[Mechanism of morphological changes of platelets upon exposure to cold].
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Blood Platelets↗
Biomedical subjects
K Miyazaki
Publications and source records attributed to K Miyazaki.
Roles of Arg231 and Tyr284 of Thermus thermophilus isocitrate dehydrogenase in the coenzyme specificity.
The coenzyme binding site of isocitrate dehydrogenase from Thermus thermophilus was analyzed by site-directed mutagenesis. The mutation analysis revealed that Arg231 and Tyr284 are involved in the discrimination between NAD and NADP, suggesting that these two residues interact with 2'-phosphate group of NADP.
Amino Acid Sequence↗
Identification of one- and two-chain forms of trypsinogen 1 produced by a human gastric adenocarcinoma cell line.
It has previously been reported that two kinds of human gastric adenocarcinoma cell lines (STKM-1 and MKN28) secrete a trypsin-like enzyme. In this study, four molecular forms of the enzyme (26, 25, 24 and 23 kDa on non-reducing SDS/PAGE) were purified from the serum-free conditioned medium of STKM-1 cells. Analysis of N-terminal amino acid sequences showed that the 26 kDa protein was a two-chain form of trypsinogen 1 which had been produced by proteolytic cleavage of the Arg107-Val108 bond of trypsinogen 1, and the 24 kDa protein was the one-chain form of trypsinogen 1. The 25 and 23 kDa proteins were the activated forms of the two-chain and one-chain trypsinogen 1 respectively. Isoelectric focusing gave pI values of 6.3 and 6.6 for the 26 kDa two-chain form and the 24 kDa one-chain form of trypsinogen 1 respectively. Comparison of the proteolytic activities indicated that the one-chain trypsin 1 had amidolytic activity about four times higher than that of the two-chain enzyme.
Adenocarcinoma↗
Effect of membrane surface potential on the uptake of anionic compounds by liposomes.
The effect of membrane surface potential on the uptake of several anionic compounds by liposomes (large unilamellar vesicles), which contain various amounts of dipalmitoylphosphatidylserine (DPPS), was investigated. The uptake amount of four tested anionic compounds (cefixime, benzyloxyindoleacetic acid (BOIAA), ceftibuten and S-1006) decreased with an increase in the DPPS content of liposomes, and was correlated with the membrane surface potential monitored using a fluorescent dye, 8-anilino-1-naphthalene sulfonate (ANS). Moreover, for all of the tested anionic compounds, a good correlation was observed between the ratio of the uptake value (5 min) by each of the liposomes comprising various amounts of DPPS to the uptake value by liposomes containing 10% DPPS and a relative membrane surface potential monitored by ANS. On the other hand, the uptake of zwitterionic compounds (enoxacin, cephradine and benzyloxytryptophan (BOTP)) was independent of DPPS content. These results suggest that the uptake of tested anionic compounds by large unilamellar lipid vesicles is dependent on the membrane surface potential which originates in the surface negative charge.
Anions↗
Expression, purification, and substrate specificity of isocitrate dehydrogenase from Thermus thermophilus HB8.
Isocitrate dehydrogenase (ICDH) from an extreme thermophile, Thermus thermophilus HB8, was overexpressed in Escherichia coli. The enzyme was easily purified to homogeneity by a combination of heat treatment (70 degrees C, 20 min) and column chromatography. The N-terminal sequence of the protein thus purified coincided with that of the protein extracted from the thermophile. The substrate specificity of the enzyme was mutationally analyzed and engineered to recognize 3-alkyl-malate as a substrate. Based on the three-dimensional structure of E. coli isocitrate dehydrogenase, Ser97 qnd Asn99 of the thermophile enzyme were speculated to participate in the substrate recognition, and these residues were replaced with threonine and leucine, respectively. Molecular recognition of the mutant enzymes, [S97T]ICDH, [N99L]ICDH, and [S97T, N99L]ICDH, were studied using isocitrate, 3-isopropylmalate, and 3-ethylmalate. The affinity toward isocitrate was reduced in the cases of [S97T]ICDH and [N99L]ICDH, confirming the importance of the residues for the reaction. Though none of the mutants acted on 3-isopropylmalate, [N99L]ICDH was competitively inhibited by 3-isopropylmalate with a higher affinity than that of the wild-type enzyme. [N99L]ICDH showed an approximately 10(3)-fold higher value of (kcat/Km)3-ethylmalate/(kcat/Km)isocitrate than the wild-type enzyme, indicating that the single mutation of Asn99 to leucine switched the substrate specificity of the enzyme away from isocitrate and toward 3-ethylmalate.
Amino Acid Sequence↗
Gelatinase A and APP.
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Amyloid Precursor Protein Secretases↗
Expression of multidrug resistance gene and localization of P-glycoprotein in human primary ovarian cancer.
Resistance to chemotherapy is the major obstacle to controlling malignant tumors. To characterize multidrug resistance phenotype in human primary ovarian cancer without chemotherapy, expressions of the mdr1 gene in 52 cases of ovarian cancer (44 common epithelial, 5 nonepithelial, and 3 metastatic cancers) were analyzed by polymerase chain reaction of RNA after reverse transcription. Furthermore, localization of P-glycoprotein, which is encoded by the mdr1 gene, was studied immunohistochemically. Although overall expression of the mdr1 gene was relatively low, its expression level was the highest in well-differentiated cancer tissues. Serous and mucinous adenocarcinomas showed higher levels of expression compared with clear cell and endometrioid carcinomas. P-glycoprotein was positive on luminal surfaces of lining cells of ovarian cancer and on those of inclusion cysts from which epithelial ovarian cancer is considered to develop. Thus, some ovarian cancer cases before chemotherapy are intrinsically multidrug resistant, which can be determined by mdr1 gene expression, and this phenotype should be taken into account for effective chemotherapy of ovarian epithelial carcinomas.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Changes in the permeation rate of organic anions through the intestinal brush-border membrane with membrane surface potential.
The effects of membrane surface potential on the uptake of anionic compounds by rat intestinal brush-border membrane vesicles were investigated. The uptake amount of all tested anionic compounds (ceftibuten, cefixime, benzylpenicillin, s-1006 and rentiapril) in the neutral medium (pH 7.5) was lower than that in the acidic medium (pH 5.5). Changes in surface potential of brush-border membrane vesicles were monitored using a fluorescence dye, 8-anilino-1-naphthalenesulfonate (ANS), and the results suggested an increase of a negative charge on the membrane surface proportional to the increase of the pH of medium. A good correlation was observed between the initial uptake rate of all tested anionic compounds and relative membrane surface potential monitored by ANS. Moreover, the uptake of cefixime by artificial liposome made from PC containing various amount of DPPS was measured. The uptake value of cefixime was decreased in proportion to an increase of DPPS content. These results suggest that the permeation of anionic compounds across intestinal brush-border membrane is dependent on surface potential originate in the surface negative charge.
Anilino Naphthalenesulfonates↗
Cell adhesion activity of a 30-kDa major secreted protein from human bladder carcinoma cells.
Human bladder carcinoma cell line EJ-1 secreted a protein of 30 kDa as a major component. This protein was purified from the serum-free conditioned medium of EJ-1. Its N-terminal amino acid sequence was identical to that of fibroblast-derived endothelial cell growth factor (f-ECGF), but there were differences in their amino acid compositions and isoelectric points. The 30-kDa protein showed no effect on the growth of bovine capillary endothelial cells, but it promoted the attachment and spreading of rat liver cell line BRL and human umbilical vein endothelial cell line ECV-304. These results suggest that the 30-kDa protein, tentatively named "tumor-derived adhesion factor (TAF)", may be related with the aberrant cell adhesion of cancer cells.
Amino Acid Sequence↗
Mucinous adenofibroma of the ovary: case report of the endocrinologic findings.
Endocrine and clinicopathologic findings in a 77-year-old woman with ovarian mucinous adenofibroma of borderline malignancy are reported. The preoperative levels of testosterone, androstenedione, estrone, and estradiol in her peripheral blood were 91 ng/ml, 3.78 ng/ml, 82 pg/ml, and 35 pg/ml, respectively, abnormally high. Those of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were < 0.5 mIU/ml and 12.1 mIU/ml, respectively. Total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. Pathologic diagnosis of the right ovarian tumor was mucinous adenofibroma of borderline malignancy with hyperthecosis. Postoperatively, the levels of sex steroids and gonadotropin returned to the normal range of a postmenopausal woman. The patient remains well 2 years and 6 months after the operation.
Adenofibroma↗
Hyaluronan, cartilage destruction and hydrarthrosis in traumatic arthritis.
The concentrations of hyaluronan (HA) and chondroitin sulfate (CS) in synovial fluids from patients with traumatic arthritis (TA) with and without hydrarthrosis were measured. The CS in synovial fluids was determined as a marker of cartilage destruction by high performance liquid chromotography. The concentration of HA in synovial fluids was lower in patients with hydrarthrosis than in healthy volunteers and patients with TA without hydrarthrosis, whereas the total amounts of HA and CS and the concentration of CS were higher in patients with hydrarthrosis. To investigate the relation between hydrarthrosis and production of HA in synovial tissues, TA synovial tissue biopsies were stained for HA with biotinylated HA binding region. The intensity of HA staining was higher in specimens from patients with hydrarthrosis than in normal and TA without hydrarthrosis specimens. Thus, there may be a correlation between hyperproduction of HA, cartilage destruction and increase in fluid volume in TA.
Adolescent↗
Glycolipids isolated from Aplysia kurodai can activate cyclic adenosine 3',5'-monophosphate-dependent protein kinase from rat brain.
Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonoglycosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGal beta 1-->3GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2](2-aminoethylphosphonyl-->6)Glc beta 1-->4Glc beta 1-->1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 microM. Activation of cAMP-kinase was maximal with 250 microM SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.
Animals↗
Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor.
Ladsin is a large cell-adhesive protein with potent cell-scattering activity, which was recently identified in the culture of a malignant human gastric carcinoma cell line [Miyazaki, K. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11767-11771]. It is a heterotrimeric protein, containing a 140-kDa subunit similar or identical to the laminin B2t chain. Ladsin is similar to the keratinocyte-derived matrix proteins, "epiligrin" and "kalinin." In the present study, the cell-adhesion and cell-migration activities of ladsin were examined in comparison with those of three cell adhesion proteins, laminin, fibronectin, and vitronectin. Ladsin showed high cell-adhesion activity toward rat liver cell line BRL at concentrations 4-20-times lower than in the case of the other three proteins. In a monolayer culture, ladsin stimulated the migration of BRL cells about 2-times more strongly than the others, as compared at the minimal concentrations required for the maximal cell-adhesion activity. In Boyden chambers, ladsin stimulated both the chemotactic and chemokinetic migration of BRL cells. When the effect of anti-integrin antibodies on the adhesion of human fibrosarcoma cell line HT1080 was examined, the adhesion to ladsin was effectively inhibited by both the anti-integrin alpha 3 and beta 1 antibodies, but not the anti-integrin alpha 6 antibody, indicating that the primary receptor of ladsin is integrin alpha 3 beta 1. These results demonstrate that ladsin is a unique extracellular matrix component which may play a major role in cell migration.
Adult↗
cDNA cloning and mRNA expression of a serine proteinase inhibitor secreted by cancer cells: identification as placental protein 5 and tissue factor pathway inhibitor-2.
A serine proteinase inhibitor was purified from conditioned medium of the human glioblastoma cell line T98G. Analysis of its partial amino acid sequences indicated that this protein was identical to placental protein 5 (PP5), a placenta-derived glycoprotein with serine proteinase inhibitor activity, the amino acid sequence of which had been partially determined. cDNA cloning of PP5 demonstrated that it belonged to the Kunitz-type serine proteinase inhibitor family, having three putative Kunitz-type inhibitor domains, and that it was identical to a recently reported inhibitor, tissue factor pathway inhibitor-2 (TFPI-2) [Sprecher et al. (1994) Proc. Natl. Acad. Sci. USA 91, 3353-3357]. PP5/TFPI-2 transcripts were highly abundant in the full-term placenta and widely expressed in various adult human tissues, such as the liver, skeletal muscle, heart, kidney, and pancreas. Several ovarian carcinoma cells as well as T98G also contained significant amounts of the transcripts.
Amino Acid Sequence↗
Chemical modification and site-directed mutagenesis of Tyr36 of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.
3-Isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was chemically modified with tetranitromethane which nitrated 1.5-2.0 Tyr residues per subunit. The nitration was biphasic and parallel to the loss of activity. The modified residue in the first phase was identified to be Tyr36, which is distantly located from the active site of the enzyme. The function of Tyr36 was investigated by site-specific replacement with Phe. The Michaelis constant for the substrate or co-enzyme was not altered by the replacement, whereas the catalytic constant decreased down to approximately 5%. X-ray analysis of the mutant enzyme revealed that Arg94 moved the largest distance among the active site residues, that is, the NH1 and NH2 of the guanidino group moved 1.11 and 1.32 A respectively. The results suggest that Arg94 is responsible for the enzyme catalysis.
3-Isopropylmalate Dehydrogenase↗
Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.
The co-enzyme specificity of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was changed from NAD to NADP by site-directed muta-genesis. Based on sequence comparison of 3-isopropylmalate dehydrogenases from various organisms with NAD- and NADP-dependent isocitrate dehydrogenases, Ser226, Ser253 and Ile279 of 3-isopropylmalate dehydrogenase were suggested as determining the co-enzyme specificity. These residues were replaced with the corresponding residues of NADP-dependent isocitrate dehydrogenases; Arg, Gly and Tyr respectively. The single-mutated enzymes, S226R and 1279Y, enhanced the activities towards NADP approximately 10- and approximately 3-fold respectively, whereas S253G reduced the activity. Among the multiple-mutated enzymes, the double-mutated S226R/I279Y increased the catalytic efficiency against NADP (approximately 5-fold) and shifted the specificity for NAD towards NADP most significantly (approximately 173-fold).
3-Isopropylmalate Dehydrogenase↗
Successful treatment of cytophagic histiocytic panniculitis with modified CHOP-E. Cyclophosphamide, adriamycin, vincristine, predonisone, and etoposide.
A 27-year-old woman developed generalized subcutaneous painful nodules, fever, abnormal liver function, a bleeding tendency, and pancytopenia. Skin biopsies revealed the lobular panniculitis with a morphologically benign histiocytic infiltration with prominent phagocytosis. Leukophagocytosis and erythrophagocytosis were also present in the bone marrow. The diagnosis of cytophagic histiocytic panniculitis was made. The patient received polychemotherapy with cyclophosphamide, Adriamycin, and vincristine on day 1, prednisone on day 1-5 (modified CHOP), with the addition of etoposide (E). This regimen was repeated 8 times every 3 weeks. The patient obtained a complete clinical remission that has lasted almost 2 years after the completion of chemotherapy. Thus we suggest modified CHOP-E chemotherapy for an effective treatment for the aggressive form of cytophagic histiocytic panniculitis.
Adult↗
High-order harmonic generation in rare gases with subpicosecond XeCl laser pulses.
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Journal Article↗