Biomedical subjects
Publications and source records attributed to Y Konno.
Two alkaline phosphatases were extracted from the membranes of Bacillus subtilis 168 stationary-phase cells and purified as homogeneous proteins by hydroxyapatite column chromatography. Alkaline phosphatases I and II differed in several properties such as subunit molecular weight, substrate specificity, thermostability, Km, pH stability, and peptide maps.
A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.
IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.
Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.
In order to examine the involvement of protein kinase C (PKC) in the transcriptional activation of genes by TPA (12-0-tetradecanoyl phorbol 13-acetate) we have constructed a series of PKC expression plasmids. Transient expression of an active fragment of PKC in rat fibroblasts resulted in the transcriptional activation of a TRE (TPA-responsive element)-CAT chimeric gene which contains various repetitions of collagenase TREs. These provide the first direct evidence that kinase activity of PKC is involved in TPA-induced transcriptional activation through TRE.
A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKC epsilon, clearly distinct from the four "conventional" PKCs [Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731-741]. We purified nPKC epsilon from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKC alpha. nPKC epsilon was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKC alpha), the only PKC expressed in COS cells. The protein kinase activity of nPKC epsilon is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificities were clearly different from PKC alpha. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10-20 mM). The activation of nPKC epsilon by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKC epsilon autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKC epsilon permit clear discrimination of nPKC epsilon from conventional PKCs.
The purpose of this study was to explore changes in body sensation through muscular relaxation by means of measurement of tactile two-point limen. This study comprised two experiments. The first experiment, in which twenty female college students participated, was carried out to examine the effect of relaxation on two-point limen, and the relationship between the acceptance of relaxation and two-point limen. The second experiment involved three physically handicapped children with cerebral palsy and hemiplegia. To determine whether they would show a decrease in two-point limen in the shoulders and forearms as a function of improvement of relaxation, they were given a week long intensive Motor Action Training, which was administered by means of the relaxation and movement training method established by Naruse. The results showed that the two-point limen decreased remarkably for both normal adult subjects and physically handicapped subjects as they relaxed muscular tension. Concerning the acceptance of relaxation, the high acceptance group showed a large decrease in the two-point limen, whereas the low acceptance group did not show any change.
Protein kinase C (PKC)-related cDNA clones encode an 84 kd protein, nPKC. nPKC contains a cysteine-rich repeat sequence homologous to that seen in conventional PKCs (alpha, beta I, beta II, and gamma), which make up a family of 77-78 kd proteins with closely related sequences. nPKC, when expressed in COS cells, confers increased high-affinity phorbol ester receptor activity to intact cells. Antibodies raised against nPKC identified a 90 kd protein in rabbit brain extract as well as in extracts from COS cells transfected with the cDNA construct. nPKC shows protein kinase activity that is regulated by phospholipid, diacylglycerol, and phorbol ester but is independent of Ca2+. The structural and enzymological characteristics of nPKC clearly distinguish it from conventional PKCs, which until now have been the only substances believed to mediate the various effects of diacylglycerol and phorbol esters. These results suggest an additional signaling pathway involving nPKC.
Three rabbit cDNA clones coding for three types of protein kinase C (PKC alpha, beta, and gamma) have recently been identified and the structures determined [Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., & Hidaka, H. (1987) Nature (London) 325, 161-166]. By use of these cloned cDNAs as hybridization probes, a fourth type (delta) of cDNA clone, which encodes a protein highly homologous to PKC alpha, beta, and gamma, was identified. PKC delta is composed of 697 amino acid residues and contains several peptide sequences determined at the protein level with the brain PKC preparation. This indicates that this molecular type (PKC delta) is, along with PKC alpha, beta, and gamma, a constituent of the brain PKC preparation. Sequence comparison among the four PKC types revealed that PKC delta is somewhat distinct from the other PKC types. PKC delta shows 99% amino acid sequence identity with rat PKC type I [Knopf, J. L., Lee, M.-H., Sultzman, L. A., Kriz, R. W., Loomis, C. R., Hewick, R. M., & Bell, R. M. (1986) Cell (Cambridge, Mass.) 46, 491-502], indicating relationship of these PKC types. The mRNA for PKC delta is exclusively concentrated in the brain.
The mechanism of acute gastric ulceration in rats with obstructive jaundice was investigated in terms of the changes in the gastric mucosal blood flow, and the effect of vagotomy on ulceration was examined. 188 Sprague-Dawley rats weighing about 250 g were prepared and divided into 4 groups as follows; control group (sham operation), jaundiced group (ligation of the bile duct), vagotomized group (vagotomy with pyloroplasty), jaundiced and vagotomized group (simultaneous ligation of the bile duct and vagotomy with pyloroplasty). Two weeks after the operation, water immersion and restraint stress procedures were performed in these 4 groups. The gravity of acute gastric ulcerations was calculated by ulcer index. The gastric mucosal blood flow was measured by hydrogen clearance technique. The following results were obtained: In control group, ulcer index was increased and gastric mucosal blood flow was decreased after the stress procedures. The jaundiced group showed significantly higher ulcer index and early significant decrease of gastric mucosal blood flow after the stress procedures compared to the control group. The vagotomized group showed significantly decreased ulcer index after the stress procedures compared to the control group, however, the gastric mucosal blood flow showed almost the same values as the control group. The jaundiced and vagotomized group showed significant decrease of ulcer index and improvement of decrease of gastric mucosal blood flow compared to the jaundiced group. These results indicated that the gastric mucosal blood flow plays an important role in the occurrence of acute gastric ulceration in rats with obstructive jaundice, and vagotomy might be useful to prevent them by maintaining gastric blood flow in obstructive jaundice.
This study was designed to identify factors which contribute to the acceptance of relaxation. Using the Dohsa Training technique, each of 58 female undergraduate students was given a relaxation procedure applied to the trunk and back. Following this relaxation procedure, subjects were asked to complete a 20 item questionnaire in which a five point rating scale served as an index of the degree of acceptance of relaxation. Factor analysis of the results led to identification of the following three factors: (I) psychological acceptance of the experimental situation; (II) differentiated and integrated bodily awareness; and, (III) feeling of generalized relaxation. A comparison of the factor scores of training and non-training experienced subjects revealed higher scores for the former on Factors I and II. On the other hand, non-training experienced subjects scored higher on Factor III. In addition, the difference in factor scores between concentrated and non-concentrated subjects indicated that volitional concentration on the relaxation process is indispensable for reorganizing self body-image.
This study elucidates changes in membrane structure and permeability due to the methods of sterilisation and the conditions under which they are carried out. Tubular dialysis membranes of regenerated cellulose having various values for porosity were sterilised by ethylene oxide gas, autoclave or gamma irradiation under varying conditions. Non-sterilised membranes were included as controls. The solute permeability of the membranes was determined using 14C-urea. The membranes tested showed no difference in clearance of urea or creatinine. Gamma-ray sterilisation under dry conditions greatly reduced the vitamin B12 clearance and hydraulic permeability of membranes with a water content of below 60%. Hydraulic permeability increased with gamma irradiation for membranes sterilised under wet conditions. A reduction in vitamin B12 clearance for membranes with a water content of above 60% resulted after autoclave sterilisation. Pore model calculation reveals that membrane shrinkage resulted from sterilisation both by gamma-rays under dry conditions, and by autoclave. Thus, the structure of dialysis membranes varies with the method of sterilisation and the conditions under which the sterilisation is carried out.
The mechanism of acute ulceration in association with obstructive jaundice was studied experimentally by measurement of gastric mucosal blood flow and energy kinetics. The effect of biliary drainage was also evaluated. Three hundred thirty nine SD male rats weighing about 250 gr were grouped as follows; control group--Sham operation, obstructive jaundice group-2 weeks after ligation and dissection of the choledochus, and biliary drainage group-- A silicone tube was inserted and opened after 2 weeks of obstructive jaundice. All the animals were stressed by water immersion and ulcer index, gastric mucosal blood flow, gastric mucosal energy kinetics and liver function were assessed. The obstructive jaundice group showed significant higher ulcer index and lower gastric mucosal blood flow and energy kinetics than the control group. These factors of the biliary drainage group started to improve soon after biliary drainage and reached to the levels of the control group. In conclusion, reduction of defensive factors caused by jaundice was proved to play an important role in acute ulceration associating obstructive jaundice. Biliary drainage improves both gastric mucosal blood flow and gastric mucosal energy kinetics so that it is preventive against acute ulceration.
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The effect of water immersing and restraint stress upon the gastric acid secretion and gastric mucosal defensive factors was investigated in rats with special reference to the influence of obstructive jaundice and vagotomy. The results obtained were as follows. Water immersing and restraint stress ulcer may be caused by increased gastric acid secretion and deteriorated gastric defensive factors. Enhanced ulceration in jaundiced rats may be caused by severely damaged gastric defensive factors, rather than by the increased gastric acid secretion. Vagotomy itself, cannot inhibit stress ulceration completely, especially under the condition of damaged gastric defensive factors.
The mechanism of acute gastric ulcerations in rats with obstructive jaundice was investigated in terms of the changes in the gastric mucosal blood flow. Further, the effect of vagotomy with or without obstructive jaundice was examined. One hundred and eighty-eight Sprague-Dawley rats weighing about 250 g were prepared and divided into 4 groups as follows; sham operation (control group), ligation of the bile duct (jaundiced group), vagotomy with pyloroplasty (vagotomized group), ligation of the bile duct and vagotomy with pyloroplasty (jaundiced and vagotomized group). After two weeks, water immersion and restraint stress procedures were performed in these 4 groups. The gravity of the gastric ulcerations was calculated by ulcer index. The gastric mucosal blood flow was measured by hydrogen clearance technique. The following results were obtained. The control group showed increased ulcer index and decreased gastric mucosal blood flow after the stress procedures. The jaundiced group showed significantly higher ulcer index and early, significant decrease of gastric mucosal blood flow compared to the control group after the stress procedures. The vagotomized group showed significantly decreased ulcer index after stress procedures compared to the control group, however, the gastric mucosal blood flow were unchanged between both groups before and after the stress procedures. The jaundiced and vagotomized group showed significant decrease of ulcer index and improvement of decrease of gastric mucosal blood flow compared to the jaundiced group. These data suggested that the significant decrease of gastric mucosal blood flow during the stress procedures was one of exacerbating factors of the acute gastric ulcerations in rats with obstructive jaundice, and vagotomy might prevent them by maintaining the gastric mucosal blood flow in obstructive jaundice.
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A direct assay system for conjugated bile acids using an enzymatic procedure and high-performance liquid chromatography was used for the analysis of urinary bile acid profiles in young infants with intrahepatic cholestasis (idiopathic neonatal hepatitis syndrome) or extra-hepatic biliary atresia. The major urinary bile acids were cholate and chenodeoxycholate conjugates, but a small amount of deoxycholate and 3 beta-hydroxy-5-cholenate conjugates were detected. Although there was no significant difference in total bile acid excretion between patients with intrahepatic cholestasis and extrahepatic biliary atresia, mean ratios of cholate to chenodeoxycholate and sulfated to total urinary bile acids were different between the two groups examined (5.63 +/- 2.83 vs. 2.50 +/- 1.25, p less than 0.05, 15.8 +/- 9.9 vs. 34.5 +/- 9.9%, p less than 0.005). The proportion of taurine-conjugated chenodeoxycholate in the sulfate fraction to the total bile acid was lower in intrahepatic cholestasis, compared with that in biliary atresia (7.7 +/- 7.5 vs 22.7% +/- 7.8%, p less than 0.005). The greater ratio of cholate to chenodeoxycholate and the reduced excretion of sulfated urinary bile acids in intrahepatic cholestasis was due to decreased taurine-conjugated chenodeoxycholate sulfate excretion.