the proliferative responses of the DNP-Pam KC extract-reactive clones and of lymph node cells from mice sensitized to different haptens. RESULTS: Lymph node cells from DNFB-sensitized mice proliferated well to hapten or to DNP-Pam KC extract. Six la(d)-restricted, alphabeta TCR-bearing, CD4+ clones were established: 4 proliferated specifically to soluble hapten (DNBS), whereas 2 proliferated in response to DNP-Pam KC extract. Surprisingly, the DNP-Pam KC extract-reactive clones proliferated as well to Pam KC extract without hapten. Lymph node cells from hapten-sensitized mice not only proliferated specifically in response to the hapten to which they were sensitized, but also proliferated to Pam KC extract without hapten. CONCLUSIONS: T cell clones generated during the induction of (CH) in mice include those reactive to hapten as well as those reactive to KC antigens independent of hapten. Analogous mechanisms in humans might account for autoreactive events such as id reactions associated with CH and angry back syndrome during patch testing.

Animals↗

PubMed2000

New technologies to prevent and treat contact hypersensitivity responses.

Allergic contact dermatitis is a common inflammatory skin disease caused by T cells that recognize environmental and industrial allergens (i.e., haptens). Langerhans' cells (LC), which are skin-specific and "immature" members of the dendritic cell (DC) family of antigen-presenting cells, play crucial roles in the induction of contact hypersensitivity (CH) responses. Upon exposure to haptens, LC migrate from the epidermis to draining lymph nodes, mature into T cell-stimulatory DC, and activate hapten-reactive T cells. Therefore, CH responses should be preventable at the sensitization phase by interfering with one of these changes that occur in LC. Our objective is to develop new technologies for the prevention and treatment of allergic contact dermatitis. In this article, we will introduce three technologies that we have recently developed. First, using a phage display strategy, we have identified a 12-mer peptide (termed "peptide 1") that binds and blocks the function of hyaluronan (HA), which is known to serve as an adhesive substrate for LC migration. Local injection of peptide 1 in mice before topical application of DNFB blocked almost completely the emigration of LC from the epidermis to the draining lymph node, where antigen presentation takes place. Peptide 1 represents a new strategy that is designed to inhibit the initial event of CH. Second, we have established an in vitro experimental system to study the terminal maturation of LC during antigen-specific interaction with T cells. This experimental system, which employs a long-term LC line and T cell clones, should provide a unique tool for the identification of new immunosuppressive agents that block LC terminal maturation selectively. Finally, under the hypothesis that LC, which are engineered to overexpress a death ligand, would deliver apoptotic signals instead of activation signals to T cells, we created a "killer" LC clone by introducing CD95L cDNA into our long-term LC line XS106. In vivo administration of DNFB-pulsed killer LC into mice, either before or after sensitization, resulted in marked suppression of CH responses to DNFB. The killer LC technology represents an entirely new immunosuppressive therapy that is designed to eliminate only the pathogenic T cells.

Animals↗

PubMed2000

A role for NF-kappaB-dependent gene transactivation in sunburn.

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-kappaB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-kappaB target genes, rather than from nonspecific changes associated with tissue damage.

Animals↗

PubMed2000

Induction of hepatocellular carcinoma with high metastatic potential in WS/Shi rats: discovery of an inbred strain highly susceptible to the liver carcinogen N-nitrosomorpholine.

We investigated the susceptibility of three inbred strains of rats to the hepatocarcinogen, N-nitrosomorpholine (NNM), to establish a spontaneous metastatic model of hepatocellular carcinoma (HCC). WS/Shi. SD/gShi, and F344/DuCrj rats were given 0.02% NNM in drinking water for 8 weeks and thereafter left without any treatment. The experiment ceased at week 20, because mortality markedly increased after this time point in WS/Shi rats. Liver weight was highest in WS/Shi rats among the three strains examined. The incidence of HCC was 15/15 (100%) in WS/Shi rats, 1/16 (6%) in SD/gShi rats, and 13/16 (81%) in F344/DuCrj rats surviving after NNM treatment. Metastasis to the lung was observed in HCC-bearing rats at an incidence of 13/15 (87%) in WS/Shi, 1/1 in SD/gShi, and 6/13 (46%) in F344/DuCrj. Four-week administration of NNM resulted in a significantly higher BrdU-labeling index of hepatocytes in WS/Shi rats than in the other strains. These findings indicated that WS/Shi is the most sensitive strain to NNM and may be the most suitable strain for use as a spontaneous metastatic model of HCC among the strains of rats examined in the present study.

Animals↗

PubMed1999

Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells.

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.

Animals↗

PubMed1999

Enhancement of amyloid beta 42 secretion by 28 different presenilin 1 mutations of familial Alzheimer's disease.

Families bearing mutations in the presenilin 1 (PS1) gene develop early onset familial Alzheimer's disease (FAD). Further, some PS1 mutants enhance secretion of the longer form of amyloid beta protein (Abeta42). We constructed cDNAs encoding human PS1 harboring 28 FAD-linked mutations, and examined the effects of the expressed PS1 mutants on Abeta42 secretion in beta amyloid precursor producing COS-1 cells. All the mutants significantly enhanced the ratio of Abeta42 to total Abeta compared with wild-type PS1. However, the increase in Abeta42 ratio in cells with each PS1 mutation did not correlate with the reported age of onset of FAD caused by that mutation. These results suggest that increased Abeta42 secretion is important for the development of Alzheimer's disease (AD), but may not be the only factor contributing to the onset of AD.

Age of Onset↗

PubMed1999

Impaired proteolytic processing of presenilin-1 in chromosome 14-linked familial Alzheimer's disease patient lymphocytes.

Many cases of early-onset familial Alzheimer's disease (FAD) are caused by mutations in the presenilin 1 (PS1) and PS2 genes. PS1 protein is generated as a 47 kDa protein and is endoproteolytically cleaved into N-terminal 28 kDa and C-terminal 19 kDa fragments in vivo. To examine whether mutated PS1 protein is abnormally metabolized, we performed immunoblot analysis of lymphoblasts from familial Alzheimer's disease patients and controls. More full-length PS1 was apparently detected in samples from PS1 mutants than those from PS2 mutant and controls. This result suggests that impaired proteolysis of PS1 may be associated with the pathogenesis of FAD. Moreover, our simple test using lymphocytes from FAD patients might be useful from a diagnostic point of view.

Alzheimer Disease↗

PubMed1999

Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium Frateuria species ANA-18.

The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA1 and catA2 encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA1 gene was clustered with catB1 encoding MC I, catC1 encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORFR1catB1C1D. The catA2 gene also constructed a gene cluster involving catB2 encoding MC II, catC2 encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORFR2catB2A2C2. The intergenic regions of ORFR1-catB1 and ORFR2-catB2 contained homologous sequences with the catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA1 and catA2 genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18.

Amino Acid Sequence↗

PubMed1999

Hepatocarcinogenic activity of N-butyl-N-(4-hydroxybutyl)nitrosamine in rats is not modified by sodium L-ascorbate.

Male F344 and Wistar Shionogi (WS) rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks and then killed at week 36 (experiment 1). Although reduction of body weight increase was found, no effects on liver weights were noted. Formalin-fixed and paraffin-embedded liver tissues from rats killed terminally were cut and stained for glutathione S-transferase placental form (GST-P) immunohistochemically. Marked elevation of quantitative values of small GST-P positive (GST-P+) foci were apparent in both strains of rat administered BBN. In experiment 2, both sexes of F344 rats were given 0.05% BBN in the drinking water for 4 weeks and then fed diet containing 0 or 5.0% sodium L-ascorbate (SA) for 32 weeks. No body and liver weight changes were evident in any group. Quantitative values for small GST-P+ foci were increased in both sexes of rats exposed to BBN but were not modified by additional SA treatment. Thus, it was confirmed that the selective bladder carcinogen BBN also acts as a liver carcinogen. These results, from the quantitative analysis of small GST-P+ foci as end point marker lesions, indicate that the liver tumor modifying potential of test chemicals can be evaluated in rats by using an initiation/promotion protocol for urinary bladder carcinogenesis.

Animals↗

PubMed1999

Dendritic cells in genetic immunization.

Both humoral and cellular immune responses are inducible by inoculation of naked plasmid DNA encoding a polypeptide antigen. This new vaccination protocol, known as genetic immunization, has been used to initiate protective immunity against a variety of infectious pathogens and tumors in experimental animals. Dendritic cells (DC) are thought to play at least three distinct roles in genetic immunization: (1) MHC class II-restricted presentation of antigens secreted by neighboring, transfected cells, (2) MHC class I-restricted "cross" presentation of antigens released by neighboring, transfected cells, and (3) direct presentation of antigens by transfected DC themselves. Several new technologies have been developed recently in an attempt to improve the overall efficacy of genetic vaccination, as well as to regulate the type and class of resulting immune responses. These technologies include modification of plasmid DNA, co-delivery of genes encoding immunoregulatory molecules, and DC targeting. We will overview some of these new technologies in genetic immunization.

Adjuvants, Immunologic↗