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Biomedical subjects

M Kikuchi

Publications and source records attributed to M Kikuchi.

At least 613 records · Page 34Linked to original sources
PubMed1993

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme.

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Animals↗
PubMed1993

Spontaneous regression in adult T-cell leukemia/lymphoma.

BACKGROUND: Spontaneous regression of adult T-cell leukemia/lymphoma (ATL) is considered to be extremely unusual. Of the 82 patients with ATL who the authors saw between 1981 and 1991, spontaneous regression occurred in 3 (3.7%), 2 of whom were previously untreated and one who had been previously treated. Surgical excisional biopsy triggered the spontaneous regression in these patients. METHODS: In two of these patients with spontaneous regression, gene analysis studies of human T-cell leukemia virus type I (HTLV-I) proviral DNA, and T-cell receptor (TCR) were carried out by Southern blot analysis in lymph node cells or peripheral lymphocytes before regression and after recurrence. RESULTS: One patient exhibited monoclonal integration of HTLV-I proviral DNA and the rearranged band of the TCR-beta gene at the same positions both before regression and after recurrence. The other patient showed them at the different positions before regression and after recurrence. CONCLUSIONS: The authors' studies indicated heterogeneity in ATL patients with spontaneous regression. Temporary spontaneous regression could occur in typical ATL and might be associated with a longer survival time than that for prototypic ATL.

Blotting, Southern↗
PubMed1993

Indication of possible post-translational formation of disulphide bonds in the beta-sheet domain of human lysozyme.

Lysozyme has two distinct folding domains, and in most molecules the alpha-helical domain folds more quickly than the beta-sheet domain in vitro [Radford, Dobson and Evans (1992) Nature (London) 358, 302-307]. In order to investigate the relationship between the formation of disulphide bonds and protein folding in vivo, we carried out cysteine scanning mutagenesis to shift positions of the disulphide bonds in both the alpha-helical and beta-sheet domains of human lysozyme. Of the constructed mutants (nine in the beta-sheet domain and 13 in the alpha-helical domain), the mutant L79CC81A, in which Leu-79 and Cys-81 in the beta-sheet domain were replaced by Cys and Ala respectively, was secreted by yeast. The rest of the mutants were retained in the insoluble fraction of the cell, probably because of a failure of folding. The distance between the two alpha-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, but analysis of the primary structure revealed that the major part of L79CC81A was secreted with a non-native disulphide bond Cys79-Cys95 and two free cysteine residues at positions 65 and 77 in the beta-sheet domain. These results suggest that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds, and the non-native folding of mutated polypeptide chains in in vivo folding. The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding two isomers. On the basis of our previous results and present study it is suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in the beta-sheet domain are post-translationally formed in vivo.

Amino Acid Sequence↗
PubMed1993

Prodrug activation via catalytic antibodies.

Prodrug activation via antibodies was examined by using the antibiotic chloramphenicol as a model drug. Based on the conformational change between substrate and product, this antibody-catalyzed reaction was designed to prevent product inhibition, thus enhancing turnover. Antibodies elicited against a phosphonate transition-state analogue were found to catalyze hydrolysis of a nonbioactive chloramphenicol monoester as a prodrug at a significantly higher rate above the uncatalyzed background reaction to regenerate chloramphenicol as a parent molecule. The antibody-catalyzed prodrug activation was tested by the paper-disc diffusion method using Bacillus subtilis as an indicator strain. The antibody 6D9 catalyzes the reaction with multiple turnover to generate enough chloramphenicol to inhibit bacterial growth, as indicated by a clear inhibitory zone after incubation with monoester. Using the same method, no inhibition was detected by incubation of either the monoester or the antibody alone. This result reveals that only the antibody hydrolytically activates the monoester, which can be expected to be a suitable prodrug, as it is resistant to the action of bacterial hydrolytic enzymes. The approach in this study demonstrates the use of catalytic antibody technology in medicine and may be applicable to drugs with undesirable effects, particularly in the field of cancer therapy.

Animals↗
PubMed1993

Characteristics of cloned cells of mixed müllerian tumor of the human uterus. Carcinoma cells showing myogenic differentiation in vitro.

BACKGROUND: To elucidate the relationship between the epithelial and mesenchymal elements of malignant mixed Müllerian tumors (MMMT), the authors examined the biologic properties of two clones of different cell types (designated as FU-MMT-2-C1 and FU-MMT-2-S1) established from a uterine MMMT cell line (FU-MMT-2), which they previously have reported. METHODS AND RESULTS: By morphologic and immunocytochemical analyses, FU-MMT-2-C1 exhibited features of adenocarcinoma cells, whereas FU-MMT-2-S1 showed characteristics of sarcoma cells with myogenic differentiation. Some FU-MMT-2-C1 cells at confluence differentiated spontaneously into myogenic mesenchymal cells in vitro. In addition, transitional-type cells between epithelial cells and sarcomatous cells were observed in the areas of mesenchymal differentiation in FU-MMT-2-C1 by light and electron microscopic study. Ultrastructurally, the transitional-type cells represented biphasic morphologic characteristics consisting of epithelial and myogenic features, which proved to coexpress epithelial, mesenchymal, and muscle markers by double immunoenzymatic staining. However, no epithelial differentiations were apparent in the sarcoma clone FU-MMT-2-S1. FU-MMT-2-C1 produced tumor in nude mice, the histologic study of which showed a mixture of adenocarcinoma and myogenic sarcomatous elements that resembled the original tumor. Cytogenetic studies demonstrated that these two clones were monoclonal in origin because of the presence of common karyotypic abnormalities in both cells. In addition, the amplified (approximately fourfold to eightfold) c-myc oncogene was found in the cloned cells and the original tumor cells. CONCLUSIONS: The current results strongly support the theory of single cell origin in uterine MMMT and suggest that the mesenchymal elements originated from primitive Müllerian epithelial cells capable of differentiating into epithelial, mesenchymal, or both elements.

Animals↗
PubMed1993

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Amino Acid Sequence↗
PubMed1993

Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin.

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

3-O-Methylglucose↗
PubMed1993

A new tumor-associated antigen defined by a monoclonal antibody directed to gastric adenocarcinoma.

BACKGROUND: To develop useful markers for the gastrointestinal neoplasms, the authors have established a new monoclonal antibody (MoAb), FU-MK-1, directed to gastric adenocarcinoma. METHODS: The MoAb was produced by a mouse hybridoma technique by immunizing a BALB/c mouse with cancerous ascites derived from a poorly differentiated adenocarcinoma of the stomach. RESULTS: FU-MK-1 did not react with the normal adult gastric mucosa, but did react with most carcinomas of the gastrointestinal tract. In addition, the MoAb recognized cholangiocarcinomas (CC), but it did not react with hepatocellular carcinomas (HCC). Furthermore, in the combined type tumor consisting of a mixture of HCC and CC, the MoAb react only with CC element, but not with pseudoglandular structures in the HCC areas. These results indicate that FU-MK-1 is a useful antigenic marker for distinguishing HCC from CC in the liver. Furthermore, because this MoAb retains its reactivity with formalin-fixed paraffin-embedded material, it may become a useful reagent for routine or retrospective immunohistologic studies. The molecular weight of the FU-MK-1 antigen was estimated to be ca. 41,000 dalton by the Western blot analysis. Periodic acid and trypsin treatment on the antigen suggested that the antigenic determinant is a glycoprotein. CONCLUSIONS: This MoAb may contribute to the histopathologic and immunologic studies of the digestive system.

Adenocarcinoma↗
PubMed1993

Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells.

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the alpha-amylases. The optimum pH, specific activity and Km value for beta-cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mM, respectively. These values were almost identical to those from B. sphaericus E-244.

Amino Acid Sequence↗
PubMed1993

Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets.

Glucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-D-glucose being reached within 15 s at 22 degrees C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the Vmax value of glucokinase activity was estimated to be 255 +/- 37 nmol.h-1.mg protein-1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high Km component of glucose utilization with a Vmax of 289 +/- 18 nmol.h-1.mg protein-1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics of MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.

3-O-Methylglucose↗
PubMed1993

Bilateral traumatic hip dislocation in a child.

Traumatic dislocation of in children occurs less frequently than in adults, and bilateral dislocation is extremely rare. Manual reduction was performed in a 3-year-old girl with bilateral traumatic hip dislocation. The recovery course has been favorable for about 1 year.

Accidental Falls↗
PubMed1993

Traumatic anterior dislocation of the shoulder in a child.

Dislocation of the shoulder joint is a rare occurrence in children. This paper reports on traumatic anterior dislocation of the shoulder in a 3-year-old boy and a 9-year-old boy, together with a discussion of the relevant literature.

Accidents↗