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T Isobe

Publications and source records attributed to T Isobe.

At least 109 records · Page 6Linked to original sources
PubMed1997

14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain.

Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with 32P-labeled recombinant IRS-1 and obtained two isoforms (epsilon and zeta) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

14-3-3 Proteins↗
PubMed1997

The 14-3-3 protein binds its target proteins with a common site located towards the C-terminus.

The 14-3-3 protein family binds a variety of proteins in cell-signaling pathways, but the structural elements necessary for the ligand binding are poorly understood. Here we demonstrate that the 'box-1' region, which spans residues 171-213 in the eta-isoform and was previously identified as the binding site of 14-3-3 to the phosphorylated tryptophan hydroxylase, plays a critical role in the interaction with many target proteins. Using a series of truncated 14-3-3 mutants, we show that the mutant 167-213 carrying box-1 binds bacurovirus-expressed Raf-1 and Bcr protein kinases to the similar extent as the full-length 14-3-3 in a phosphorylation-dependent manner, while the mutants lacking this region abolish the binding activity. Furthermore, the box-1 region also appears essential for binding of 14-3-3 to more than 40 phosphoproteins found in the brainstem extract. These results suggest that the box-1 region, consisting of helices 7 and 8 in the tertiary structure, is a common structural element whereby the 14-3-3 protein binds many, if not all, target proteins.

14-3-3 Proteins↗
PubMed1997

Expression of alpha and beta genes of human chorionic gonadotropin in lung cancer.

To confirm the ectopic production of human chorionic gonadotropin (hCG) in lung cancer, we attempted to detect the presence of mRNA transcripts of the alpha and beta genes for hCG in lung cancer tissues obtained from surgical operations. Although we were able to show the presence of hCG beta mRNA transcripts in lung cancer tissue by Northern blot, the sensitivity of the assay was too low for a precise analysis of hCG beta mRNA transcripts in most lung cancers. Using reverse transcription PCR (RT-PCR) and Southern blot analysis, however, various amounts of mRNA transcripts of hCG beta genes 3, 5, 7 and 8 were demonstrated in 9 of the 14 lung cancer tissues examined, while no mRNA transcripts were detectable in 12 normal lung tissues from the same patients. Our results are consistent with a clear difference in serum and urinary hCG beta levels observed between normal subjects and lung cancer patients. The expression of the hCG alpha gene, however, was detected in normal lung tissues more frequently than in lung cancer tissues using RT-PCR Southern blot. Our results strongly suggest the production of hCG beta as being part of the phenotype of malignantly transformed lung cells and further strengthen its superior specificity over intact hCG or hCG alpha as a tumor marker for lung cancers.

Aged↗
PubMed1997

Propolypeptide of von Willebrand factor is a novel ligand for very late antigen-4 integrin.

We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) promotes melanoma cell adhesion in a beta1 integrin-dependent manner. In this report, we identified the alpha subunit of the cell adhesion receptor for pp-vWF as alpha4. Human leukemia cell lines that express alpha4beta1 integrin (very late antigen-4, VLA-4), but not cell lines which lack VLA-4, attached well to pp-vWF substrate and these adhesions were completely inhibited by anti-alpha4 integrin monoclonal antibody HP2/1. Adhesion of mouse melanoma expressing alpha4 integrin was also inhibited by anti-mouse alpha4 mAb PS/2. Furthermore, transfection of human alpha4 cDNA into alpha4(-) Chinese hamster ovary cells resulted in an acquisition of adhesive activity to pp-vWF, indicating that pp-vWF is a ligand for VLA-4 integrin. Using a recombinant fragment of pp-vWF, the cell attachment site was shown to be located within amino acid residues 376-455 of pp-vWF. A series of synthetic peptides covering this region were tested for the ability to promote cell attachment and a 15-residue peptide designated T2-15 (DCQDHSFSIVIETVQ, residues numbered 395-409) promoted VLA-4 dependent cell adhesion. The peptide was also capable of inhibiting cell adhesion to pp-vWF, suggesting that this sequence represents the cell attachment site. By affinity chromatography using peptide T2-15-Sepharose, it was found that alpha4beta1 integrin complex from extracts of surface iodinated B16 cells specifically bound to the peptide. These results strongly suggest that pp-vWF is a novel physiological ligand for VLA-4.

Animals↗
PubMed1997

Salivary drug monitoring of irinotecan and its active metabolite in cancer patients.

To assess the clinical usefulness of salivary monitoring of irinotecan (CPT-11) and its active metabolite (SN-38), we examined the clinical pharmacological profile of both drugs in 9 patients with thoracic malignancies who received 60 mg/m2 CPT-11 (21 courses). Plasma and unstimulated whole saliva were collected over a 24-h period, and concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography. Both CPT-11 and SN-38 were detectable in saliva, and the concentration-time curves in plasma and saliva showed a very similar pattern. A good correlation was observed between the saliva concentration (C3) and the plasma concentration (Cp) for both CPT-11 and SN-38 (r = 0.732, P < 0.0001 and r = 0.611, P < 0.0001, respectively). The area under the concentration-time curve calculated for saliva (AUCs) correlated with that generated for plasma (AUCp) for both CPT-11 and SN-38 (r = 0.531, P = 0.012 and r = 0.611, P = 0.0025, respectively). These results suggest that it may be feasible to use saliva instead of plasma for pharmacokinetics/pharmacodynamics studies of CPT-11.

Aged↗
PubMed1997

Structural interlock between ligand-binding site and stalk-like region of beta1 integrin revealed by a monoclonal antibody recognizing conformation-dependent epitope.

Integrin activation and sebsequent ligand binding to it are regulated by intracellular mechanisms called inside-out signaling, which are not fully understood and are accompanied by dynamic structural changes of the integrin molecule itself. A monoclonal antibody recognizing a conformation-dependent epitope on human beta1 integrin was produced and characterized in detail. This antibody, AG89, reacted with human integrin beta1 chain regardless of the alpha subunit. AG89 can recognize resting state beta1 integrin on the cells, but the reactivity is increased approximately 2-fold upon integrin activation by activating anti-beta1 antibodies and approximately 3-fold by Mn2+. Furthermore, occupation of the ligand-binding pocket by a soluble ligand (RGD peptide for alpha(v)beta1 and CS-1 peptide for alpha4beta1) resulted in maximum binding of AG89, indicating that the epitope for AG89 is exposed during the conformational changes of beta1 integrin upon activation/ligation. Epitope mapping by using interspecies chimeric beta1 revealed that the epitope for AG89 lies within residues 426-587, which corresponds to the cysteine-rich repeat structure located in the middle of the beta1 chain. The fact that binding of AG89 itself could activate the resting beta1 integrin indicates that exposure of the AG89 epitope in the membrane-proximal stalk-like domain and "opening" of the ligand-binding pocket at the outermost domain are physically linked. We propose that the integrin "signaling" is mediated by this direct physical transduction of conformational information along the integrin molecule.

Antibodies, Monoclonal↗
PubMed1997

Pathogenesis of reduced egg production and soft-shelled eggs in laying hens associated with Leucocytozoon caulleryi infection.

The pathogenesis of reduced egg production with soft-shelled eggs in laying hens naturally infected with Leucocytozoon caulleryi was investigated. Many large schizonts of L. caulleryi schizonts were seen in the ovary and oviducts of chickens. Granulomatous and lymphocytic inflammation, edema, and pressure atrophy were associated with these schizonts. The uterine region that secretes the egg shell exhibited the most severe damage. These lesions in the reproductive organs may explain the mechanism for causing the reduced egg production and soft-shelled eggs in laying hens infected with L. caulleryi.

Animals↗
PubMed1997

Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction.

A method was developed for the discrimination of 8 Eimeria species of chickens, i.e., E. acervulina, E. brunetti, E. mitis, E. maxima, E. necatrix, E. praecox, E. tenella, and E. hagani using the 2-step polymerase chain reaction (PCR). In the first PCR, the small subunit ribosomal RNA (srRNA) gene was amplified from the parasite genome using conserved sequences for the Apicomplexa srRNA gene as the primers. The srRNA gene amplified from the parasite genome was discriminated in the second step by random-amplified polymorphic DNA (RAPD) PCR using 10 arbitrary primers. Each arbitrary primer produced species-specific RAPD patterns that provided a simple method for species identification from the srRNA genes of the 8 Eimeria species. This method should be useful for discrimination of the parasite species for diagnosis or epidemiological surveys of chicken coccidiosis.

Animals↗
PubMed1996

Structural organization and chromosomal assignment of the human 14-3-3 eta chain gene (YWHAH).

14-3-3 protein, a brain-specific protein, is thought to be a multifunctional protein involved in the activation of tyrosine and tryptophan hydroxylases, the inhibition or activation of protein kinase C, and the activation of signal transduction. The human 14-3-3 eta chain gene was isolated and its structure was determined. It is composed of two exons separated by one long intron (approximately 8 kb) and spans about 10 kb. A transcription initiation site was identified by a combination of S1 nuclease mapping, primer extension analysis, and RACE methods. In the 5'-flanking region, we found four GC box sequences, four anti-GC box sequences, a TATA box-like sequence, CAAT box-like sequences, a C/EBP element, two AP-2 sequences, an AP-3 sequence, an Oct-6-like sequence, six E boxes, and a CRE sequence. FISH with DNA probes of the human 14-3-3 eta chain gene mapped the 14-3-3 eta chain gene to chromosome 22q12.1-q13.1.

14-3-3 Proteins↗
PubMed1996

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Application to cerebellar protein mapping.

A method is presented for the structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 x 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.

Amino Acid Sequence↗
PubMed1996

A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride membrane after thin-layer chromatography blotting and in situ mass spectrometric analysis of the ligands.

A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride (PVDF) membrane has been developed. Glycosphingolipids were separated on a high-performance thin-layer chromatography (HPTLC) plate and transferred onto a PVDF membrane by TLC blotting [Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221, 312-316]. The PVDF membrane was blocked with phosphate-buffered saline containing 4% casein and 0.1% methionine and overlaid with 35S-labeled Escherichia coli possessing K99 fimbriae (E. coli K99) at 37 degrees C for more than 2 h. Binding of the 35S-labeled E. coli K99 was detected with a bioimaging analyzer. Radioactivities were located on the bands corresponding to N-glycolylneuraminic acid containing glycosphingolipids such as sialylparagloboside, GM2, and GM3 with hydroxy fatty acid in ceramide moiety, and a weak binding was detected on the band of N-acetylneuraminylparagloboside. Furthermore, an in situ mass spectrometric analysis of the ligand glycosphingolipids on the membrane was demonstrated. The present method has several advantages compared with the overlay binding assay on the HPTLC plate as follows: (i) the method is simple and rapid; (ii) the membrane is easy to handle; (iii) binding is clear with low background; (iv) a small amount of [35S]methionine is required; and (v) sensitive ligand characterization can be done by in situ mass spectrometric analysis.

Bacterial Adhesion↗
PubMed1996

High-performance liquid chromatography of peptides on a microspherical carbon column.

The properties of a microspherical carbon column for the separation of peptides and proteins are described. One hundred and thirty-three peptides, with from 1 to 148 amino acid residues, were applied on a carbon column and eluted with a linear gradient of acetonitrile [10-70% (v/v), 30 min] in 0.1% (v/v) trifluoroacetic acid solution. The elution behaviour of the peptides on the carbon column and on an octadecylsilica (ODS) column were compared under the same elution conditions. Relationships between the logarithm of the hydrophobicity of peptides and their capacity factors were approximately linear on both columns, but some exceptions were observed with peptides with aromatic residues, indicating stronger adsorption on the carbon than on the ODS column. These results suggest that the major factor for the separation of peptides on both the carbon and ODS columns is hydrophobic interactions. The stronger adsorption of aromatic side-chains in peptides on the carbon column could be understood by an interaction based on the aromatic or graphitic nature of the surface of the microspherical carbon packings.

Amino Acid Sequence↗
PubMed1996

Reversed-phase liquid chromatography on a microspherical carbon column at high temperature.

The chromatographic conditions for the use of a spherical carbon column at high temperatures up to 160 degrees C are described. The column heating system and the eluate cooling system are also described. Under the conditions studied, it was found that the carbon column at elevated temperature was stable and could be applicable to any compounds that are durable under these conditions. The peptide bonds at this high temperature and at this acidity were still stable enough for analysis, but the indole residues showed poor stability.

Amino Acid Sequence↗
PubMed1996

Ultrastructural evidence for colocalization of kappa light chain- and beta 2-microglobulin-derived amyloids using double labelling immunogold electron microscopy.

In systemic amyloidosis, it is essential to decide what type of amyloid protein is deposited in tissues before the therapy can be selected and the prognosis assessed in each patient. We examined tissues affected by amyloid deposition from a patient with multiple myeloma by immunohistochemistry and double labelling immunogold electron microscopy and demonstrated colocalization of kappa light chain- and beta 2-microglobulin-derived amyloids in the same tissue. beta 2-Microglobulin-derived amyloid had ultrastructurally characteristic features that distinguished it from kappa light chain-derived amyloid. This is the first report of the colocalization of two different amyloids by immunoelectron microscopy.

Amyloid↗
PubMed1996

Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation.

We examined the gene expression of V-1, a novel soluble protein with the cdc10/SWI6 motif, in pseudopregnant rat ovaries. Northern blot analysis on days 1, 5, and 11 of pseudopregnancy revealed an approximately 2-fold increase in the V-1 messenger RNA (mRNA) expression level on day 5 to that on day 1, and no significant change was observed between those on day 5 and day 11. An injection of human CG on days 5 further increased th V-1 mRNA level to about 1.6-fold of that of the untreated control. Western blot analysis showed higher V-1 protein expression on days 5 and 11 of pseudopregnancy than that on day 1. In situ hybridization and immunohistochemistry with ovaries on day 3 of pseudopregnancy showed that luteal cells of corpora lutea and also cells of the coexisting follicles including oocytes express V-1 mRNA and the protein, with apparent rank order of the expression: oocytes > luteal cells > follicular cells >> atretic follicular cells including oocytes. These data indicate the dynamic change in the V-1 gene expression in the ovarian steroidogenic cells and oocytes and suggest potential roles of the V-1 protein in ovarian functions including corpus luteum formation and folliculogenesis.

Animals↗