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Biomedical subjects

T Utsumi

Publications and source records attributed to T Utsumi.

At least 19 recordsLinked to original sources
PubMed1992

Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor.

The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Neutrophil priming by granulocyte colony stimulating factor and its modulation by protein kinase inhibitors.

Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-.) generation, phagocytosis enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca(2+)-and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2-. generation in PMN was enhanced by the priming of recombinant human granulocyte colony stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2-. by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2-. generation from G-CSF were 0.5 and 5 microM, respectively. In contrast, O2-. generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genistein and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2-. might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2-. generation of neutrophils.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.

Adenosine Triphosphate↗
PubMed1992

The role of amino functions in recombinant human tumor necrosis factor in expression of biological activity.

The role of amino functions in the expression of the biological activity of recombinant human TNF (rHuTNF) was studied by chemical modification. rHuTNF is a homotrimer of 17 kD subunits, each of which contains an N-terminal valine and six lysyl residues: two of these lysyl residues are known to be involved in intra- or intersubunit interactions. Chemically reactive amino functions were modified with the N-hydroxysuccinimide ester of acetic acid; modification of amino groups to amide, and the concomitant loss of charge, was monitored by native PAGE. When rHuTNF was reacted with the active ester at increasing mole ratios, up to 12 amino groups per trimer could be modified. When the biological activity of acetylated rHuTNF was determined, a strong correlation between the extent of modification and loss of biological activity was observed. One to three amino groups per trimer could be modified with nearly complete retention (approximately 80-95%) of biological activity; activity was essentially completely destroyed at the highest levels of modification. These results reveal important functions for the amino groups of rHuTNF and significant constraints on strategies involving their modification in development of second-generation-TNF variants.

Acetylation↗
PubMed1992

Structural organization and expression of the gene for bovine myosin I heavy chain.

Brush border myosin I heavy chain (MIHC), known previously as the brush border 110-kDa protein, contains an amino-terminal sequence which is highly homologous to the globular head domain of conventional myosin II heavy chain (MIIHC). The carboxyl-terminal sequence of MIHC completely diverges from that of MIIHC and functions as calmodulin-binding and membrane-interaction sites. In this investigation, we determined the structural organization of the bovine MIHC by isolating a set of genomic segments containing the whole MIHC gene. The bovine MIHC gene is 26 kilobase pairs long and consists of 28 exons. At the homologous amino-terminal portion of MIHC, many introns are located at positions equivalent to those of the rat MIIHC gene and the amoeba MIHC gene. At the carboxyl-terminal sequence of MIHC, the putative calmodulin-binding and membrane-interacting domains are specified by discrete sets of exons. These findings support the view that the amino-terminal head portions of MIHC and MIIHC evolved from a common ancestral origin and also that the MIHC protein was generated as a result of fusion of discrete genomic segments encoding different functional and structural protein domains. Analysis of tissue expression of the MIHC mRNA was also extended in this investigation, and the results indicated that this mRNA is expressed in some tissues other than the intestines.

Amino Acid Sequence↗
PubMed1992

Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

[Inhibitory effects of calphobindins on phospholipase A2 and phospholipase C].

Calphobindins (CPBs) I, II and III which we isolated and characterized from placenta belong to a family of calcium and phospholipid binding proteins represented by lipocortins known to inhibit phospholipase A2(PLA2). The amino acid sequence of three CPBs shares much homology with lipocortins I and II, so we investigated the functions of CPBs in affecting the enzymatic activities of PLA2 and also phospholipase C(PLC). In our experiments CPBs I, II and III showed an apparent dose-dependent inhibition of PLA2 and PLC activities, and all three proteins had a more potent inhibitory effect than lipocortins. The inhibition was overcome by high phospholipid substrate concentrations, for example, in the presence of 2.9 x 10(-9)M PLA2 and 2.6 x 10(-7)M CPB I, the inhibition decreased from 85 to 9% as phosphatidylethanolamine was increased from 10 to 100 microM. A similar result was observed in the case of PLC also. The evidence strongly suggests that the inhibitory effect of CPBI results from binding of the proteins to the phospholipid substrate rather than from a direct action on the phospholipase itself.

Annexins↗
PubMed1992

[Age effects of topical tyramine and cocaine on the pupil].

In order to obtain the age distribution of normal values of the effects of topical tyramine or cocaine on the pupil, we measured the pupillary light reflexes of 50 normal volunteers before and 45 minutes (tyramine) or 90 minutes (cocaine) after the topical instillation in right eyes. Left eyes were used as controls. An infrared videopupillogram (HTV-C301) was used. Topical tyramine as well as cocaine caused mydriasis, increase of maximum %-velocity of dilatation (%-VDmax), reduction of %-amplitude of constriction (%-A) and decrease of maximum %-velocity of constriction (%-VCmax). These alternations of pupillary parameters coincided with the changes induced by topical epinephrine. The effects of tyramine or cocaine significantly increased as the age of the subjects increased, though the increase of %-VDmax caused by cocaine was only related to age. Age-related augmentation of pupillary changes were also been observed in a previous study using topical epinephrine or topical pilocarpine which concluded that the effects of any topical autonomics were increased by aging. No physiological senile Horner's syndrome was detected. It was concluded that the age-related augmentation of pupillary changes caused by topical autonomics probably resulted from the increment of corneal permeability in the aged which was confirmed in the previous fluorophotometric study by the authors following topical fluorescein administration. It is possible to assess the pupillary sensitivity to topical adrenergics of the patients with bilateral Horner's syndrome by comparing the age-related normal values obtained in this study.

Adolescent↗
PubMed1992

[A pupillographical study on the effects of cataract surgery on the contralateral eye].

In order to estimate the effect of cataract surgery on the contralateral eye, we examined pupillary light reflex and performed laser flare-cell measurement. The subjects were nine patients who underwent extracapsular cataract extraction (ECCE) and fourteen patients who underwent ECCE with intraocular lens implantation. Pupillary light reflexes of the contralateral eyes were examined using a computerized videopupillogram (HTV C-301). Aqueous cells and flare of both eyes were measured by a laser flare-cell meter (KOWA FC 100). The author performed five successive measurements before and 1, 3 days, 1, 2 weeks and 1, 2, 3 months after surgery and statistically estimated the consensual effect of cataract surgery. As a result of contralateral cataract surgery, pupils became significantly miotic from the first postoperative day for more than 3 months without any alteration of other parameters of light reflex. On the contrary aqueous cells and flare significantly raised from the first postoperative day and disappeared within 3 months. Consensual miosis was considered to be sensitive indicator of postoperative inflammation of cataract surgery as well as contralateral increase of aqueous cells and flare.

Adult↗
PubMed1991

Preparation and characterization of liposomal-lipophilic tumor necrosis factor.

The liposomal delivery of recombinant human tumor necrosis factor (rHuTNF) has been shown to be effective in reducing its toxic effects and in its targeting of organs rich in cells of the reticuloendothelial system. However, native recombinant human TNF shows only poor affinity for liposomes, presumably due to its low hydrophobicity. In an attempt to increase the efficiency of association with liposomes, we have modified TNF to increase its hydrophobicity by selective substitution of its amino groups with fatty acid side chains. N-hydroxysuccinimide esters of saturated fatty acids ranging from C8 to C18 were reacted with recombinant human TNF. Modification with esters of C8 to C14 acids occurred as determined by consumption of positively charged amino groups monitored by native polyacrylamide gel electrophoresis; however, esters of longer chain lengths (C16, C18) were much less capable of introducing these chains via amide linkages, and thus these adducts were not further characterized. Biological assays revealed that retention of activity was inversely dependent both on the number of chains introduced and on their length; activity was most conserved (greater than 50%) in a TNF preparation modified with only approximately 1-2.5 caprylic acid (C8) residues/trimer. This preparation was found to bind with high efficiency (approximately 50%) to preformed dipalmitoylphosphatidylcholine-small unilamellar vesicles. The extent of binding closely paralleled both the number of chains introduced and their length; binding was even more efficient (80-90%) for TNF modified either with approximately 3.5 caprylic acid residues/trimer or with approximately 1.5 residues of myristic acid (C14). However, the biological activity of these acylated TNFs was further reduced by this more extensive chemical modification (less than 50% activity for C8 and less than 10% for C14). The biological activity of dipalmitoylphosphatidylcholine-small unilamellar vesicle-C8-TNF was found to be comparable to that of the nonliposomal C8-TNF. Thus, biologically active preparations of liposomal-lipophilic TNF can be prepared with high efficiency.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Inhibition of 12-O-tetradecanoyl phorbol-13-acetate promoted tumorigenesis by cepharanthine, a biscoclaurine alkaloid, in relation to the inhibitory effect on protein kinase C.

In two-stage mouse skin carcinogenesis initiated by 7,12-dimethylbenz[alpha]anthracene (DMBA), cepharanthine inhibited the tumor promoting activity of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Since Ca2(+)-phospholipid-dependent protein kinase (PKC) was shown to be an intracellular target of TPA, effects of cepharanthine on the activity of this enzyme were investigated Cepharanthine also inhibited the phosphorylation of H1 histone by PKC in a concentration dependent manner. While cepharanthine inhibited the association of H1 histone with phospholipid vesicles, autophosphorylation of PKC was not inhibited by this drug. Cepharanthine also inhibited TPA-stimulated phosphorylation of some cytoplasmic proteins of mouse skin epidermis. These results indicated the possibility that anti-tumor promoting action of cepharanthine was the result of inhibition of PKC dependent cytoplasmic protein phosphorylation through the reduction of the interaction of these proteins with the plasma membrane.

Alkaloids↗
PubMed1991

Targeting SOD by gene and protein engineering and inhibition of free radical injury.

Although oxygen toxicity of tissues can be decreased by a variety of antioxidants and some enzymes, such as SOD and catalase, their protective effect on tissue injury in various diseases are fairly small predominantly because of their unfavorable in vivo behavior. To minimize oxidative stress in various diseases, such as ischemic myocardial injury, circulatory disturbance and corneal inflammation, we synthesized three types of SOD derivatives by gene and protein engineering technique. One type of SOD (SM-SOD covalently linked with hydrophobic anions) circulates bound to albumin with a half life of 6 h and accumulates in tissues whose local pH is decreased. The other type of SOD (AC-SOD covalently linked with long chain fatty acids via the epsilon-amino group of lysyl residues) anchors onto membrane/lipid bilayers of various cells. The last type of SOD (HB-SOD synthesized by constructing a fusion gene coding human CuZn-type SOD and a C-terminal heparin-binding domain) binds to heparin-like proteoglycans on vascular endothelial cell surface. Intravenous administration of either SM-SOD or HB-SOD markedly inhibited postischemic reflow arrhythmias in the rat. When the left anterior descending artery was occluded permanently, about 65% of animals died within 30 min predominantly due to irreversible ventricular fibrillation; the motality of animals decreased to 15% by administering SM-SOD either before or after occlusion. Topically administered AC-SOD bound to the corneal epithelial cell surface and polymorphonuclear leukocytes and efficiently dismutated superoxide radicals at their cell surface. Thus, endotoxin-induced keratitis was inhibited markedly by topical instillation of AC-SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

[A follow-up study of pupillary dynamics in patients with ocular hypertension and primary open angle glaucoma].

The authors previously reported light reflex abnormalities and adrenergic supersensitivity to topical epinephrine (DPE; dipivalyl epinephrine) of the pupils in patients with ocular hypertension (OH) and those with primary open angle glaucoma (POAG). In this investigation, we attempted to reconfirm pupillary light reflex abnormalities we reported previously, and to investigate the relationship between the normotensive effect of 0.1% DPE and the pupillary light reflex abnormalities in OHs and POAGs. A total of 11 OHs and 11 POAGs under good oculotensive control with neither mydriatics nor miotics were examined. They were measured by an open-loop photically stimulated infrared videopupilogram, and were neurologically diagnosed by comparing the simulated patterns of the light reflex made by topical autonomic agents. When we considered the progress of the stage of POAG, both OHs and POAGs showed satisfactory reproducibility of pupillary light reflex abnormalities. Other cases in which light reflexes altered, showed worsening of the visual field deficit, which was prominent when OHs developed into POAG. OHs with significant reduction of intraocular pressure (IOP) after topical administration of 0.1% DPE showed various kinds of abnormal pupillary light reflexes. On the contrary, cases with little reduction of IOP after 0.1% DPE instillation showed normal pupillary light reflexes in OHs and afferent pupillary defect on POAGs.

Adult↗
PubMed1991

[Balloon occlusion pulmonary angiography and anticoagulant-antithrombotic therapy in ARDS-associated pulmonary vascular thrombosis].

Balloon occlusion pulmonary angiography was used to assess the frequency of pulmonary vascular thrombosis in five patients suffering from ARDS of diverse causes. Pulmonary hypertension, elevated pulmonary vascular resistance and disseminated intravascular coagulation were observed in all patients. Pulmonary artery filling defects (PAFD) were found in four of five patients. The results suggest that PAFD can be detected in a large proportion of patients in ARDS. It was therefore speculated that the detection of PAFD is a sign of severe lung injury. Urokinase, heparin and protease inhibitor were infused to test the potential reversibility of pulmonary vascular thrombosis in four patients. After infusion, we found angiographic evidence of clearing of obstruction in arteries in three of four patients. We conclude that anticoagulant-antithrombotic therapy can improve hemodynamics in ARDS-associated pulmonary vascular thrombosis.

Adult↗
PubMed1990

[Investigation of where schizophrenic patients receive treatment].

Schizophrenic patients receiving treatment in Kagawa Prefecture were studied as to where treatment was obtained. Analysis showed that there was a tendency by schizophrenic patients to seek treatment in institutions distant from their place of residence. The direction in which a patient proceeded to seek treatment varied and showed no pattern. Analysis by age groups showed that patients in the 15 to 39 years age group were most likely to seek treatment in locations other than where they lived and traveled the longest distance. The tendency to seek treatment away from their place of residence decreased with increasing age. However even among the elderly there were patients who traveled long distances for treatment. Factors associated with geographical or social relationships between the cities/towns could not explain the tendency observed. Neither was there a relationship to specialized medical care. Factors such as the related to the mental health care system, including prejudice and lack of societal understanding were inferred to be associated to this problem. Results of this study indicate a need to organize the community mental health system to serve districts with wider areas than that established for other diseases. However in the future there should be a gradual narrowing of the district area, a broadening of services offered, and a removal of societal prejudice against psychiatric patients.

Adolescent↗
PubMed1990

Corneal permeability in patients with tonic pupil. A reevaluation of its cholinergic supersensitivity.

Cholinergic supersensitivity of the iris sphincter helps to make the diagnosis of "tonic pupil." Because uncertain responses sometimes occur with mecholyl 2.5%, dilute solutions of pilocarpine are often used. There has always been some question as to whether individual variation in corneal permeability was one of the factors contributing to the variability in pupillary constriction to topical cholinergics in these patients. In this study, the degree of intraocular penetration of topical fluorescein was compared with the degree of pilocarpine-induced miosis in six tonic pupils and in seven age-matched normal controls. A noncontact fluorophotometer and a computerized videopupillogram were used. The patients showed a significant cholinergic supersensitivity in their affected tonic pupils and had normal intraocular penetration of fluorescein through the cornea in both eyes. It is concluded that the cholinergic supersensitivity of the iris in tonic pupils is not the result of increased corneal permeability but instead results from an increased sensitivity of the cholinergic receptors in iris sphincter muscles.

Adult↗