Biomedical subjects

T Itoh

Publications and source records attributed to T Itoh.

At least 73 records · Page 4Linked to original sources

PubMed2001

Rectal immunization with antigen-containing microspheres induces stronger Th2 responses than oral immunization: a new method for vaccination.

The rectum as an effective site for induction of systemic and local immunity has received little attention. Rectal immunization with microspheres-containing ovalbumin (MS-OVA) was tested for its ability to elicit systemic and mucosal immune responses. Rectal immunization with MS-OVA enhanced both Th2 dominant OVA-specific IgG levels in the serum and OVA-specific IgA levels in fecal extracts more prominently than did oral immunization. Cytokine analysis of CD4(+) T cells indicated a predominant induction of Th2-type responses compared to Th1-type responses following rectal immunization compared to oral immunization. These results demonstrate that rectal immunization with microspheres could be an effective new vaccination method.

Administration, Oral↗

PubMed2001

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Amino Acid Sequence↗

PubMed2001

Phosphoinositides, key molecules for regulation of actin cytoskeletal organization and membrane traffic from the plasma membrane.

Phosphoinositide plays a critical role not only in generating second messengers, such as inositol 1,4,5-trisphosphate and diacylglycerol, but also in modulating a variety of cellular functions including cytoskeletal organization and membrane trafficking. Many inositol lipid kinases and phosphatases appear to regulate the concentration of a variety of phosphoinositides in a specific area, thereby inducing spatial and temporal changes in their availability. For example, local concentration changes in phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to extracellular stimuli cause the reorganization of actin filaments and a change in cell shape. PI(4,5)P(2) uncaps the barbed end of actin filaments and increases actin nucleation by modulating a variety of actin regulatory proteins, leading to de novo actin polymerization. PI(4,5)P(2) also plays a key role in membrane trafficking processes. In endocytosis, PI(4,5)P(2) targets clathrin-associated proteins to endocytic vesicles, leading to clathrin-coated pit formation. On the contrary, PI(4,5)P(2) must be dephosphorylated when they shed clathrin coats to fuse endosome. Thus, through regulating actin cytoskeleton organization and membrane trafficking, phosphoinositides play crucial roles in a variety of cell functions such as growth, polarity, movement, and pattern formation.

Actin Cytoskeleton↗

PubMed2001

Abnormal regulation of DDB2 gene expression in xeroderma pigmentosum group E strains.

A damage-specific DNA binding protein (DDB) activity is absent from a subset (DDB(-)) of cells from individuals initially classified as group E of xeroderma pigmentosum (XP), a hereditary, photosensitive disease with a high incidence of skin malignancies. In these cases, mutations have been identified in the DDB2 gene (DDB2(-)) that codes for the small subunit, p48, of the DDB heterodimer. In four DDB2(- )strains, neither p48 nor DDB activity were observed before or after UV-irradiation, despite an unusually strong up-regulation of DDB2 mRNA levels after UV-irradiation. In a fifth strain, XP82TO, p48 was detectable and both DDB2 mRNA and p48 levels were more up-regulated after UV-irradiation than in normal primary cells. Moreover, DDB activity also became apparent after irradiation. XP82TO showed very mild clinical manifestations compared with the other DDB(-) patients. These results, coupled with our findings that most, if not all DDB(+) cells classified as XP-E were misclassified, suggests a direct correlation between DDB2 levels and the XP-E phenotype.

Cells, Cultured↗

PubMed2001

Synthesis of a muscarinic receptor antagonist via a diastereoselective Michael reaction, selective deoxyfluorination and aromatic metal-halogen exchange reaction.

An efficient synthesis of a structurally unique, novel M(3) antagonist 1 is described. Compound 1 is conveniently disconnected retrosynthetically at the amide bond to reveal the acid portion 2 and the amine fragment 3. The synthesis of key intermediate 2 is highlighted by a ZnCl(2)-MAEP complex 19 catalyzed diastereoselective Michael reaction of dioxolane 7 with 2-cyclopenten-1-one (5) to establish the contiguous quaternary-tertiary chiral centers and a subsequent geminal difluorination of ketone 17 using Deoxofluor in the presence of catalytic BF(3).OEt(2). The synthesis of the amine moiety 3 is highlighted by the discovery of a novel n-Bu(3)MgLi magnesium-halogen exchange reaction for selective functionalization of 2,6-dibromopyridine. This new and practical metalation protocol obviated cryogenic conditions and upon quenching with DMF gave 6-bromo-2-formylpyridine (26) in excellent yield. Further transformations afforded the amine fragment 3 via reductive amination with 35, Pd-catalyzed aromatic amination, and deprotection. Finally, the highly convergent synthesis of 1 was accomplished by coupling of the two fragments. This synthesis has been used to prepare multi-kilogram quantities of the bulk drug.

Amides↗

PubMed2001

Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity.

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

Amino Acid Sequence↗

PubMed2001

Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines.

Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.

Acid Phosphatase↗

PubMed2001

Oral absorption of cephalosporins is quantitatively predicted from in vitro uptake into intestinal brush border membrane vesicles.

In order to establish a method to predict oral absorption of drugs, which are absorbed by the oligopeptide transporter (PepT1), fraction absorbed (F) of cephalosporin antibiotics was predicted from in vitro uptake into rat intestinal brush border membrane vesicles (BBMV). Using in vitro uptake data, F values of cephalosporins in humans were predicted using the equation derived from the complete radial mixing (CRM) model, which was proposed by Amidon et al. (Amidon et al., J. Pharm. Sci. 69 (1980) 1369). In the present study, uptake into BBMV was measured at 25 and 4 degrees C in the presence of an H+ -gradient, and the uptake clearance (CLuptake) was calculated. Clearance for the uptake mediated by PepT1 (DeltaCLuptake) was then calculated as CLuptake at 25 degrees C minus that at 4 degrees C. When DeltaCLuptake and F values were analyzed according to the present equation, fairly good correlation between DeltaCLuptake and F was observed. It was further demonstrated that the present method may be able to quantitatively predict F values of cephalosporins by using several cephalosporins as standards.

Absorption↗

PubMed2001

Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

Animals↗

PubMed2001

Stereoselective binding and degradation of sulbenicillin in the presence of human serum albumin.

Binding of sulbenicillin (SBPC) isomers to human serum albumin (HSA) was stereoselective. There were at least two classes of binding sites on HSA for SBPC isomers. At the stereoselective high affinity site, binding was in favor of R-SBPC, the binding constant of R-SBPC being approximately 2.3-fold greater than that of S-SBPC. By using site marker ligands, it was revealed that the stereoselective site was Site I (warfarin binding site). Affinity for the low affinity (nonstereoselective) site was similar for the diastereomers, approximately 7--30-fold lower than for the stereoselective site. R-SBPC and S-SBPC appeared to displace each other competitively at both binding sites. On the other hand, R-SBPC was degraded much faster than S-SBPC in the presence of HSA, with a degradation rate constant approximately 7-fold greater for R-SBPC than for S-SBPC. The degradation of R-SBPC was inhibited in the presence of warfarin and dependent on the concentration of R-SBPC bound to Site I. The results demonstrate that Site I is responsible for the stereoselective degradation.

Anti-Bacterial Agents↗

PubMed2001

AT oligonucleotides inducing B lymphocyte activation exist in probiotic Lactobacillus gasseri.

This study determined oligonucleotide sequences of mitogenic DNA derived from lactic acid bacteria (LAB). The chromosomal DNA, which was purified from 12 out of 16 strains of Lactobacillus acidophilus group LAB, induced proliferation of splenic lymphocytes. When DNA from L. gasseri JCM1131T was cloned and amplified using PCR, the mitogenic activities of B lymphocytes were significantly increased by 108 of 321 DNA clones. Ten high homologous nucleotide sequences were found as possible DNA sequences of mitogens, and were then chemically synthesized (sOL-LG1 to sOL-LG10). Two nucleotide sequences (sOL-LG7 and sOL-LG10) that consist of only A and T nucleotides (AT oligonucleotides) were characterized as B lymphocyte specific mitogens because they resulted in proliferation of B lymphocytes but not of T lymphocytes. sOL-LG7 preferentially bound to large B lymphocytes and enhanced the expression of the CD86 antigen more than the CD69 antigen on B lymphocytes. The findings show that mitogenic AT oligonucleotides are likely to restrict pre-activated subsets of B lymphocytes. This study demonstrated that novel AT oligonucleotides triggering B lymphocyte mitogenic responses exist in the nucleoids of L. gasseri and proposed that they have potential as applicants for the production of new functional foods, "Bio-Defense Foods".

Animals↗

PubMed2001

Redox-sensitive transactivation of epidermal growth factor receptor by tumor necrosis factor confers the NF-kappa B activation.

Cross-communication between different signaling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. In this paper we have explored the possibility that tumor necrosis factor (TNF) receptor signal cross-talks with epidermal growth factor (EGF) receptor signal on the nuclear factor-kappa B (NF-kappa B) activation pathway. We have demonstrated that overexpression of the EGF receptor (EGFR) in NIH3T3 cells significantly enhances TNF-induced NF-kappa B-dependent luciferase activity even without EGF, that EGF treatment has a synergistic effect on the induction of the reporter activity, and that this enhancement is suppressed by AG1478, EGFR-specific tyrosine kinase inhibitor. We also have shown that TNF induces tyrosine phosphorylation and internalization of the overexpressed EGFR in NIH3T3 cells and the endogenously expressed EGFR in A431 cells and that the transactivation by TNF is suppressed by N-acetyl-l-cysteine or overexpression of an endogenous reducing molecule, thioredoxin, but not by phosphatidylinositol 3-kinase inhibitors and protein kinase C inhibitor. Taken together, this evidence strongly suggests that EGFR transactivation by TNF, which is regulated in a redox-dependent manner, is playing a pivotal role in TNF-induced NF-kappa B activation.

Animals↗

PubMed2001

Gene organization of bovine BCNT that contains a portion corresponding to an endonuclease domain derived from an RTE-1 (Bov-B LINE), non-LTR retrotransposable element: duplication of an intramolecular repeat unit downstream of the truncated RTE-1.

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion. The 3' UTR of bovine bcnt cDNA also contains an approximately 300-bp portion homologous to the 3'-part of RTE-1. We examined the bovine bcnt genomic DNA sequence to understand how the bovine bcnt gene has been organized. The sequence of 3' UTR homologous portion was found to more closely resemble the Art2 element than the bovine RTE-1. By PCR screening a bovine/hamster hybrid somatic cell panel, the bovine bcnt gene was mapped to chromosome 18, syntenic human chromosome 16q on which human BCNT is located. The bcnt genomic DNA sequence corresponding to the cDNA downstream of a RTE-1 derived portion reveals that each IR unit is flanked by both 5'-side and 3'-side introns and that 3'-UTR consists of one exon. The alignment of the above sequence with a bovine RTE-1 did not show any significant homology downstream of the endonuclease domain. On the other hand, the alignment of the intron sequences with each other revealed that the six sequential homologous segments ranging in size from 40 to 453 bp existed over a 1 kb long sequence between both the 5'- and 3'-side introns flanking each bovine IR unit. In addition, both the 174-bp of 5'-side intron and 80-bp of 3'-side intron neighboring each 120-bp IR exon are significantly homologous among the two bovine IRs, human IR and mouse IR. These results suggest that a truncated bovine RTE-1 was inserted into the intron upstream of an IR unit of an ancestor bcnt gene and that a duplication of a relatively long region that includes IR occurred in the bovine genome.

3' Untranslated Regions↗

PubMed2001

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo.

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.

Animals↗

PubMed2001

Initiation of polyoma virus origin-dependent DNA replication through STAT5 activation by human granulocyte-macrophage colony-stimulating factor.

Several lines of evidence indicate that transcriptional activation is coupled with DNA replication initiation, but the nature of initiation of DNA replication in mammalian cells is unclear. Polyoma virus replicon is an excellent system to analyze the initiation of DNA replication in murine cells because its replication requires an enhancer, and all components of replication machinery, except for DNA helicase large T antigen, are supplied by host cells. This system was used to examine the role of signal transducer and activator of transcription (STAT5) in replication initiation of polyoma replicon in the mouse lymphoid cell line BA/F3. The plasmid with tandem repeats of consensus STAT5 binding sites followed by polyoma replication origin was replicated by stimulation with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in the presence of polyoma large T antigen in BA/F3 cells. Mutation analysis of the hGM-CSF receptor beta subunit revealed that only the box1 region is essential, and the C-terminal tyrosine residues are dispensable for the activity. Addition of the tyrosine kinase inhibitor genistein suppressed this replication without affecting transcriptional activation of STAT5. Because deletion analysis of STAT5 indicates the importance of the C-terminal transcriptional activation domain of STAT5 for the initiation of replication, the role of this region in the activation of replication was examined with a GAL4-STAT5 fusion protein. GAL4-STAT5 activated replication of the plasmid containing tandem repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells. Mutation analysis of GAL4-STAT5 indicated that multiple serine residues coordinately have a role in activating replication. This is the first direct evidence indicating the potential involvement of STAT5 in replication.

Animals↗

PubMed2001

cDNA cloning and expression of swine IL-7 from neonatal intestinal epithelium.

Swine IL-7 (sIL-7) cDNA has been isolated from neonatal intestinal epithelium by reverse transcription polymerase chain reaction (RT-PCR) with designated primers based on the conserved sequences of the other mammalian IL-7. Recombinant sIL-7 was able to induce the bone marrow cell proliferation like human IL-7. The complete open reading frame contains 531 bp coding deduced 176 amino acid residues, with a calculated molecular mass of 20.1 kDa. The observed similarity between swine and human IL-7 sequences (66% identical and 89% conserved) suggests that a study on the immune responses correlated with IL-7 in the gut of swine will be valuable to understand their mechanisms in human. By RT-PCR using the constructed specific primers to sIL-7, sIL-7 mRNA was confirmed to be expressed in swine intestine, thymus, kidney and skin but not in the heart.

Amino Acid Sequence↗

PubMed2001

Down-regulation of promoter 1.3 activity of the human aromatase gene in breast tissue by zinc-finger protein, snail (SnaH).

Aromatase (estrogen synthetase) is expressed in breast cancer tissue, and in situ expression of the enzyme stimulates breast cancer growth. Promoter I.3 is one of the major promoters that control the expression of aromatase in breast cancer tissue. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that the zinc-finger transcriptional factor Snail (SnaH) interacted with a regulatory region near promoter I.3 of the human aromatase gene. DNA mobility shift assays and mutation analyses using recombinant SnaH protein expressed in Escherichia coli have revealed that this protein interacts with a segment, 5'-CTGATGAAGT-3', which is between 66 and 76 bp upstream from the transcriptional start site of promoter I.3. Using mammalian cell transfection experiments, SnaH was found to act as a repressor of promoter I.3 activity. Site-directed mutagenesis experiments have revealed that the NH2-terminal SNAG domain is important for the repressor activity of SnaH. To demonstrate the inhibitory activity against aromatase expression, a stable SnaH-expressing MDA-MB-231 breast cancer cell line was generated, and the aromatase RNA messages in the SnaH-transfected cell line were found to be 30% of those in the vector-transfected cell line. Reverse transcription-PCR analysis on RNAs isolated from 12 cell lines has confirmed that SnaH is expressed at a higher level in normal breast epithelial cell and stromal fibroblast cell lines than in breast cancer cell lines. In addition, SnaH mRNA was detected in only 16 of 55 breast cancer specimens. On the other hand, aromatase mRNA was detected in 54 of the 55 specimens. Our results indicate that SnaH acts as a repressor that down-regulates the expression of aromatase in normal breast tissue by suppressing the function of promoter I.3. A reduction of the expression of SnaH in breast cancer tissue further suggests a cancer-protective role for this protein in normal breast tissue.

Amino Acid Sequence↗

PubMed2001

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.

Adaptor Proteins, Vesicular Transport↗