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Biomedical subjects

M Murakami

Publications and source records attributed to M Murakami.

At least 757 records · Page 42Linked to original sources
PubMed1993

Histidyl-proline diketopiperazine. Novel formation that does not originate from thyrotropin-releasing hormone.

We asked whether cyclo(His-Pro) may arise from a substance other than the initially found progenitor thyrotropin-releasing hormone (TRH). Incubation of TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor, with brain hypothalamic cytosols caused significant formation of His-Pro-Gly followed by the generation of histidyl-proline diketopiperazine (cyclo(His-Pro)), whereas TRH was not formed. The addition of purified pyroglutamyl aminopeptidase resulted in the transformation of TRH-Gly to His-Pro-Gly and cyclo (His-Pro) but not to TRH. Neither TRH nor cyclo(His-Pro) was produced from TRH-Gly in the presence of alpha-amidating enzyme under the present conditions. Nonenzymatic conversion of His-Pro-Gly to cyclo(His-Pro) occurred. The present results provide evidence that cyclo(His-Pro) can emanate from a direct predecessor form of TRH by pyroglutamyl aminopeptidase action, not through TRH formation.

Amino Acid Sequence↗
PubMed1993

The bcl-2 gene product inhibits clonal deletion of self-reactive B lymphocytes in the periphery but not in the bone marrow.

To test whether the product of the bcl-2 proto-oncogene blocks clonal deletion of self-reactive B cells, we have generated transgenic mice carrying the bcl-2 gene and the immunoglobulin genes for the anti-erythrocyte 4C8 antibody. In these transgenic mice, clonal deletion of self-reactive immature B cells in the bone marrow was not inhibited in spite of expression of the bcl-2 gene. In contrast, self-antigen-induced clonal deletion of mature self-reactive Ly-1 B (B1) cells in the peritoneal cavity was inhibited in the transgenic mice. These results indicate that the mechanism for clonal deletion of immature self-reactive B cells in the bone marrow differs from that of mature self-reactive B cells in the periphery.

Animals↗
PubMed1993

Pituitary adenomas of patients with acromegaly express thyrotropin-releasing hormone receptor messenger RNA: cloning and functional expression of the human thyrotropin-releasing hormone receptor gene.

We cloned a human thyrotropin-releasing hormone receptor (TRH-R) gene and its pituitary cDNA, and examined whether TRH-R mRNA may be expressed and function in the GH-secreting pituitary adenomas of patients with acromegaly. The human TRH-R consists of 398 amino acids and is a member of the G protein-coupled receptor family. The gene has a single intron in the coding sequence. An addition of TRH caused an obvious increase in intracellular calcium concentration in chinese hamster ovary cells expressing this receptor. Although highly homologous to the rat and mouse TRH-Rs, the human TRH-R has several differences in the carboxyl-terminal structure. The sequence and functioning analyses showed the same expression of TRH-R mRNA in the pituitary adenomas as that in the normal pituitaries, indicating that 1) some adenomas of patients with acromegaly express an intact and functional TRH-R, and that 2) the paradoxical response of GH secretion to TRH found in the patients might be a direct effect of TRH on TRH-R expressed in the adenoma.

Acromegaly↗
PubMed1993

An enzyme immunoassay for carcinoembryonic antigen (CEA) with homogeneous reactivity to different CEA preparations and low cross-reactivity with CEA-related normal antigens.

A new sandwich-type solid-phase enzyme immunoassay (EIA) for carcinoembryonic antigen (CEA) was established by using two selected monoclonal antibodies (MAbs). One MAb, F82-61, which is immobilized on polystyrene beads, reacts with an epitope present on the N-terminal domain (N) of the CEA molecule and the other, F11-39, which is conjugated to horseradish peroxidase (HRP), recognizes an epitope present on the C-terminal domain (B3) of the CEA molecule. The assay consists of incubating 0.05 ml of test serum both with F82-61-coated beads and HRP-labeled F11-39 at room temperature for 2 h. The assay system, referred to as New EIA, showed very homogeneous reactivity with purified CEA preparations from different tumors and could discriminate CEA from four CEA-related normal antigens tested; nonspecific cross-reacting antigen (NCA), NCA-2, normal fecal antigen-1 (NFA-1) and NFA-2. The assessment of technical qualities, such as reproducibility, recovery and dilution tests, demonstrated the excellent performance of New EIA. Serum samples (n = 604) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by New EIA and by two commercial CEA immunoassays which both showed heterogeneous reactivity with different CEA preparations and reacted to a greater or lesser extent with NCA-2 and NFA-2, and it was found that New EIA significantly increased the sensitivity and specificity of tumor diagnosis as compared with the commercial assays.

Animals↗
PubMed1993

IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase.

The biological functions of interleukin-6 (IL-6) are mediated through a signal-transducing component of the IL-6 receptor, gp130, which is associated with the ligand-occupied IL-6 receptor (IL-6R) protein. Binding of IL-6 to IL-6R induced disulfide-linked homodimerization of gp130. Tyrosine kinase activity was associated with dimerized but not monomeric gp130 protein. Substitution of serine for proline residues 656 and 658 in the cytoplasmic motif abolished tyrosine kinase activation and cellular responses but not homodimerization of gp130. The IL-6-induced gp130 homodimer appears to be similar in function to the heterodimer formed between the leukemia inhibitory factor (LIF) receptor (LIFR) and gp130 in response to the LIF or ciliary neurotrophic factor (CNTF). Thus, a general first step in IL-6-related cytokine signaling may be the dimerization of signal-transducing molecules and activation of associated tyrosine kinases.

Antigens, CD↗
PubMed1993

An analysis of intracellular 23Na relaxation using the double-quantum filtered NMR signal from the perfused rat salivary gland.

The intracellular sodium of the perfused rat mandibular salivary gland was measured by double-quantum filtered 23Na-NMR spectroscopy at 2.34, 4.7 and 8.45 T. Biexponential relaxation of the intracellular 23Na signal was observed, and its intensity was increased by administration of acetylcholine with ouabain at 25 degrees C. The transverse and longitudinal relaxation rate constants were determined by the 'transverse experiment' (D-90 degrees-tau/2-180 degrees-tau/2-90 degrees-delta-90 degrees-acquire) and the 'longitudinal experiment' (D-180 degrees-tau-54.7 degrees-delta-90 degrees-acquire), respectively. From observed dependencies on B0 and temperature (5-37 degrees C), a possibility of exchange between two populations of intracellular Na+ was suggested. A small fraction of Na+ is in the slow-motion condition (with a quadrupole coupling constant of approx. 1.75 MHz and a correlation time of 6 x 10(-8) s). The major portion of intracellular Na+ is in the extreme narrowing condition with a transverse relaxation rate constant of approx. 100 s-1, which corresponds to a viscosity of approx. 5 cP.

Animals↗
PubMed1993

Biological consequences of overexpression of a transfected c-erbB-2 gene in immortalized human bronchial epithelial cells.

In order to examine the effects of the overexpression of c-erbB-2 (HER-2, neu) on human bronchial epithelial cells, a human c-erbB-2 expression vector was introduced into the simian virus 40 large T-antigen-immortalized human bronchial epithelial cell line BEAS-2B. Isolation of multiple clonal cell lines after selection revealed a wide range of expression of the gene product gp185erbB-2. While three of six clones tested expressed gp185erbB-2 at levels detectable by immunocytochemistry, only one, B2BE6, induced adenocarcinoma-like tumors in athymic nude mice. Both a nontumorigenic clone, B2BE2, and a tumorigenic clone, B2BE6, expressed comparable amounts of gp185erbB-2, which became phosphorylated on tyrosine in response to treatment with the c-erbB-2 ligands gp30 and p75. These data suggest that overexpression of c-erbB-2 in human bronchial epithelial cells can contribute to, but is not sufficient for, induction of tumorigenicity in this human model system.

Animals↗
PubMed1993

[Clinical utility of Gd-DTPA subtraction MR imaging for spinal bone metastasis].

Based on reports that Gd-DTPA contributes to the detection of tumors, we used it in 31 cases (97 lesions) of spinal bone metastases. The result was that Gd-DTPA increased the intensity of tumors and the surrounding bone marrow to almost the same level in 53%. To show the metastases clearly, an existing subtraction command system was utilized. The technique included the pixel-by-pixel method, to obtain a Gd-DTPA T1-weighted image (T1WI) subtracted by the original T1WI. The detectability of the subtraction image was improved up to 96%, but was less than the original T1WI (99%). Because of the different imaging rationale between two methods, a means to assess the quality of diagnosis must be proposed. To check the normal background, the same kind of postprocessing was performed in 21 patients without malignancy. Gd-DTPA perfusion was highest in the paravertebral veins, moderate in muscles and epidural fat, and lowest in the spinal cord, intervertebral disk and bone cortex. Gd-DTPA enhanced subtraction MR imaging provides a new diagnostic tool to detect and to assess bone metastasis.

Adult↗
PubMed1993

Possible role of mammalian secretory group II phospholipase A2 in T-lymphocyte activation: implication in propagation of inflammatory reaction.

Both 2-lysophosphatidylcholine and cis-unsaturated fatty acids were previously shown to intensify agonist-induced cellular responses by enhancing the diacylglycerol-dependent activation of protein kinase C. Consistent with these observations, extracellular, secretory group II phospholipase A2, when added directly to human resting T lymphocytes, greatly potentiates their activation that was induced by a membrane-permeant diacylglycerol and ionomycin, as determined by the expression of the alpha subunit of the interleukin 2 receptor and thymidine incorporation into DNA. Diacylglycerol and ionomycin were essential for this cellular response, and phospholipase A2 alone showed no effect. The amount of 2-lysophosphatidylcholine produced in these cells by the exogenous phospholipase A2 was greatly increased in the presence of diacylglycerol and ionomycin, suggesting that the membrane phospholipids became susceptible to the phospholipase A2 when protein kinase C was activated. The results suggest that phospholipase A2 secreted into inflammatory sites plays a role in the propagation of cellular responses. Protein kinase C may function in the hydrolysis of membrane phospholipids by the exogenous phospholipase A2, and the products of this phospholipid hydrolysis enhance agonist-induced protein kinase C activation, thereby intensifying cellular responses.

Cell Membrane Permeability↗
PubMed1993

Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2.

A lysate of unstimulated human umbilical vein endothelial cells (HUVEC) exhibited phospholipase A2 (PLA2) activity, which hydrolyzed phospholipids bearing arachidonate more preferentially than those bearing linoleate at the sn-2 position. An anti-rabbit cytosolic PLA2 monoclonal antibody absorbed the activity, whereas an anti-human type II PLA2 monoclonal antibody did not. HUVEC treated with thrombin generated prostaglandin I2 (PGI2), and the PLA2 activity of the thrombin-stimulated cells was absorbed almost completely by the anti-cytosolic PLA2 antibody. HUVEC treated with tumor necrosis factor (TNF) also generated PGI2. PGI2 generation by TNF-treated cells was suppressed partially by extracellular addition of the anti-type II PLA2 antibody. PLA2 activity in a lysate of TNF-stimulated cells was increased about 2-3-fold, and about half of the increased activity was suppressed by the anti-type II PLA2 antibody. Addition of heparin together with TNF resulted in release of type II PLA2 in the medium. Thus, both cytosolic and type II PLA2s may be involved in agonist-stimulated PGI2 synthesis in HUVEC. Furthermore, exogenously added type II PLA2 was bound to the cell surface and synergistically enhanced PGI2 generation in TNF-stimulated HUVEC. This binding was blocked by either heparin or a monoclonal antibody recognizing the heparin-binding domain of type II PLA2. Taken together, type II PLA2 generated endogenously as well as added exogenously may be captured on the HUVEC surface via heparan sulfate proteoglycan and may contribute to cellular arachidonate metabolism.

Antibodies, Monoclonal↗
PubMed1993

Structure-function analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130.

Here, we report the analysis of the structure-function relationship of the extracellular region of human interleukin 6 receptor (IL-6R). Upon binding of IL-6, IL-6R becomes associated extracellularly with a non-IL-6-binding but signal transducing molecule, gp130, and the IL-6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin-like domain, was responsible both for IL-6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL-6R (sIL-6R) could bind IL-6 and mediate IL-6 functions through gp130, amino acid substitutions were introduced into sIL-6R by site-directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL-6 binding have a tendency to be distributed to the hinge region between the two 'barrel'-like fibronectin type III modules and to the same side of these two 'barrels'. Amino acid residues, of which substitutions barely affected the IL-6-binding but did abolish the IL-6 signalling capability of sIL-6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL-6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL-6.

Amino Acid Sequence↗
PubMed1993

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands.

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In 1-day-old chickens, three types of C cells were distinguished on the basis of their granule size. Type-I cells were filled with large secretory granules (200-600 nm in diameter). These elements represented a majority of the C-cell population. Type-II cells contained medium-sized granules (100-280 nm in diameter). Type-III cells displayed small secretory granules (60-200 nm in diameter). The latter cells were elongate or irregular in shape and frequently extended cytoplasmic processes into the connective tissue stroma or contacted other C cells. Enkephalin-immunoactivity was revealed by dense deposits of immunogold particles on the secretory granules of type-II and type-III cells. There were only a few type-I cells showing immunoreactivity for enkephalin. A double immunogold labeling procedure demonstrated that calcitonin and enkephalin were colocalized in the same secretory granules of type-I and type-II cells. Type-III cells were devoid of immunoreactivity for calcitonin. Enkephalin-immunoreactive nerve fibers were characterized by the presence of granular vesicles, 60-160 nm in diameter, and frequently established direct contact with the surface of C cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around beta-turn potentials: data-based prediction.

Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins. By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these substrate specificities of trypsin, L-lactate dehydrogenase, aspartate aminotransferase, beta-lactamase, and cytochrome P-450 are found to exist within regions predicted as beta-turns. The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations. Inhibitory specificities of bovine pancreatic trypsin inhibitor and alpha 1-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures. Occurrence of beta-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme. These findings will have relevance to work on protein engineering and enzyme evolution.

Amino Acid Sequence↗
PubMed1993

(14C-methyl)-L-methionine uptake in rat brain tumors before and after treatment with the protein synthesis inhibitor cycloheximide.

Autoradiographic study of (14C-methyl)-L-methionine with brain tumor bearing rats aimed at an elucidation of the mechanism of tracer accumulation in the protein synthesis of tumor. Twice as much tracer accumulated in the tumors compared as in the contralateral gray matter (nontumor region) at 90 min post intravenous injection. The protein-bound fraction of the tumors, expressed as acid-insoluble fraction (AIF), was 1.7 +/- 0.6 (mean +/- standard deviation, n = 6), significantly higher than that (0.8 +/- 0.2) of the nontumor region (p < 0.05 by the Mann-Whitney test). The tumor AIF comprised 82.3 +/- 9.2% of the total amount of the tracers accumulated in the tumors. The protein synthesis inhibitor cycloheximide reduced the tracer uptake and the AIF of the tumors to an almost same level as the nontumor region. These findings indicate that metabolic acceleration of protein synthesis may be a main reason for the high accumulation of (14C-methyl)-L-methionine in tumor.

Animals↗