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Biomedical subjects

J Okuda

Publications and source records attributed to J Okuda.

At least 91 records · Page 5Linked to original sources
PubMed1995

Thyroid-stimulating antibodies in sera from patients with Graves' disease are heterogeneous in epitope recognition.

Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354-14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354-14 and five others were absorbed by both peptides to an almost similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1995

Presence of heterogeneous thyroid-stimulating antibodies in sera from individual Graves' patients as shown by synthesized thyrotropin receptor peptide application: evidence showing two independent epitopes and a possible recognition of two epitopic regions by one antibody molecule.

To define the epitope(s) of stimulating thyrotropin receptor antibody (TSH-R-Sab), we synthesized 19 oligopeptides covering almost all amino acids of the extracellular domain of the human TSH-R and studied these effects on the inhibition of one TSH-R-Sab activity. Four of the 19 peptides encompassing residues 31-50 (P31-20), 91-119 (P91-29), 287-304 (P287-18) and 354-367 (P354-14) were found to show significant TSH-R-Sab inhibition and to have similar effects on the other three Graves' immunoglobulins. When these peptides were applied in combination with P354-14 only P287-18 revealed additional effects but the other two combinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequential peptide-affinity gel studies were then performed. Most of the TSH-R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbed to a subsequent P354-14 gel and the eluted fraction from P287-18 mostly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by the subsequent P354-14 gel. When a P354-14 affinity gel-purified TSH-R-Sab immunoglobulin was labeled and evaluated for its binding to FRTL-5 cells, additions of original immunoglobulin, P354-14 and P91-29 resulted in significant inhibition of the binding but P287-18 did not affect either. From these results, it was concluded that most of the individual Graves' immunoglobulins contain at least two heterogeneous moieties with TSH-R-Sab activity, one of which binds P354-14 and the other binds P287-18. Further, P354-14 and P91-29 were indicated to bind the same molecule of TSH-R-Sab immunoglobulin.

Absorption↗
PubMed1994

Evidence for glucokinase translocation by glucose in rat hepatocytes.

By an immunohistochemical technique, we examined changes in the subcellular distribution of rat liver glucokinase in response to external stimuli. Glucokinase immunoreactivity was found predominantly in the nucleus of hepatocytes. In situ perfusion of the liver with 20 mM glucose for 10 min caused a marked decrease in nuclear immunoreactivity and an increase in cytoplasmic immunoreactivity. Insulin (10 nmol/l) potentiated this glucose effect. However, no change took place when 5 mM glucose was perfused. These results indicate that glucokinase is translocated from the nucleus to the cytoplasm in response to a high concentration of glucose.

Animals↗
PubMed1994

Glucagon responses in rabbits with obstructive jaundice and a low energy status in the liver.

Glucagon has a choleretic effect and also stimulates energy-consuming reactions, such as gluconeogenesis and ureogenesis in the liver. The effect of glucagon on energy metabolism in the liver was analyzed in rabbits with obstructive jaundice that had severe liver damage, indicated by significantly lower hepatic energy charge levels. The gluconeogenetic responses to glucagon administration, as shown by increases in the plasma glucose levels, were usually observed in normal rabbits, but these responses were completely depressed after glucagon administration in rabbits with obstructive jaundice. The energy charge levels in the latter animals decreased even further after glucagon administration. Even though the energy consumption for gluconeogenesis after glucagon administration was limited in the jaundiced rabbits, it was sufficient to cause a deterioration in the hepatic energy balance. The above findings show that the administration of glucagon increases the amount of energy expended in the liver following a reduction in the hepatic energy charge in rabbits with obstructive jaundice. Therefore, even though glucagon has a choleretic effect, it should be administered carefully to patients with obstructive jaundice, especially in those with severe liver damage, such as in patients with cholangitis.

Adenine Nucleotides↗
PubMed1994

Purification and characterization of triokinase from porcine kidney.

In order to be able to use triokinase for the enzymatic assay of tissue glyceraldehyde, we purified the enzyme to homogeneity from porcine kidney and characterized its biochemical properties. The purification was performed by polyethylene glycol fractionation, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic chromatography, and gel filtration. The enzyme was purified 937-fold from the crude extract with an overall yield of 28%. It had a molecular weight of 122,000 and was a dimer composed of identical subunits. The optimal pH and optimal temperature were 7.0 and 60 degrees C, respectively. This enzyme was stable when incubated at pH 7.0 at 40 degrees C for 1 h in the presence of 0.1 mg/ml bovine serum albumin. No loss of activity occurred for at least 1 month when the enzyme was stored at 4 degrees C in the presence of 1 mM dithiothreitol and 15 mM NaN3 under N2. Only three compounds, i.e., D-glyceraldehyde, dihydroxyacetone, and glycolaldehyde, acted as the substrate of the enzyme, having Km's of 11, < 5, and 260 microM, respectively. The Km for ATP-Mg2+ was 68 microM. These results indicate that porcine kidney triokinase has properties advantageous for the glyceraldehyde assay using glyceraldehyde-3-phosphate dehydrogenase as a coupling enzyme.

Amino Acid Sequence↗
PubMed1994

Purification of rat liver glucokinase.

A new purification method for rat liver glucokinase was developed. Glucokinase was purified to homogeneity in a yield of 70% in 5 days. The procedure consists of DEAE-cellulose ion-exchange chromatography, QAE-Toyopearl ion-exchange chromatography, glucosamine-Sepharose affinity chromatography, and HiLoad Superdex 200 gel filtration. Purified glucokinase had a specific activity of 200 units/mg protein and was highly stable in the presence of 100 mM glucose, 300 mM KCl, and 20% glycerol. We found that some of the methionine residues of glucokinase were oxidized to methionine sulfoxide residues during dialysis in the presence of glucose. It would appear that this oxidation is caused by formation of hydroxyl radicals in the presence of glucose and contaminating transition metal(s).

Animals↗
PubMed1994

Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal.

Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.

Animals↗
PubMed1994

Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema.

Anti-TSH receptor antibodies (TSH-R Ab), which have been detected in the serum of some patients with primary myxedema, are themselves considered to induce hypothyroidism. These are termed blocking-type TSH-R Ab (TSH-R BAb), because they inhibit adenylate cyclase stimulation by TSH on thyrocytes or nonthyroidal cells transfected with TSH-R complementary DNA. We prepared monoclonal TSH-R BAb and characterized them. Peripheral lymphocytes from three patients with primary hypothyroidism and potent TSH-R BAb were transformed by Epstein-Barr virus, and the culture supernatants were screened by TSH binding inhibitor immunoglobulin (TBII) assay. Twenty positive and 7 negative lymphocyte clones were obtained; their monoclonality was confirmed by Southern blot analysis, using an immunoglobulin (Ig) JH probe. These monoclonal antibodies were then tested for TSH-R BAb activity. TSH-R BAb activity ranged from 24.1-58.5% (normal range, < 24%) in all 20 TBII-positive clones and in 2 of 7 TBII-negative clones. An enzyme-linked immunosorbent assay showed that the Ig isotypes of these clones with TBII and/or TSH-R BAb activity were IgG in 8 and IgM in 14. Another enzyme-linked immunosorbent assay and Southern blot analysis of the light chains revealed that 13 clones had kappa-chains, whereas the light chains could not be determined in the other 9 clones. To summarize, 1) we obtained 22 clones that produced monoclonal TSH-R BAb, including 8 IgG-type clones. 2) The clones exhibited dominant usage of the kappa-chain. 3) Although all TBII clones had TSH-R BAb activity, their TBII and TSH-R BAb activities were not significantly correlated, and two TSH-R BAb clones did not show TBII activity.

Animals↗
PubMed1994

Recent progress in TSH receptor studies with a new concept of "autoimmune TSH receptor disease".

Recent progress in the studies of epitope analysis of the TSH-R against TSH-R Ab was reviewed extensively. Using both site-directed mutagenesis and synthetic TSH-R peptide, binding and/or action sites of TSH-R Ab have been known to be multiple and discontinuous, but the significance of two unique and TSH-R specific regions has been implicated. Further, the possible existence of heterogeneity among stimulatory TSH-R Ab has also been indicated. As for immunogenetic factors related to autoimmune thyroid diseases, studies on HLA analysis, TSH-R specific T lymphocyte analysis and VH analysis of TSH-R Ab were discussed. Of note was the negative association of HLA-DP w2 antigen in Japanese patients with Graves' disease and hypothyroidism due to blocking TSH-R Ab, and we proposed a new concept of autoimmune TSH receptor disease within autoimmune thyroid disease and emphasized possible critical roles of HLA-DP. Recent evidence of restricted usage of the Ig VH gene in cloned B lymphocytes from Graves' patients producing TSH-R Ab has also been presented.

Autoimmune Diseases↗
PubMed1993

Stimulatory effect of phospholipase A2 treatment on glucose utilization in human erythrocytes.

We examined whether modification of membrane phospholipids of human erythrocytes by hydrolysis with phospholipase A2 (PLA2 from bee venom) would affect glucose utilization, chosen as a typical model of intracellular metabolism, and, if so, intended to clarify the mechanism of the alteration of glycolysis. Treatment of erythrocytes with PLA2 induced a marked shape change (i.e., crenation) and significantly increased the rate of lactate production from glucose. Available evidence indicated that there is no relevance of this cell-shape change to the alteration of glycolysis. The lack of a detectable effect of papain treatment on glycolysis in PLA2-treated cells suggested that the increase in glycolysis by PLA2 treatment might not be caused by the conformational change of band-3 protein through modulation of membrane phospholipids. The result of the measurement of lactate production in the presence and absence of ouabain did not support the idea that hydrolysis of phospholipids by PLA2 treatment makes plasma membranes leaky to Na+ and consequently enhances glycolysis through activation of Na+/K(+)-ATPase. The action of PLA2 on glycolysis was abolished by extraction of free fatty acids in the cell membrane with bovine serum albumin. Loading erythrocytes with free fatty acid (oleic acid, linoleic acid, or arachidonic acid) caused a significant increase in glycolysis. Analysis of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of 6-phosphofructokinase. The data, thus, indicate that treatment of human erythrocytes with PLA2 significantly accelerates glucose utilization and suggest that the stimulation of glycolysis is caused by activation of 6-phosphofructokinase through liberation of free fatty acids of membrane phospholipids by PLA2.

Adult↗
PubMed1993

Purification and some properties of a Vero toxin 2 (Shiga-like toxin II) variant (SLT-IIva) of Escherichia coli isolated from infantile diarrhea.

A variant of Vero toxin 2 (VT2), or Shiga-like toxin II (SLT-II) was purified from a cloned strain of Escherichia coli carrying a gene that encodes SLT-IIva, which is most closely related to SLT-IIv (VT2vp, VTe) in its nucleotide sequence. The purified SLT-IIva showed a slight difference from the purified SLT-IIv (VT2vp) in mobility on polyacrylamide gel disc electrophoresis and SDS-polyacrylamide gel slab electrophoresis and in pI, but both showed a similar potency in biological activities. Ouchterlony double diffusion test revealed that the purified SLT-IIva and SLT-IIv (VT2vp) formed a spur against anti-SLT-IIva and anti-SLT-IIv (VT2vp) antisera.

Animals↗
PubMed1993

Alteration of anomeric preference of glucose-induced insulin secretion by glyceraldehyde.

Glucokinase activity in pancreatic islets was dose-dependently inactivated by D-glyceraldehyde, whereas islet hexokinase activity was not altered. In untreated islets, alpha-D-glucose stimulated insulin secretion more efficiently than beta-D-glucose at a glucose concentration of 10 mM. However, glyceraldehyde highly attenuated the insulin-secretory response of pancreatic islets to alpha-D-glucose compared with that to beta-D-glucose. Thus, there was apparently no anomeric preference of glucose-induced insulin secretion in glyceraldehyde-treated islets. Glyceraldehyde affected neither the alpha-preference of glucose phosphorylation by glucokinase nor the beta-preference of glucose phosphorylation by hexokinase. Our study suggests that defective discrimination of glucose anomers by glyceraldehyde-treated islets may be caused by inactivation of glucokinase.

Animals↗
PubMed1993

Extraction and quantitation of paraquat and diquat from blood.

Trace amounts of paraquat and diquat in blood were extracted with phenol after deproteinization of blood protein with a chloroform:ethanol mixture and ammonium sulfate. The color reaction of paraquat was achieved by addition of alkaline sodium dithionite to the phenolic extract. The blue paraquat radical produced was determined directly by measurement of the absorption of the phenol layer. The assay of paraquat (> or = 0.5 micrograms) in 1.0 ml of blood (recovery, 93.4%) could be performed within 30 min. Furthermore, simultaneous analysis of paraquat and diquat in the phenolic extract of the sample could be achieved by use of second-derivative spectroscopy within 30 min.

Ammonium Sulfate↗
PubMed1993

Immunoglobulin G that interferes with thyroid-stimulating antibody measurements can be eliminated specifically by incubation with synthetic peptides corresponding to partial sequences of the human thyrotropin receptor.

Two IgG preparations out of more than 100 tested, distinct from the typical Graves' disease IgG, were shown specifically to enhance the cAMP production of FRTL-5 cells by the addition of a synthetic peptide, P-218, corresponding to the partial amino acid sequence from No. 354 to 367 of the h thyroid-stimulating hormone (TSH) receptor. IgG obtained from a patient with Graves' disease revealed a serial alteration of the enhancement; negative in July, 1989, potent in January, 1991, and weak in September 1991. During this time there was no remarkable change in the patient's serum protein components or TSH receptor antibody activities. A peptide with a completely reverse sequence of P-218 showed little effect, and P-218 in combination with bTSH or forskolin did not affect cAMP production by these ligands, and did not alter the inhibitory activity of thyroid-stimulation-blocking antibody. High concentrations of P-218 resulted in reduction of such enhancing effects of cAMP by thyroid-stimulating antibody. P-218 affinity chromatography showed almost complete absorption and recovery of thyroid-stimulating antibody and P-218 reactivity. In the 15 synthesized peptides with proximal sequences of P-218 (from 338 to 378), regions thought to be involved with the enhancement were defined as follows: 354-367 (P-218) is a critical unit; 354-357 and 364-367 are considered to be the essential sites; several amino acid extensions on both N- and C-terminal sides of P-218 show additional enhancement. In conclusion, evidence was shown to indicate the presence of IgG that interferes with thyroid-stimulating antibody measurements.

Amino Acid Sequence↗
PubMed1993

Inhibition of glucose-induced insulin secretion through inactivation of glucokinase by glyceraldehyde.

D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37 degrees C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.

Amino Acids↗
PubMed1993

[History of professionalization of modern clinical pharmacist (1): pharmaceutical practices in hospice before the French Revolution].

The times around the French Revolution, as E. H. Ackerknecht pointed out, were especially epochmaking in that "the clinical medicine" or 'medicine in the hospital' was born and bred through the days by discarding the medicine of court physicians and the metaphysical medicine that had been dominant for centuries until then. This change is, therefore, very important from the viewpoints of sociopolitical, cultural and medical histories. The medicine practised and taught mainly in Paris in those days based politically on the newborn nationalistic centralization; technically on pathologicoanatomy and biostatistics; and conceptually on the modern medical epistemologoy that made it possible to abstract and see a 'disease' from the sick by observation, description and classification of their common signs and symptoms, and then to ignore a suffering specific to each individual patient instead. Thus those who could believe in the être (entity) of a 'disease' the clinical medicine made it 'visible' were trained for medical professionals such as modern physicians, surgeons and clinical pharmacists, which finally realized the professional monopolization of the medicine. The aim of this paper is to report and analyze the historical process how there came out the modern clinical pharmacist who was permitted by the nation as the only occupation to deal in ethical drugs in the hospital, while excluding many other people who had been treating 'traditional medicines' from the modern pharmacies of hospitals.

Clinical Medicine↗