[A portable measurement system for automatic transcutaneous blood-glucose monitoring].
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Biosensing Techniques↗
Biomedical subjects
M Kikuchi
Publications and source records attributed to M Kikuchi.
Current status of PACAP as a regulator of insulin secretion in pancreatic islets.
PACAP-27 and PACAP-38 as low as 10(-13) M stimulate insulin release from rat islets in a glucose-dependent manner. PACAP also glucose dependently increases cAMP and [Ca2+]i in rat islet beta cells. The [Ca2+]i and insulin secretory responses to PACAP exhibit a similar concentration-response relationship, exhibiting a peak at 10(-13) M. When the [Ca2+]i response is abolished by nitrendipine, a blocker of L-type Ca2+ channels, the insulin response is also inhibited. Insulinotropic peptides glucagon, GLP-1, and VIP also increase [Ca2+]i in beta cells, but only in the nanomolar concentration range. PACAP is 4 logs more potent that VIP, a peptide that exhibits 68% amino acid homology and shares the type II PACAP receptor with PACAP. Immunoreactivity for the type I PACAP receptor is demonstrated in rat islets. Furthermore, PACAP immunoreactivity is demonstrated in nerve fibers and islets in rat pancreas. Based on these findings, we can draw the following conclusions: (1) PACAP is localized in pancreatic nerve fibers and islets; (2) PACAP in the subpicomolar range stimulates insulin release from islets; (3) the stimulation of insulin release is mediated by the cAMP-dependent increase in [Ca2+]i in beta cells; (4) all the PACAP effects are glucose-dependent; (5) PACAP is the most potent insulinotropic hormone known, and (6) the type I PACAP receptor appears to mediate the action of PACAP in the subpicomolar range. Finally, we hypothesize that PACAP is a pancreatic peptide of both neural and islet origin and functions as an intrinsic potentiator of glucose-induced insulin secretion in pancreatic islets (FIG 6).
Animals↗
Construction of biotinylated firefly luciferases using biotin acceptor peptides.
A cDNA for a thermostable mutant of Luciola lateralis (a Japanese firefly) luciferase was fused with either a gene for an artificial biotin acceptor peptide No. 84 [P. J. Schatz (1993) Bio/Technology 11, 1138-1143] or a gene for the carboxyl-terminal 87 residues of Escherichia coli biotin carboxyl carrier protein. The fused genes, when introduced into separate E. coli, directed the expression of luciferases that were able to bind with streptavidin. We purified them and showed that their specific activities and thermal stabilities remained unchanged. We also found that more than 95% of each fusion protein was biotinylated, suggesting that the biotin holoenzyme synthetase in the host cells worked efficiently. Using the biotinylated luciferases, we developed a highly sensitive bioluminescent enzyme immunoassay system.
Animals↗
Integrin-specific tissue-type plasminogen activator engineered by introduction of the Arg-Gly-Asp sequence.
To tailor tissue-type plasminogen activator (tPA) to possess an affinity for the integrins, several mutants were constructed by introducing the Arg-Gly-Asp (RGD) sequene into the tPA molecule. These mutants were expressed in COS-1 cells and partially purified by lysine-Sepharose chromatography. The RGD-dependent binding of the mutants to platelet integrin, integrin alpha IIb beta 3, was evaluated by subtracting the nonspecific binding in the presence of 10 mM EDTA (or 1 mg/ml GRGDSP). The binding assay showed that two tPA mutants possess high affinity for the integrin in an RGD-dependent manner. One mutant is 148RGD-tPA with RGDS in place of DRDS (residues 148 to 151) in the loop region of the kringle 1 domain of tPA, and the other is 270RGD-tPA with RGDS in place of SQPQ (residues 270 to 273) in the linker region between the kringle 2 and protease domains. Using the chromogenic substrate Spectrozyme tPA, the 148RGD-tPA mutant was shown to possess amidolytic activity comparable with that of native tPA, while the 270RGD-tPA mutant exhibited several-fold lower activity. In addition, the 148RGD-tPA exhibited full tPA activity even when interacting with the integrin alpha IIb beta 3. These results suggest that the bifunctional 148RGD-tPA molecule might be useful as an improved thrombolytic agent specific for the platelet integrin, the integrin alpha IIb beta 3.
Amino Acid Sequence↗
Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae.
We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64,000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30,000 and 33,000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300,000.
Amino Acid Sequence↗
Epithelioid sarcoma with an 18q aberration.
Epithelioid sarcoma is a peculiar soft-tissue neoplasm of uncertain origin, which is characterized by an epithelioid morphology of tumor cells coexpressing epithelial (keratin) and nonepithelial (vimentin) antigens. We herein report a new cytogenetic abnormality with der(22)t(18;22)(q11;p11.2) in a case of epithelioid sarcoma that occurred in the elbow of a 75-year-old man. Histologically, the tumor demonstrated a multinodular proliferation of epithelioid cells, with positive immunostaining for keratin, epithelial membrane antigen (EMA), and vimentin. Cultured tumor cells obtained from fresh surgical materials were frozen in plastic ampules and stocked in a liquid nitrogen freezer. Six years after surgery, the cells were recovered from the freezer and utilized for both morphologic and cytogenetic analyses. These cultured cells both before and after the freezing exhibited essentially the same epithelioid morphology and immunophenotypes as those of the original tumor. A chromosome analysis, together with fluorescence in situ hybridization (FISH), demonstrated a 61-67 modal population, and a characteristic clonal abnormality with der(22)t(18;22)(q11;p11.2). Other clonal abnormalities included numerical (-3, -4, +7, -13, -14, -16, -18, +20, -22) and structural (8p+, 9p+, 12p+, i(21q)) aberrations. Some variant clones also demonstrated i(18q). Since the breakpoint at 18q11 is similar to that reported in synovial sarcoma, this finding may support the presence of a histogenetic relationship between epithelioid sarcoma and synovial sarcoma. Our study thus indicates that the storage of frozen cells is useful for both morphologic and cytogenetic analyses of soft tissue tumors.
Aged↗
Lack of the t(2;5) or other mutations resulting in expression of anaplastic lymphoma kinase catalytic domain in CD30+ primary cutaneous lymphoproliferative disorders and Hodgkin's disease.
The t(2;5) (p23;q35) chromosomal translocation has been found in a high proportion of lymph node-based CD30+ large cell lymphomas of T-cell lineage. This translocation is believed to result in the expression of a fusion protein containing the catalytic domain of anaplastic lymphoma kinase (ALK) under the control of the promoter for nucleophosmin, a nucleolar phosphoprotein. Expression of ALK activity, which does not normally occur in lymphocytes, is postulated to be involved in the pathogenesis of lymphomas bearing the t(2;5) translocation. Several primary cutaneous lymphoproliferative disorders and Hodgkin's disease are also known to contain CD30+ large lymphoid cells. To determine the role of the t(2;5) translocation in these diseases, we developed a DNA-based polymerase chain reaction (PCR)/Southern blot assay to detect this translocation at the genomic level in lymphomatoid papulosis (14 cases), primary cutaneous CD30+ large cell lymphoma of T-lineage (10 cases) and Hodgkin's disease (13 cases). Two cases of pityriasis lichenoides were also studied. The t(2;5) translocation was not present in any of these specimens. To determine if some other somatic mutation might have resulted in inappropriate expression of ALK catalytic domain, we devised an RNA-based reverse transcriptase-PCR assay to detect transcripts encoded by this ALK region. None were found in the six additional cases of lymphomatoid papulosis that were studied. In aggregate, these results strongly suggest that inappropriate expression of ALK is not involved in the pathogenesis of these CD30+ lymphoproliferative disorders, and that lymph node-based CD30+ large cell lymphoma is a disease that is biologically distinct from skin-based CD30+ lymphoproliferative disorders and Hodgkin's disease. Using methods developed for this report, we also cloned and sequenced the t(2;5) genomic junctional sequences present in the SUP-M2 and SU-DHL-1 cell lines. These intron sequences will be useful for mapping t(2;5) breakpoint clusters.
Adult↗
63/65Cu/203/205Tl NMR study on the antiferromagnetic phase of the Tl-based high-Tc oxide TlBa2YCu2O7.
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Journal Article↗
Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.
To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.
3T3 Cells↗
Possible involvement of C5/C5a in the efferent and elicitation phases of contact sensitivity.
The elicitation of 24-h contact sensitivity (CS) in mice requires a serotonin-dependent, 2-h response called CS initiation. We studied the role of complement (C) by comparing CS in DBA/1 (C5-normal) vs DBA/2 (C5-deficient) mice and found impaired 2-h, but not 24-h, CS. We showed previously that 2-h responses represent CS initiation and are required for elicitation of 24-h responses. Treatment of C5-deficient mice with absent macroscopic responses (ear swelling) with a selective serotonin antagonist inhibited 24-h CS, suggesting that C5-deficient mice had submacroscopic (i.e., microscopic), serotonin-dependent CS initiation. When normal mouse serum was used as a source of C5 to reconstitute C5-deficient mice, significant 2-h responses were restored. Furthermore, heat treatment of normal mouse serum to inactivate C abrogated restoration of 2-h responses. Thus, C5 was suggested to be involved in CS initiation. Using a suboptimal immunizing dose of Ag revealed an impaired 24-h component of CS in DBA/2 mice, but not in DBA/1 mice, and also in C5-deficient B10.D2/o mice compared with C5-normal B10.D2/n mice with a suboptimal eliciting dose of Ag. Again, reconstitution of B10.D2/o mice with normal mouse serum restored deficient 24-h CS responses. Thus, 2-h and classical 24-h CS probably depend in part on C5. These results imply that C5 may play a role in the elicitation of 24-h CS, probably via required preceding CS initiation.
Animals↗
Detection of human T-lymphotropic virus type-I DNA and mRNA in the lymph nodes; using polymerase chain reaction in situ hybridization (PCR/ISH) and reverse transcription (RT-PCR/ISH).
To examine the relationship between human T-lymphotrophic virus type I (HTLV-I) proviral DNA and its expression in the lymph nodes, HTLV-I DNA and tax/rex mRDA were directly amplified by polymerase chain reaction in situ hybridization (PCR/ISH), and reverse transcription (RT)-PCR/ISH [RT-PCR/ISH]. We studied 24 lymph nodes from patients with adult T-cell leukemia/lymphoma (ATLL), incipient ATLL (I-ATLL), and HTLV-I associated lymphadenitis dermatopathic type (HAL-D) and enlarged paracortical type (HAL-EP). In ATLL, 40-60% of the nucleated cells were positive for for HTLV-I proviral DNA by PCR/ISH, while in I-ATLL and HAL, respectively 5-20% and less than 1-5% of cells were positive. The number of mRNA expressing cells was smaller than that of the proviral DNA-positive cells. The mRNA-expressing cells varied in number among the ATLL and I-ATLL cases, while they were only rarely observed in HAL-D and HAL-EP. These results show that HTLV-I infection and activation might increase with malignant transformation of the target T helper cells.
Adult↗
Dual actions of nitric oxide in N-methyl-D-aspartate receptor-mediated neurotoxicity in cultured retinal neurons.
This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) receptor-mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. The NOS activity measured by monitoring the conversion of [3H]arginine to [3H]citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. The concomitant addition of an inhibitor of NOS, Nomega-nitro-L-arginine (300 microM), with glutamate or NMDA reduced cell death by 70%. A brief exposure of the cells to sodium nitroprusside (SNP, 500 microM) or S-nitrosocysteine (SNOC, 500 microM), NO-generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA, or NO generating agents. Fifty microM SNOC alone had no effect on the cell viability. However, pretreatment with 50 microM SNOC as well as simultaneous application of 50 microM SNOC with NMDA inhibited cell death induced by NMDA. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO, interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.
Animals↗
Random suppression of T cells that bear specific T cell receptor V beta sequences in adult T cell leukemia/lymphoma (ATLL) patients at each clinical stage: carrier, smoldering, chronic, and acute.
Human T cell leukemia virus type I (HTLV-I) is associated with adult T cell leukemia/lymphoma (ATLL), which is well known as a T cell malignancy. In order to clarify whether HTLV-I plays a role as a virus-encoded superantigen in the neoplastic process, we examined the TCR V beta families in the peripheral blood at four different clinical stages: carrier, smoldering leukemia, chronic leukemia, and acute leukemia. An increased number of CD4 T cells was found in each of the four clinical stages. However, we found neither uniform specific losses nor uniform clonal expansion of particular TCR V beta gene families in any case from the four clinical stages. However, a suppression of the random TCR V beta families was found. Our data did not therefore directly suggest the existence of a common superantigen model of HTLV-I which induces an increase in CD4 T cells. The random suppression in the TCR V beta repertoire is most likely caused by the influence of HTLV-I neoplastic pathogenesis rather than by virus-encoded superantigens. In the patients with acute leukemia, one or two families of the V beta repertoires were very strongly expressed, while in chronic leukemia, no such repertoire of strong expression was observed. The immunological reaction of the hosts might thus be different between the above described groups.
Acute Disease↗
Epstein-Barr virus infection and associated products (LMP, EBNA2, vIL-10) in nodal non-Hodgkin's lymphoma of human immunodeficiency virus-negative Japanese.
Sixty cases of B-cell nodal non-Hodgkin's malignant lymphoma (B-ML), and 46 cases of T-cell nodal lymphoma (T-ML) were surveyed for Epstein-Barr virus (EBV) genomes, RNA, and associated proteins. We used a Southern blot analysis, polymerase chain reaction (PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the presence of EBV. We performed an immunohistochemical study on EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 (EBNA-2), latent membrane protein (LMP), and viral interleukin-10 (vIL-10). In addition, we also analyzed the terminal repetitive sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin's lymphomas were grouped into three types by number of EBV-infected cells: I) almost all lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed an EBV presence; and III) only a few cells showed such a presence, which was probably due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infected cells were found; 7 were type I, 1 was type II, and 17 were type III. In 27 of 46 T-MLs, EBV-infected cells were found; no cases were type I, 5 cases were type II, and 22 cases were type III. Seven B-MLs and 3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-2/LMP-positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was detected in almost all lymphoma cells; in T-MLs, however, LMP was found in only a small portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In summary, it was thus speculated that EBV infection was associated with the various states of lymphomagenesis.
Antigens, Viral↗
Hemorrhage from abdominal non-Hodgkin's lymphoma treated successfully by emergency transcatheter arterial embolization.
A 49-year-old Japanese woman with follicular lymphoma who presented with severe abdominal and back pain is reported. She was known to have malignant lymphoma and had been previously treated with combination chemotherapy. An abdominal tumor occurring at the root of the mesentery and involving the superior mesenteric artery (SMA) had been diagnosed by computed tomography (CT), magnetic resonance imaging, and abdominal angiography. Emergent ultrasonography and CT findings showed intraperitoneal bleeding from the abdominal tumor. Selective SMA angiography revealed extravasation from a small branch originating from the dorsal pancreatic artery, which was embolized through a catheter by using platinum coils. It should be noted that a large tumor of malignant lymphoma, involving large vessels, may bleed, and in such a case selective transcatheter arterial embolization may be one of the effective modalities for hemostasis.
Abdominal Neoplasms↗
Detection of human T lymphotrophic virus type I (HTLV-I) DNA and mRNA in individual cells by polymerase chain reaction (PCR) in situ hybridization (ISH) and reverse transcription (RT)-PCR ISH.
Human T lymphotrophic virus type I (HTLV-I) proviral DNA and mRNA in the blood obtained directly from HTLV-I infected adult T cell leukemia (ATL) patients were amplified by the polymerase chain reaction (PCR), and reverse transcription (RT)-PCR, and then were hybridized to fluorescein-labelled probes by means of in situ hybridization (ISH). Before the cytospin samples were prepared, heterogenous cell populations were reproducibly resolved into HTLV-I-positive and -negative distributions. Immunohistochemical staining was performed, using anti-fluorescein monoclonal antibody. Microscopic observations demonstrated a preserved cellular morphology. The intranuclear localization of amplified DNA products of proviral HTLV-I by PCR/ISH, and intracytoplasmic localization of amplified DNA of HTLV-I tax/rex mRNA by RT-PCR/ISH were maintained. In this study, about one in 10 HTLV-I provirus integrated cells expressed low copies of tax/rex mRNA. In HTLV-I-negative cell lines, amplified DNA was not observed by either PCR/ISH or RT-PCR/ISH. With the use of this technique it is thus possible to detect single-copy DNA and a few copies of mRNA, and it is therefore possible to study, not only suspended materials, but also other tissue materials for further characterization, in association with the localization of the HTLV-I infected cells.
DNA, Viral↗
Classical Hodgkin and Reed-Sternberg cells demonstrate a non-clonal immature B lymphoid lineage: evidence from a single cell assay and in situ hybridization.
Hodgkin and Reed-Sternberg (H & RS) cells are generally considered to be the neoplastic cells of Hodgkin's disease (HD), however such cells are only found in a minority of the lesions. Recently in a few studies on HD, the clonality of H & RS cells was examined, using a single-cell polymerase chain reaction (PCR) examination. To clarify the lineage and clonality of H & RS cells, we performed single cell PCR and in situ hybridization (ISH), and nine cases of classical HD were thus studied. By ISH, the immunoglobulin J chain, and the kappa and lambda light chain were rarely expressed in the H & RS cells, however, no T-cell markers could be detected. The expression of the recombination activating genes (RAG-1, 2) could be determined in the H & RS cells. We isolated CD30+ H & RS cells, CD3 + T cells and CD20 + B cells from suspended materials using a mechanical sorter. We performed single cell PCR in a sorted individual cell, to amplify the complementarity determining region of the Ig heavy chain (IgH) gene and T-cell receptor gamma chain (TCR gamma) gene. In all cases, TCR gamma could be frequently amplified in the T cells, but was only rarely amplified in the H & RS and B cells. In contrast, the IgH was frequently amplified in the H & RS and B cells, but not in the T cells. In addition, the PCR production of the H & RS cells all showed different lengths. The results therefore support the polyclonal nature and immature B lymphoid cell origin of H & RS cells.
B-Lymphocyte Subsets↗
Tissue polypeptide antigen production in a uterine carcinosarcoma cell line in a serum-free culture.
Carcinosarcomas (malignant mixed Mullerian tumors, MMMT) of the female genital tract are uncommon neoplasms, most of which show a highly aggressive behavior. However, the biological characteristics of MMMT, especially the producibility of tumor-related substances in vitro, have yet to be clarified. Using a serum-free culture model system which was newly established from an FU-MMT-2 cell line originating from an MMMT of the human uterus, the biological and immunocytochemical characteristics of MMMT cells were examined in vitro. As a control model, the uterine fibroblasts were also cultured in the same conditions. Moreover, several tumor markers were also measured by a radioimmunoassay in the serum of five patients with MMMT. FU-MMT-2 produced and secreted tissue polypeptide antigen (TPA), a tumor marker closely related to the keratin family, under serum-free culture conditions. Moreover, TPA was also immunocytochemically demonstrated in carcinoma cells as well as sarcoma cells in FU-MMT-2. On the other hand, the uterine fibroblasts showed no detectable producibility of the substances examined in vitro. An elevation of the serum TPA levels was also detected in four of five (80%) patients with MMMT. This is the first serum-free culture study of MMMT, which appears to provide a useful model for further studies of the tumor biology in MMMT of the female genital tract. An evaluation of the serum TPA level thus appears to be useful in determining the optimum management of patients with MMMT. In addition, the demonstration of TPA in sarcoma cells of FU-MMT-2 in vitro supports both the theory of a single cell origin and the current concept of MMMT as a "metaplastic carcinoma," as substantiated in our previous study.
Aged↗