Search PubMed⌕ Search

Biomedical subjects

T Ohkubo

Publications and source records attributed to T Ohkubo.

At least 217 records · Page 12Linked to original sources
PubMed1994

Heparin Binding Sites Are Located in a 40-kD gamma-Chain and a 36-kD beta-Chain Fragment Isolated from Human Fibrinogen.

Objective: We have previously shown that (125)I-fibrinogen binds to heparin sepharose CL-6B. To identify the localization of the heparin binding domain in human fibrinogen, reduced and alkylated fibrinogen was digested by limited-Staphylococcus aureus V8 protease. Methods/Results: Two fragments have now been isolated and purified to apparent homogeneity by heparin-affinity chromatography. These fragments, denoted the 40-kD and 36-kD fragments, contain NH(2)-terminal sequences of Ala-Ser-Ile-Leu-Thr-Hb;-Asp and Thr-Val-Asn-Ser-Asn-Ile-Pro, respectively. These fragments established the positions of these peptides within the gamma chain of fibrinogen as beginning with the residue tentatively designated 124 and within the beta chain as beginning with the residue designated 186. Binding of (125)I-fibrinogen to heparin-sepharose CL-6B was completely inhibited by a mixture of these fragments, with an IC(50) of 3.2 µM. The synthetic peptide of the gamma chain carboxy-terminal 15 residues (GQQHHLGGAKQAGDV;G15) partially inhibited fibrinogen binding. The mixture of these fragments partially inhibited the ADP-induced aggregation of platelets. Conclusions: These data indicate that the domains for heparin binding may be present on both the gamma chain and the beta chain of fibrinogen, and that the domain on the gamma chain may be close to the binding domain on the carboxy terminus of the fibrinogen gamma chain to glycoprotein IIb-IIIa.

Journal Article↗
PubMed1994

An evaluation of a new lactic acid polymer drug delivery system: a preliminary report.

A new drug delivery system, made of a mixture of DL-lactic acid polymer and a copoly (L-lactic acid/delta-valerolactone) polymer (LAP) containing varying concentrations of Ofloxacin, has been evaluated. Results revealed that a LAP drug delivery system containing 0.03% Ofloxacin inhibited the in vitro growth of clinically derived Staphylococcus aureus and Pseudomonas aeruginosa. Further, in an in vivo situation in rats, at 1 week after the wound was inflicted, no infection was macroscopically seen in split-thickness wounds that had been covered by LAP drug delivery system containing 0.5 or 0.03% Ofloxacin, and more than 80% of the wound area had epithelialised.

Animals↗
PubMed1994

"Bridged" peptide of fibrinogen binds to platelets activated by RGD peptide as well as by ADP.

We have evaluated the binding of a bridged peptide of Y-HHLGGAKQAGDV(G)9RGDS to RGDS-activated platelets. The bridged peptide saturably bound to platelets activated by RGDS peptide and by ADP with binding sites of 38,600 +/- 4800 molecules/platelet (Kd = 8.9 +/- 1.3 x 10(-7) M) and 43,600 +/- 9,200 molecules/platelet (Kd = 3.1 +/- 0.5 x 10(-7) M), respectively. Native fibrinogen inhibited the binding of the bridged peptide to platelets activated by RGDS peptide and by ADP with IC50s of 1.2 +/- 0.4 microM and 1.8 +/- 0.6 microM, respectively. In addition, the RGDS peptide and the dodecapeptide (gamma 400-411; HHLGGAKQAGDV) completely inhibited the binding of the bridged peptide to platelets activated by RGDS peptide with IC50s of 32 +/- 2.6 microM for RGDS and 102 +/- 4.1 microM for gamma 400-411, respectively. The anti-GPIIb/IIIa monoclonal antibody, LJ-CP8, also inhibited these bindings. The IC50s were 75 +/- 8.6 micrograms/ml for RGDS activation and 56 +/- 6.3 micrograms/ml for ADP stimulation. These results suggest that the flexible hinge region may allow assumption of the appropriate structure in the presence of the receptor complex. It indicates that both RGDS and agonist stimulation may play an important role in inducing activation of platelets that results in further platelet aggregation and tight thrombus formation.

Adenosine Diphosphate↗
PubMed1994

Autoimmune antibody in a patient with idiopathic thrombocytopenic purpura reacted to the platelet low molecular weight glycoproteins and activated platelets.

The authors characterized antiplatelet membrane antibodies in the sera of patients with idiopathic thrombocytopenic purpura (ITP) and cirrhosis of the liver. Antibodies were detected in five of the 22 patients with chronic ITP and in none of the eight patients with cirrhosis of the liver. The authors report a patient with chronic ITP in complete remission. Antibody to platelet glycoproteins (GP), with molecular weights of 55 and 49 kDa, was detected in the serum. The patient's immunoglobulin G (IgG) alone could cause the aggregation of platelet-rich plasma. Anti-GPIIb/IIIa antibody (LJ-CP8) inhibited the aggregation of platelet-rich plasma induced by the patient's IgG in a dose-dependent manner. The F(ab')2 of the patient's IgG had a synergetic effect on the aggregation of PRP induced by adenosine 5-diphosphate. This demonstrates that in ITP, the binding of IgG via its fragment of antigen binding site portion may activate platelets.

Aged↗
PubMed1994

The determination of ofloxacin in human skin tissue by high-performance liquid chromatography and correlation between skin tissue concentration and serum level of ofloxacin.

A simple high-performance liquid chromatographic (HPLC) assay was developed for the measurement of ofloxacin in skin tissue, and the correlation between serum levels and skin tissue levels of ofloxacin was determined. The homogenized skin samples were directly introduced into an HPLC system after filtering through a Molcut II membrane filter to remove proteins. The filtrate was concentration on a pre-column using a phenyl-stationary phase and was then introduced to an analytical column using an ODS-stationary phase by column-switching techniques. Ofloxacin and lomefloxacin as an internal standard were detected by UV absorbance at 300 nm. Determination was possible for ofloxacin over the concentrated range 250-3000 ng (g skin tissue)-1; the limit of detection was 100 ng g-1. The absolute recovery of ofloxacin added to skin tissue homogenate was over 72% with a coefficient of variation of less than 4.1%. This method is applied to determination of skin tissue level of ofloxacin in patients after treatment with ofloxacin. The correlation between serum levels and skin tissue levels of ofloxacin was determined for 30 patients after oral administration of ofloxacin. Good correlation was obtained and the coefficient of correlation was 0.84.

Administration, Oral↗
PubMed1994

Differential regulation of prolactin receptor mRNA expression in rat liver and kidney by testosterone and oestradiol.

Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Animals↗
PubMed1994

Primary empty sella in association with superior sagittal sinus thrombosis and dural arteriovenous malformation. Case report.

A 61-year-old woman under treatment with oral medroxyprogesterone acetate following surgery for breast cancer developed diplopia and papilledema due to raised intracranial pressure (ICP). Radiological studies disclosed an empty sella turcica, a dural arteriovenous malformation (AVM), and superior sagittal sinus thrombosis. The sinus thrombosis was considered to have been a side effect of the medroxyprogesterone acetate medication. The association between a primary empty sella and raised ICP is briefly discussed, along with the cause-and-effect relationship between a dural AVM and venous sinus thrombosis.

Arteriovenous Malformations↗
PubMed1994

A model of ergonomics intervention in industry: case study in Japan.

This paper presents the results of an ergonomics survey in four different Japanese work places. The survey consisted of two parts. In the first part, the physical and psycho-social problems of the female workers were investigated. Questionnaire techniques were used to assess musculoskeletal disorders in various parts of the body and of psycho-social stress at work as well as in the daily life situation. Furthermore, work posture analysis, task analysis, simple clinical tests and flexibility tests were conducted. In the second part, the ergonomics intervention practices in different types of Japanese work places were examined. Three different questionnaires were developed and used to collect information from representatives of employees, management and responsible governmental agents of the Department of Industrial Safety and Health, Ministry of Labor, with regard to ergonomic interventions at work places. Management, employees, and government representatives expressed desire for cooperation and participation for arrangement of the ergonomics intervention program. A model for appropriate ergonomics intervention in industry is developed with regard to physical and psycho-social problems at work. Factors influencing worker participation and the establishment of a dynamic system of ergonomics intervention at work places are discussed. Practical ways for improving the working conditions of female workers are: (i) cooperation between managers, workers, and government officers is regarded as vital for the ergonomics intervention program, (ii) worker participation, (iii) appropriate training course with regard to ergonomics education, and (iv) managerial support.

Adult↗
PubMed1994

[A case of esophageal cyst resected by thoracoscopic surgery].

A 59-year-old woman was admitted with back pain and an abnormal chest shadow in the left hilar region. Her chest CT scan showed a posterior mediastinal mass surrounding the descending aorta. By transesophageal ultrasonography, a diagnosis of mediastinal cyst was made. She underwent thoracoscopic resection under general anesthesia with new endoscopic devices including endo-scissors, endoscopic hemoclips and electrocautery. Pathologically, the tumor was an esophageal cyst lined by ciliated columnar and squamous epithelium overlying smooth muscle layers. She made an uneventful recovery. Her back pain subsided following the operation. Thoracoscopic resection is a useful technique for patients with mediastinal cystic tumor.

Esophageal Cyst↗
PubMed1993

Fibronectin inhibits platelet aggregation independently of RGD sequence.

Fibronectin binds to platelet membrane glycoprotein (GP) IIb-IIIa in Arg-Gly-Asp (RGD)-dependent and -independent manner. We have isolated and characterized the 29-kDa dispase fragment of fibronectin. Binding of 125I-fibronectin to thrombin-stimulated platelets was inhibited by the 29-kDa fragment and the GRGDSPA peptide with IC50 values of 1.5 +/- 0.4 and 8.1 +/- 0.9 microM, respectively. The NH2-terminal sequence of this fragment gave this result: Ala-Val-Thr-Thr-Ile-Pro-Ala-Pro-Thr-Asp. This established the position of this peptide within fibronectin as beginning with the residue tentatively designated 1597. Neither the RGDS sequence nor the RGD-independent binding domain of fibronectin (Bowditch, R. D., Halloran, C. E., Aota, S., Obara, M., Plow, E. F., Yamada, K. M., and Ginsberg, M. H. (1991) J. Biol. Chem. 266, 23323-23328) was contained in this fragment. The 29-kDa fragment inhibited ADP-induced aggregation of platelets and binding of fibrinogen to activated platelets. The fragment bound to immobilized GPIIb-IIIa. The 125I-labeled 29-kDa fragment directly bound to thrombin-stimulated platelets with 98,000 +/- 4,600 molecules/platelet (Kd = 4.6 +/- 0.5 x 10(-6) M). Direct binding was inhibited by the unlabeled 29-kDa fragment but was not blocked by either the GRGDSPA peptide or the monoclonal anti-GPIIb-IIIa antibody. These results indicate that the additional RGD-independent binding domain(s) to GPIIb-IIIa are present on fibronectin. This additional binding domain(s) on fibronectin may prevent thrombus formation by interfering with the interaction of fibrinogen with GPIIb-IIIa.

Adenosine Diphosphate↗
PubMed1993

Myocardial contractility and energetics in cardiac hypertrophy and its regression.

The changes in myocardial contractility and ventricular myosin isoenzymes were investigated in rats with pressure-overload cardiac hypertrophy as well as during its regression. Hypertrophic myocardium was obtained from rats with renovascular hypertension (Goldblatt rats), rats with abdominal aortic constriction (AC), and spontaneously hypertensive rats (SHR). Regression of cardiac hypertrophy was induced by lowering the blood pressure through nephrectomy on the affected side in Goldblatt rats, by opening the clip which constricted the abdominal aorta in AC rats, and by the administration of antihypertensive agents to SHR. The isometric developed tension of isolated left ventricular papillary muscles and the maximum rate of increase in the tension (dT/dtmax) were measured. Left ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis. Isometric developed tension remained unchanged, but dT/dtmax was decreased in hypertrophic myocardium, although it recovered along with the regression of cardiac hypertrophy. The left ventricular myosin isoenzyme pattern was shifted towards V3 in hypertrophic myocardium, and shifted back again towards V1 with the regression of cardiac hypertrophy. These results indicate that relief of hemodynamic overload is one of the most important elements in the regression of cardiac hypertrophy and the associated physiological or biochemical alterations. However, other factors such as neurohumoral influences must also be taken into consideration.

Animals↗
PubMed1993

Folding topology and DNA binding of the N-terminal fragment of Ada protein.

Three amino terminal fragments of Escherichia coli Ada protein (39 kDa) with different molecular masses (14 kDa, 16 kDa and 20 kDa) were prepared in large quantities from an E. coli strain harboring plasmids constructed for the overproduction of the truncated proteins. The three fragments can be methylated to an extent similar to that of the intact molecule. The methylated 16 kDa fragment specifically binds to the ada box on a DNA duplex. NMR analyses revealed that the 14 kDa fragment comprises two alpha-helices and a beta-sheet with parallel and anti-parallel mixed strands. A comparison of the 15N-1H HMQC spectra of the fragments has led to the conclusion that this tertiary structure within the 14 kDa fragment is retained in the larger 16 kDa and 20 kDa fragments.

Amino Acid Sequence↗
PubMed1993

Fibrinogen binds to heparin: the relationship of the binding of other adhesive proteins to heparin.

The binding of heparin to fibrinogen was characterized. We have used an assay employing heparin coupled to Sepharose CL-6B to show that 125I-fibrinogen binds to heparin in a time-dependent, divalent ion-independent, saturable, and reversible manner. A total of 1.75 mg of fibrinogen could bind to 1 ml of heparin-Sepharose CL-6B with a Kd of 4.03 x 10(-7) M. Binding was completely inhibited by other adhesive proteins including fibronectin, von Willebrand factor (vWF), and vitronectin with IC50s of 7.2-8.8 microM. The binding of 125I-fibrinogen to heparin-Sepharose CL-6B was blocked by a monoclonal antibody (52k-2) and a polyclonal antibody (FK-2). 52k-2 inhibits the binding of vWF, and FK-2 blocks the binding of fibronectin to heparin-Sepharose CL-6B. It is therefore likely that fibrinogen has a heparin-binding site(s) which may have some overlap with those of other adhesive proteins, although the physiological role of the fibrinogen heparin-binding domain is uncertain at present.

Antibodies, Monoclonal↗
PubMed1993

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

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

Amino Acid Sequence↗
PubMed1993

Determination of chlorpromazine in human breast milk and serum by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of chlorpromazine in serum and human breast milk. Chlorpromazine in serum and human breast milk was extracted by a rapid and simple procedure based on C18 bonded-phase extraction, and a reversed-phase HPLC separation technique was developed. Chlorpromazine and levomepromazine as the internal standard were detected by ultraviolet absorbance at 254 nm. Determination was possible for chlorpromazine in the concentration range 10-300 ng/ml. The recoveries of chlorpromazine added to serum and human breast milk were 80.1-87.6 and 80.3-84.4%, respectively, with coefficients of variation of less than 10.2 and 7.8%. The method is applicable to drug level monitoring in the serum and human breast milk of patients treated with chlorpromazine.

Chlorpromazine↗
PubMed1993

Effects of hybrid peptide analogs to receptor recognition domains on alpha- and gamma-chains of human fibrinogen on fibrinogen binding to platelets.

We synthesized a series of hybrid peptides that correspond to the gamma-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAKQAGDV(G)nRGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 +/- 1.6 microM, 15 +/- 2.1 microM, and 87 +/- 6.8 microM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 +/- 0.14 microM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 +/- 0.6 microM. When the peptide Y-HHLGGAKQAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (Kd = 3.1 +/- 0.5 x 10(-7) M) to stimulated platelets and 12,500 molecules/platelet (Kd = 1.4 +/- 0.2 x 10(-7) M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or gamma-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Assignment of 1H, 15N, and 13C resonances, identification of elements of secondary structure and determination of the global fold of the DNA-binding domain of GAL4.

Almost complete assignments of the 1H, 15N, and aliphatic 13C resonances of the 62-residue N-terminal DNA-binding domain of GAL4 [GAL4(62)] have been obtained using a combination of two-dimensional homonuclear and two- and three-dimensional double- and triple-resonance heteronuclear NMR methods. The sequential NOE connectivities, amide proton exchange measurements, and 13C alpha chemical shift data indicate the presence of two short alpha-helices in the N-terminal half of the polypeptide. Residues 1-9 and 41-62 appear to be unstructured and flexible in solution. Analysis of the 13C alpha chemical shifts also revealed a significant downfield shift of approximately +3 ppm, relative to random-coil values, for the four nonbridging Zn(II) ligands, Cys 14, 21, 31, and 38. Interestingly, no such correlation was observed for the two bridging ligands, Cys 11 and 28. Preliminary structure calculations using a subset of distance restraints derived from three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra are consistent with the recently reported solution structures of Zn(II)2GAL4(7-49) [Kraulis, P., et al. (1992) Nature 356, 448-450] and of Cd(II)2GAL4(65) [Baleja, J. D., et al. (1992) Nature 356, 450-453].

Amides↗