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T Isobe

Publications and source records attributed to T Isobe.

At least 19 recordsLinked to original sources
PubMed1995

Primary structure of a gamma subunit of G protein, gamma 12, and its phosphorylation by protein kinase C.

We have determined the primary structure of a novel gamma subunit (gamma 12, previously designated gamma S1) of G protein purified from bovine spleen. The mature gamma 12 protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from gamma 12 cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against gamma 12 showed that gamma 12 is present in all tissues examined. Among various gamma subunits (gamma 1, gamma 2, gamma 3, gamma 7, and gamma 12), gamma 12 has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser1 (or Ser2). The phosphorylated beta gamma 12 associated with Go alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of gamma 12. Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of gamma 12. The extent of phosphorylation of beta gamma 12 in vitro was suppressed by a complex formation with Go alpha, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that gamma 12 is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.

Amino Acid Sequence↗
PubMed1995

Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase.

The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

14-3-3 Proteins↗
PubMed1995

Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes.

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

3T3 Cells↗
PubMed1995

Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb.

In the two-stage model of controlling cellular senescence in cultured human fibroblasts, retinoblastoma (Rb) and p53 proteins may be key factors regulating the mortality stage 1 mechanism. In addition, the critical loss of telomeric DNA due to the end-replication problem may result in the mortality stage 2 mechanism. Cells which acquire telomerase activity can overcome the M2 mechanism by stabilizing telomere length and thus become immortal (telomere hypothesis). At present it is known whether cellular immortality is a prerequisite for all human cancers. To investigate this question and the applicability of the two-stage model to human cancers, we analysed the relationship between alterations of telomere length and other genetic changes in lung cancer. Among 60 primary lung cancer tissues, telomere length alterations were observed in 16 tumors (26.7%) including 14 with short and two with elongated telomeres. Ten of them revealed allelic loss of both p53 and Rb genes, and remaining six showed no abnormalities in both genes. We propose that inactivation of both p53 and Rb genes may promote cell divisions causing telomere shortening in lung cancer as in the two-stage model, while there may be another pathway to overcome both M1 and M2 mechanisms, especially for adenocarcinoma.

Base Sequence↗
PubMed1995

Combination assay of urinary beta-core fragment of human chorionic gonadotropin with serum tumor markers in gynecologic cancers.

Ectopic production of the immunoreactive beta-subunit of human chorionic gonadotropin (IR-hCG beta) by gynecologic malignancies has been well recognized, but IR-hCG beta has not yet been established as a clinically useful tumor marker, except for germ cell tumors. We measured the concentrations of IR-hCG beta-related molecules, intact hCG, free hCG beta, and beta-CF, in the sera and urine of patients with various gynecologic cancers (cervical, endometrial, and ovarian cancers) to assess their clinical usefulness as a tumor marker in comparison with serum tumor markers such as CEA, SCC, CA125, and CA19-9. The highest incidence of IR-hCG beta was obtained in the assay for beta-CF in the urine, with positive rates of 47.7% (94 of 197) for cervical, 37.8% (14 of 37) for endometrial, and 84.4% (38 of 45) for ovarian cancers with a cut-off value of 0.2 ng/mg of creatinine. In cervical cancer, there was no significant correlation between the concentrations of urinary beta-CF and serum SCC, and 57.9% (114 of 197) of the patients were detected by the combination assay of these tumor markers. Serial determination in 22 cervical cancer patients with elevated urinary beta-CF level prior to therapy showed that its level decreased after successful treatment, but 4 of 5 patients with persistent or recurrent disease had elevated levels of urinary beta-CF. All of the ovarian cancer patients examined were detected by the combination assay of urinary beta-CF and serum CA125. The levels of urinary beta-CF showed little correlation with those of the serum tumor markers, indicating the usefulness of the combination assay of urinary beta-CF with serum tumor markers for detecting cervical and ovarian cancers.

Biomarkers, Tumor↗
PubMed1995

Establishment of a myeloid leukaemic cell line (SKNO-1) from a patient with t(8;21) who acquired monosomy 17 during disease progression.

A novel cell line SKNO-1 was established from the bone marrow cells of a 22-year-old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 17 monosomy. SKNO-1 has been maintained for more than 36 months as a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent line. Morphologically, SKNO-1 cells were myeloblasts somewhat matured. The cells grow in suspension with a doubling time of 48-72 h. The survival and growth of SKNO-1 cells was absolutely dependent on granulocyte-macrophage colony stimulating factor (GM-CSF). SKNO-1 cells possessed t(8;21) and monosomy 17 which were observed in original leukaemic cells. We confirmed that the AML1 gene, located on chromosome 21, was rearranged and the AML1-MTG8 fusion transcript was expressed in SKNO-1 cells. Over-expression and mutation of the p53 gene were also detected in SKNO-1. It is likely that alterations of AML1 or MTG8 gene and p53 gene contribute to a disease progression in this case. Since t(8;21) translocation is a common chromosome abnormality in AML, and inactivation of the p53 gene may play a crucial role in disease progression in AML, SKNO-1 would be a useful tool for analysing the molecular mechanisms in myeloid leukaemogenesis.

Adult↗
PubMed1995

[Application of molecular diagnosis to human lung cancer].

Recent developments in the molecular genetics of human cancers shows the importance of multiple genetic alterations in the pathogenesis of these lesions. DNA diagnostic techniques are being introduced rapidly into the clinical laboratory setting. 1) In lung cancer, several oncogenes and tumor suppressor genes, such as ras, myc, p53, RB, allelic loss of chromosomes, play very important roles. These genetic changes are being applied to cancer diagnosis, prediction of prognosis or disease metastasis, or response to treatment. 2) Drug resistance is one of the major problems of current lung cancer chemotherapy. Identification of the molecular marker for drug resistance, like DNA topoisomerase gene mutation, in clinical samples will be of great help for choosing chemotherapy regimens. 3) Interindividual differences in susceptibility to lung cancer may be screened using genotyping of the P450IA1 and GSTmu genes. To develop newer diagnostic and therapeutic approaches, detailed investigation of the molecular pathogenesis of lung cancer using clinical samples is essential. I review the present status on these applications of genetic markers to lung cancer diagnosis in this article.

DNA Topoisomerases, Type I↗
PubMed1995

[AL amyloidosis].

AL amyloidosis is one type of systemic amyloidosis. AL represents Amyloid of Light chains (of Ig's) type. Diagnosis of amyloid must be carried out by patho-histological Congo-red stains of the biopsied materials under suspicion of amyloid deposits from patients with various clinical manifestations, including macroglossia, cardiac abnormalities, ECG abnormalities, GI tract problems or carpal-tunnel syndrome. Al amyloidosis may be also termed as plasma cell dyscrasia (PCD) associated amyloidosis, or monoclonal gammopathy associated amyloidosis. As to classification, AL amyloidosis consists of primary (or idiopathic) amyloidosis, and myeloma-associated (and then macroglobulinemia-associated) amyloidosis. As similarly to myeloma, patients with AL amyloidosis have monoclonal Ig's in the serum and-or in the urine. Furthermore, there have been known to be a higher incidence of Bence Jones proteins as well as more lambda than kappa among patients with AL amyloidosis compared with distributions of monoclonal Ig's among myeloma patients. It is also important for clinicians to recognize a data of a median survival of 12 months (only 4 months if cardiac amyloid occurred) in all patients with AL amyloid VS, a data of a median survival of about 36 months in myeloma patients. An attempt to clarify a way to an early diagnosis of AL amyloidosis should be investigated.

Amyloidosis↗
PubMed1994

A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.

A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons.

Aging↗
PubMed1994

Synergistic activation by Ras and 14-3-3 protein of a mitogen-activated protein kinase kinase kinase named Ras-dependent extracellular signal-regulated kinase kinase stimulator.

We have identified, in Xenopus oocyte cytosol, a protein kinase named REKS (Ras-dependent extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) stimulator), which phosphorylates and activates recombinant ERK2 through recombinant MEK in a recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ras-dependent manner. We show here that this REKS activity is synergistically enhanced by a combination of mammalian recombinant GTP gamma S-KiRas and 14-3-3 protein purified from rat brain. 14-3-3 protein is known to activate tyrosine and tryptophan hydroxylases, to modulate the protein kinase C activity, to stimulate secretion, and to show phospholipase A2 activity per se. 14-3-3 protein did not affect the MEK activity. 14-3-3 protein neither interacted with Ki-Ras nor affected the neurofibromin activity to stimulate the GTPase activity of Ki-Ras under the conditions where the recombinant N-terminal fragment of c-Raf-1 inhibited it. These results suggest that 14-3-3 protein has an additional function in the regulation of the Ras-MEK-ERK cascade pathway through the activation of REKS.

14-3-3 Proteins↗
PubMed1994

Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH-EP)

A plant cysteine endopeptidase, designated SH-EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies. Here we show that the 43 kDa intermediate of SH-EP formed in the endoplasmic reticulum is transported to protein bodies and processed to the 33 kDa mature form during transport or thereafter, and that the COOH-terminal propeptide of 10 amino acid residues containing a KDEL sequence, which is known as a retention signal for the endoplasmic reticulum lumen, is processed to form the mature SH-EP.

Amino Acid Sequence↗
PubMed1994

Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate determination proteins.

We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells.

Amino Acid Sequence↗
PubMed1994

Covalent structure of bovine brain calreticulin.

The covalent structure of bovine brain calreticulin, a major Ca(2+)-binding protein in the lumen of the endoplasmic reticulum, was determined by analysis of the purified protein. The protein consisted of 400 amino acids, with an N-linked oligosaccharide attached to the polypeptide chain. The polypeptide sequence determined was compatible with the sequence of calreticulin deduced from cDNA of different sources, with a number of differences presumably due to species-specific amino acid substitutions. The protein retained the C-terminal tetrapeptide, KDEL, involved in retention of proteins resident in the endoplasmic reticulum, whereas the N-terminal signal peptide predicted from the cDNA sequence had been removed in the purified protein. The bovine brain protein contained a high-mannose type of oligosaccharide attached to Asn162, which is typical of resident endoplasmic reticulum proteins. The carbohydrate moiety was heterogeneous and had the composition GlcNAc2Man4-9, of which GlcNAc2Man5 was the most abundant in the bovine brain preparation. Glycosylation of calreticulin, however, appeared to be a species-specific modification, as Asn162 is replaced by Asp in the sequences already determined for a number of species. Analysis of the purified protein also identified an intramolecular disulphide bridge between Cys120 and Cys146.

Amino Acid Sequence↗
PubMed1994

A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues.

Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 omega, one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 omega activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 omega binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 omega does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.

14-3-3 Proteins↗
PubMed1994

Plasma cell leukemia with myelofibrosis.

We describe a case of plasma cell leukemia associated with myelofibrosis. A 60-year-old woman was admitted due to lumbago and monoclonal hypergammaglobulinemia. Peripheral blood showed about 40% of plasma-cell-like cells. A bone marrow aspiration was dry tap. The patient was diagnosed as having plasma cell leukemia with myelofibrosis by bone marrow biopsy. Plasma cell leukemia as well as myelofibrosis improved with combination chemotherapy using vincristine, pirarubicin, and dexamethasone. However, when plasma cell leukemia became resistant to chemotherapies, myelofibrosis also reappeared. This case strongly suggests the pathogenetic relationship between plasma cell leukemia and myelofibrosis.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Analysis of schizont antigen of Leucocytozoon caulleryi by SDS-PAGE and western blot.

To investigate the molecular weight of the schizont antigen of Leucocytozoon caulleryi, the schizont antigen was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Thirty to 35 protein bands were detected by SDS-PAGE under reduced condition and ranged from 25 to 300 kDa. Most of them reacted with the sera of infected chickens by Western blot. The bands corresponding to polypeptides of 44, 58, 67 and 180 kDa reacted strongly, however, the bands of 30, 50 and 78 kDa did not react.

Animals↗