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Biomedical subjects

S Kimura

Publications and source records attributed to S Kimura.

At least 19 recordsLinked to original sources
PubMed1992

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.

Adenocarcinoma↗
PubMed1992

Interaction of melittin derivatives with lipid bilayer membrane. Role of basic residues at the C-terminal and their replacement with lactose.

Melittin possesses an amphiphilic property in the primary sequence in which hydrophilic residues are located at the C-terminal region from Lys-21 to Gln-26. A part of the hydrophilic sequence was cleaved off by endopeptidase Arg-C to obtain melittin 1-22. The affinity of melittin 1-22 for neutral phospholipid membrane was reduced to 1/3 that of melittin, indicating that the basic residues, Lys-23 and Arg-24, are important in binding of melittin to the membrane. The melittin 1-22 was extended toward the C-terminal end by connection of lactose (melittin-lac), the membrane affinity of which was slightly higher than the melittin 1-22, but lower than melittin. The leakage experiment of 5,6-carboxyfluorescein encapsulated in DPPC liposomes showed that the activities of melittin 1-22 and melittin-lac in membrane lysis were much lower than melittin. However, the melittin 1-22 formed a voltage-dependent ion-channel in an azolectin bilayer membrane. It is thus considered that Lys-23 and Arg-24 residues of melittin play an important role in binding to the polar region of membrane for lysis, but not for ion-channel formation.

Amino Acid Sequence↗
PubMed1992

Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.

Amino Acid Sequence↗
PubMed1992

Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.

Amino Acid Sequence↗
PubMed1992

Effect of sphingosine and its N-methyl derivatives on oxidative burst, phagokinetic activity, and trans-endothelial migration of human neutrophils.

Neutrophils display three major functions: (i) oxidative burst, (ii) phagokinetic activity, and (iii) trans-endothelial migration. Sphingosine (SPN) is known to inhibit oxidative burst in human neutrophils via inhibition of protein kinase C (PKC). SPN is metabolically converted into N,N-dimethylsphingosine (DMS) in some tissues and cell lines. In previous studies, we have demonstrated that the PKC-inhibitory effect of DMS is stronger than that of SPN, and that of the synthetic analogue N,N,N-trimethylsphingosine (TMS) is even stronger. Therefore, in the present study, we compared the effects of SPN, DMS, and TMS on the neutrophil functions mentioned above. These three compounds, at 10-20 microM, showed equal inhibition of phorbol 12-myristate 13-acetate (PMA)-dependent superoxide (O2-) production and O2 consumption. They and other known PKC inhibitors (H-7, staurosporine, calphostin C), at 1-5 microM, showed equal inhibition of the phagokinetic activity of neutrophils. On the other hand, trans-endothelial migration of neutrophils was suppressed by SPN, DMS, and TMS at 5-10 microM, but was relatively unaffected by the other PKC inhibitors. All of these compounds inhibited PMA-induced phosphorylation of major neutrophil proteins with a M(r) of 60 and 47 kDa; this effect is ascribable to inhibition of PKC. Despite the similar effects of SPN, DMS, and TMS on neutrophil function, TMS was considerably less cytotoxic to neutrophils under the same experimental conditions. Furthermore, SPN and DMS at 10-20 microM caused obvious morphological changes of the endothelial cells, but TMS did not. SPN undergoes rapid metabolic conversion to various sphingolipid compounds, but TMS is stable. In view of all these findings, TMS appears to be a superior pharmacological agent, compared to SPN derivatives or other PKC inhibitors, for suppression of neutrophil overfunction associated with inflammatory processes and tissue injury.

Cell Movement↗
PubMed1992

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.

Base Sequence↗
PubMed1992

Autophosphorylation of beta-connectin (titin 2) in vitro.

Phosphorylation of beta-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [gamma-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK), cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate beta-connectin in vitro under optimal conditions. Thus it appears that beta-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.

Adenosine Triphosphate↗
PubMed1992

A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini.

Two human liver UDP-glucuronosyltransferase (transferase) cDNAs, HUG-Br1 and HUG-Br2, were previously isolated (Ritter, J. K., Crawford, J. M., and Owens, I. S. (1991) J. Biol. Chem. 266, 1043-1047), and each was shown to encode a bilirubin transferase isozyme which catalyzes the formation of all physiological conjugates of bilirubin IX alpha following expression in COS-1 cells. Sequence data showed that the cDNAs contained identical 3' ends (1469 base pairs in length) to each other and to that of the human phenol transferase cDNA, HLUG P1 (Harding, D., Fournel-Gigleux, S., Jackson, M. R., and Burchell, B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8381-8385). Here we report that the two corresponding bilirubin transferases and the phenol transferase are encoded by a novel locus, UGT1, which is also predicted to encode three other bilirubin transferase-like isozymes all having identical carboxyl termini. The transcriptional arrangement utilizes six nested promoter elements, each of which is positioned upstream of a unique exon 1. Each exon 1 encodes the NH2-terminal domain (286 amino acids) and confers the substrate specificity of the isoform. The 3' end of the locus contains 4 common exons which encode the identical carboxyl termini (246 amino acids). It is predicted that six nested primary transcripts are synthesized and that each exon 1 is differentially spliced to the 4 common exons to produce six unique, mature mRNAs. Although the gene organization is present as a single copy, it provides the flexibility of independent regulation of each isoform which is known to occur in the case of bilirubin and phenol transferase activities. With an understanding of the gene structure, lethal, as well as the nonlethal defects, associated with bilirubin transferase activity can now be determined.

Amino Acid Sequence↗
PubMed1992

Correlation between killing activity towards the murine L929 cell line and expression of membrane-associated lymphotoxin-related molecule of human lymphokine-activated killer cells.

Human lymphokine-activated killer (LAK) cells expressed a membrane-associated lymphotoxin-related molecule (mLT) which was detected by flow cytometric analysis with anti-lymphotoxin antibody. Upon removal of exogenous interleukin-2 from LAK cell culture medium and another 24 h cultivation, the expression of mLT was decreased. Corresponding to the decrease of mLT expression, the killing activity of LAK cells towards L929 cells was remarkably reduced and killing of MIA PaCa-2 and U937 cells was moderately reduced, whereas killing of Daudi and K562 cells was fully restored. The supernatant of mLT-expressing LAK cells had no cytotoxic activity towards L929 cells in the absence of actinomycin D. Moreover, not only the killing of L929 cells but also that of human tumor cell lines (MIA PaCa-2, U937) by mLT-expressing LAK cells was partially inhibited in the presence of anti-lymphotoxin antibody. These results suggest an involvement of mLT in the killing of some tumor target cells by LAK cells.

Animals↗
PubMed1992

High blood pressure in transgenic mice carrying the rat angiotensinogen gene.

Transgenic mice were generated by injecting the entire rat angiotensinogen gene into the germline of NMRI mice. The resulting transgenic animals were characterized with respect to hemodynamics, parameters of the renin angiotension system, and expression of the transgene. The transgenic line TGM(rAOGEN)123 developed hypertension with a mean arterial blood pressure of 158 mmHg in males and 132 mmHg in females. In contrast, the transgenic line TGM(rAOGEN)92 was not hypertensive. Rat angiotensinogen was detectable only in plasma of animals of line 123. Total plasma angiotensinogen and plasma angiotensin II concentrations were about three times as high as those of negative control mice. In TGM(rAOGEN)123 the transgene was highly expressed in liver and brain. Transcripts were also detected in heart, kidney and testis. In TGM(rAOGEN)92 the brain was the main expressing organ. In situ hybridization revealed an mRNA distribution in the brain of TGM(rAOGEN)123 similar to the one in rat. In TGM(rAOGEN)92 the expression pattern in the brain was aberrant. These data indicate that overexpression of the angiotensinogen gene in liver and brain leads to the development of hypertension in transgenic mice. The TGM(rAOGEN)123 constitutes a high angiotensin II type of hypertension and may provide a new experimental animal model to study the kinetics and function of the renin angiotensin system.

Angiotensinogen↗
PubMed1992

Pharmaceutical evaluation of ibuprofen fast-absorbed syrup containing low-molecular-weight gelatin.

Two kinds of ibuprofen syrups were prepared and evaluated. One was a suspending syrup, prepared by using hydroxypropyl methylcellulose (HPMC) as a dispersing agent, and the other was a dry syrup containing low-molecular-weight gelatin (LM gelatin). The dissolution behaviors of ibuprofen from syrups were studied, and both syrups showed size-dependent dissolution; the smaller particles exhibited faster dissolution. The in vivo absorption behaviors of the syrups were compared with that of commercial tablets of ibuprofen in beagle dogs and human volunteers. The absorption rates following oral administrations of syrups were much greater than those following administration of commercial tablets. Moreover, both syrups reduced the bitter taste and irritation of the oral mucosa caused by ibuprofen; the dry syrup markedly masked these side effects. These results suggest that the dry syrup containing LM gelatin improves some of the pharmaceutical properties of ibuprofen, and that the LM gelatin may be used in a variety of oral dosage forms.

Administration, Oral↗
PubMed1992

BRM activities of low-toxic Bordetella pertussis lipopolysaccharides.

A low-toxic lipopolysaccharide (BP-LPS) was isolated from killed Bordetella pertussis (Tohama strain). LD50 of BP-LPS was about 0.8 mg/mouse which was about 10-fold higher than the LD50 of E. coli-LPS(80 micrograms/mouse). Toxicity measured by decrease in body weight of BP-LPS-injected mice was similarly low. BP-LPS had strong antitumor activities against various murine syngeneic tumors, and its systemic administration caused clear regression of such as MM46 mammary carcinoma and Meth A fibrosarcoma. It is noteworthy that a tolerable dosage of BP-LPS (375 micrograms/mouse) showed clear antitumor activity against MH134 hepatoma, which is known to be insusceptible to usual types of BRM including bacterial LPS. These findings suggest that BP-LPS is a promising candidate as an antitumor agent for clinical use. Biological activities of BP-LPS were examined and compared with those of toxic LPS extracted from Escherichia coli and other enterobacteria. Activation or stimulation of macrophages and lymphocytes by these LPS, including TNF induction, was found to be similar. However, activation of human or murine neutrophils, as estimated by neutrophil-adherence assay in vitro, though induced by all other toxic LPS tested, was not induced by BP-LPS. This inability of BP-LPS to activate neutrophils is assumed to be related to its low toxicity.

Animals↗
PubMed1992

Changes in the productivity of cytokines and active-oxygen in peripheral blood cells following surgery.

An investigation was carried out on the productivity of cytokines and active-oxygen by peripheral blood cells during the pre- and post-operative periods. While the preoperative production of tumor necrosis factor (TNF) and interleukin-1 alpha (IL-1 alpha) was elevated, that of lymphotoxin (LT) and interferon-gamma (IFN-gamma) were slightly suppressed. In the postoperative period the peak TNF and IL-1 alpha and active-oxygen productivity was elevated, while LT and IFN-gamma productivity was suppressed in patients with an intraoperative bleeding volume of more than 1,000 ml compared to those with that of less than 1,000 ml. Thus, stress stimulates the TNF and IL-1 alpha and active-oxygen producing system, that is, the macrophage-neutrophil system, and suppresses the LT and IFN-gamma system, being, the inflammatory helper T cell system, in the early postoperative period.

Adult↗
PubMed1992

Diagnosis and surgical treatment of five cases of triple shunt (ASD + VSD + PDA).

Five patients with triple shunt (atrial septal defect, ventricular septal defect, and patent ductus arteriosus) were treated surgically during the past twelve years (1978-1989). They developed severe cardiac and respiratory failure in the neonatal period and/or in early infancy. Simultaneous repair of the triple shunt was performed, but initially two patients suffering severe cardiac failure were lost immediately after operation. The following three patients were operated on relatively soon after establishing a definitive diagnosis and a successful outcome was able to be achieved. Repeated evaluation of the patient with echocardiography was necessary for diagnosis. Early primary operation is, therefore, considered to be the treatment of choice after confirming the diagnosis.

Cardiac Catheterization↗
PubMed1992

Characterization and localization of alpha-connectin (titin 1): an elastic protein isolated from rabbit skeletal muscle.

A simplified procedure to isolate alpha-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate alpha-connectin is introduced. Separation of alpha-connectin from beta-connectin is introduced. Separation of alpha-connectin from beta-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of alpha-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of alpha-connectin (MW, approximately 3 x 10(6)) were similar to those of its proteolytic product, beta-connectin (MW, approximately 2 x 10(6)). Circular dichroic spectra suggested that both alpha- and beta-connectin consist of 60% beta-sheet and 30% beta-turn. It thus appears that the whole elastic filament of connectin has a folded beta-strand structure. Proteolysis of alpha-connectin by calpain resulted in formation of beta-connectin and smaller peptides. The alpha-connectin interacted with both myosin and actin filaments similarly to beta-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with alpha-connectin (titin 1, TI) but only weakly with beta-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SM1 whose position depends on sarcomere length. It thus appears that beta-connectin extends from the edge of M-line to the above epitope region in the I-band.

Actins↗
PubMed1992

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Actin Cytoskeleton↗
PubMed1992

Embolotherapy of massive duodenal hemorrhage.

Eleven patients with massive duodenal hemorrhage were treated by emergent embolization. Bleeding originated from duodenal ulcer in three patients, from duodenal tumor in one, from ruptured pancreaticoduodenal artery pseudoaneurysm in three, and from ruptured gastroduodenal artery pseudoaneurysm in four. Complete hemostasis was obtained immediately after embolotherapy in all cases. Three of these patients died during the hospitalization period, one of whom from duodenal infarction and pancreas necrosis induced by embolization. In three patients with duodenal ulcer, complete hemostasis was obtained only by the gastroduodenal artery embolization with Gelfoam particles. Seven patients with pseudoaneurysms of the gastroduodenal artery or its branches required not only blockage of blood flow from the celiac artery but also the superior mesenteric artery for complete hemostasis. Therefore, in patients presenting with duodenal hemorrhage, the possibility of dual blood supply to the duodenum should be considered. Emergent embolization represents a useful alternative to surgery for massive duodenal hemorrhage, but it carries a risk of complications in patients with previous gastroduodenal surgery or significant visceral atherosclerosis.

Adolescent↗