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Biomedical subjects

E Doi

Publications and source records attributed to E Doi.

At least 19 recordsLinked to original sources
PubMed1999

High Prevalence of Hepatitis C Virus Infection in Chronic Hemodialysis Patients.

Hepatitis C virus (HCV) infection is very common among hemodialysis (HD) patients. Transmission of infection in this setting has been related to the number of blood transfusions, the duration of hemodialysis and to nosocomial transmission of virus in the dialysis unit. We conducted a study of 74 HD patients to determine the frequency of HCV at a single point in time (cross-sectional analysis), and to evaluate the association between HCV infection and patients' demographic, clinical and biochemical features. Serum samples were tested for anti-HCV antibodies using a third-generation enzyme-linked immunosorbent assay (ELISA). In the case of a positive result, third-generation recombinant immunoblot and HCV RNA detection by polymerase chain reaction (PCR) tests were performed. Collected data included the patient's age, gender, time on HD, number of blood transfusions and serum alanine aminotransferase (ALT) activity. Twenty-nine patients (29/74.4%) were found to be HCV positive using a third-generation ELISA assay. Of these 29 patients, 27 were also positive by recombinant immunoblot assay and 2 patients had indeterminate results. In the anti-HCV ELISA-positive subgroup, 20 (69%) of the 29 patients had detectable HCV RNA. The HCV RNA-positive patients had received more blood transfusions (15&plusm;3 vs. 5&plusm;1 units of packed red blood cells, p<0.0001) and had been on HD for a longer period of time than the HCV RNA-negative patients (65&plusm;32 vs. 32&plusm;29 months, p<0.0001). Mean serum ALT levels were significantly higher in the HCV RNA positive group (30&plusm;18 vs. 15&plusm;9, p<0.0001). We were unable to determine the most likely mode of transmission in our unit, but these results emphasize the need for strict adherence to blood collecting and handling precautions, careful attention to hygiene in the dialysis units, and sterilization of dialysis machines in order to properly combat this frequent infection.

Journal Article↗
PubMed1997

Temperature control for kinetic refolding of heat-denatured ovalbumin.

The folding of heat-denatured ovalbumin, a non-inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat-denatured at 80 degrees C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat-denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat-denatured molecules assumed the metastable non-native conformations that were susceptible to trypsin. The non-native species were marginally stable for several days at a low temperature, but the molecules were transformed slowly into the native conformation. Considering data from size-exclusion chromatography and from analyses of CD, intrinsic tryptophan fluorescence, and adsorption of the dye 1-anilinonaphthalene-8-sulfonate, we postulated that the non-native species that accumulated upon rapid cooling were compact but structureless globules with disordered side chains collectively as a folding intermediate. Temperature-jumped CD experiments revealed biphasic kinetics for the refolding process of heat-denatured ovalbumin, with the features of increasing and subsequently decreasing amplitude of the rapid and the slow phases, respectively, with the decrease in folding temperature. The temperature dependence of the refolding kinetics indicated that the yield of renaturation was maximal at about 55 degrees C. These findings suggested the kinetic partitioning of heat-denatured ovalbumin between alternative fates, slow renaturation to the native state and rapid collapse to the metastable intermediate state. Analysis of disulfide pairing revealed the formation of a scrambled form with non-native disulfide interactions in both the heat-denatured state and the intermediate state that accumulated upon rapid cooling, suggesting that non-native disulfide pairing is responsible for the kinetic barriers that retard the correct folding of ovalbumin.

Animals↗
PubMed1993

Dynamic light scattering of aqueous solutions of linear aggregates induced by thermal denaturation of ovalbumin.

Dynamic light scattering measurements were performed on dilute aqueous solutions of native ovalbumin (OA) and on those of linear OA aggregates induced by thermal denaturation at low ionic strength and neutral pH. The weight-average molecular weight MW of four aggregates tested ranged from 1,700,000 to 5,500,000. The translational diffusion coefficient D0 of native OA at infinite dilution was estimated as 8.70 x 10(-7) cm2/s, which gave 56.0 A as the diameter of the rigid spherical particle. The intensity autocorrelation function of linear OA polymers was analyzed with the cumulant method to obtain the first cumulant gamma e. The dependence of gamma e on the scattering vector q at very low polymer concentration was found intermediate between those of a flexible chain and a rigid rod. The translational diffusion coefficient Dtr [identical to (gamma e/q2)q-->0] was in proportion to M-0.55W, and the magnitude was in good agreement with a value calculated from the wormlike cylinder model with values of three parameters determined in an earlier study, ML = 1600 A-1, d = 120 A, and Q = 230 A, where ML, d, and Q are the molecular weight per unit length, diameter, and persistence length, respectively. Based on these results, a new model, to be called as the dimer model, was proposed to interpret the formation mechanism of linear OA polymers induced by thermal denaturation.

Animals↗
PubMed1993

Toxicity evaluation of the mycotoxins, citrinin and ochratoxin A, using several animal cell lines.

1. The cytotoxicities of the nephrotoxic mycotoxins, citrinin and ochratoxin A were assayed on HeLa, C3H/10T1/2, NIH/3T3, MDCK (canine kidney), and HeLa P3 cell lines, using the MTT colorimetric assay. 2. Citrinin was less toxic than ochratoxin A in all of the cell lines examined. 3. The MDCK cells were more susceptible to both citrinin and ochratoxin A, in comparison with other cell lines. 4. Dose-responses, as measured by activities of leucine aminopeptidase and alkaline phosphatase of MDCK cells, were less sensitive than MTT colorimetric assay, indicating that these enzymes were not specifically inhibited in MDCK cells. 5. The LD50 of both toxins, calculated at 72 hr incubation, was in the same order as those reported from animal experiments using rats and mice.

Alkaline Phosphatase↗
PubMed1991

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin.

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.

Amino Acid Sequence↗
PubMed1990

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.

Acetylcholine↗
PubMed1990

Freezing denaturation of ovalbumin at acid pH.

The effects of rapid freezing and thawing at acid pH on the physiochemical properties of ovalbumin were examined. At low pH (around 2), UV difference spectra showed microenvironmental changes around the aromatic amino acid residues; elution curves by gel permeation chromatography showed decreasing numbers of monomers after neutralization. These changes depended on the incubation temperature (between -196 and -10 degrees C) and the protein concentration (0.5-10 mg/ml), and a low concentration of ovalbumin incubated at around -40 degrees C suffered the most damage to its conformation. With freezing and then incubation at -40 degrees C, three of the four sulfhydryl groups in the ovalbumin molecule reacted with 2,2'-dithiodipyridine. The CD spectra showed these changes in the secondary structure, but they were smaller than those when guanidine hydrochloride was used for denaturation. Supercooling at -15 degrees C or freezing at -196 degrees C had little or no effect on the conformation of the ovalbumin molecule. Thus, irreversible conformational changes of ovalbumin were caused under the critical freezing condition at an acid pH. These changes arose from partial denaturation and resembled those with thermal denaturation of ovalbumin at neutral pH.

Chromatography, Gel↗
PubMed1990

Toxicity of dimethyl sulfoxide as a solvent in bioassay system with HeLa cells evaluated colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.

The toxicity of dimethyl sulfoxide (Me2SO) was examined in HeLa cells cultured at 37 degrees C for up to 72 hr. The growth of the cells was measured by a colorimetric method with the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which gave good correlation between the cell number and the color development from the reduction of MTT under suitable conditions. When the initial number of cells was 3 x 10(4)/ml, Me2SO at 1% or less had no apparent effect on proliferation for up to 48 hr of incubation, but in longer incubations, cell growth was repressed. When the initial number of cells was 3 x 10(5)/ml, the effect of Me2SO was similar.

Animals↗
PubMed1989

Effects of anion binding on the conformations of the two domains of ovotransferrin.

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.

Anions↗
PubMed1988

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation.

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.

Alkylation↗
PubMed1988

Conformational changes in ovalbumin at acid pH.

Several physicochemical parameters of ovalbumin were examined at acid pH. The intrinsic viscosity and far UV-CD spectrum at pH 2 did not differ from those at pH 7. But the near UV-CD spectrum, difference absorption spectrum around 250-320 nm, and fluorescence spectrum showed micro-environmental changes around the aromatic amino acid residues in acid solution. The reactivity of one of the four sulfhydryl groups with 2,2'-dithiodipyridine increased at pH below 5. The rate of denaturation by urea and that of surface tension decay were high in the low pH range. We concluded that at low pH (around 2), ovalbumin molecules kept their native globular conformation, but that their chain flexibility increased and they were very susceptible to denaturation. This state might be equivalent to the molten-globule state observed with some globular proteins in acidic region.

Circular Dichroism↗
PubMed1988

Amino-terminal and carboxyl-terminal half-molecules of ovotransferrin: preparation by a novel procedure and their interactions.

The amino- (N-) and carboxyl- (C-)terminal half-molecules of ovotransferrin were prepared by a novel procedure. The trypsin-nicked ovotransferrin (Ikeda et al. (1985) FEBS Lett. 182, 305-309), in which the two half-molecules interact non-covalently forming a stable dimer, was purified by gel filtration and anion-exchange column chromatography. By subsequent cation-exchange chromatography, the nicked form was distinctly separated into an equivalent amount of the N-terminal and C-terminal half-molecules. Analyses of the N-terminal and C-terminal sequences indicated that the N-terminal and C-terminal half-molecules comprised the alignments of residues 1-332 and 342-686 of ovotransferrin, respectively. Anion-exchange chromatography, gel filtration chromatography, and non-denaturing polyacrylamide gel electrophoresis revealed that the isolated half-molecules had the ability to re-associate in solution. The contents of alpha-helix and beta-sheet of the two half-molecules, as determined by circular dichroism (CD) spectra, were very similar to those of intact ovotransferrin. No prominent alteration in the secondary structure of the two half-molecules was induced by the re-association.

Amino Acids↗
PubMed1986

[Stereotactic evacuation of hypertensive thalamic hematomas using plasminogen activator (urokinase)].

In the past five years, the authors have performed stereotactic evacuation of hypertensive thalamic hematoma using Komai's CT-stereotactic apparatus in 44 cases. Liquefied hematoma was aspirated through a stereotactic cannula, and solid hematoma difficult to aspirate was dissolved by a plasminogen activator (Urokinase) and drained out through a silastic catheter. The mean ratio of the total volume of evacuated hematoma to the estimated volume by CT image was 83.8%. The recovery from motor paresis and consciousness disturbance was observed during the early postoperative days in most patients. Functional outcome (ADL) at 3 months after operation was as follows: 24 cases (54.5%) recovered to a full or partial (self-cared) social life, 15 (34.1%) required partial care at home and 2 (4.5%) remained bedridden. Although 3 patients died within 1 month after operation, the cause of death in these patients was admitted to have no direct relation to operative procedure or to rebleeding due to the Urokinase injection. Postoperative functional prognosis was not affected by the timing of the operation. The important factors affecting ADL were preoperative neurological grade and CT classification. This stereotactic method apparently exceeded the conventional craniotomy method in the functional outcome.

Adult↗
PubMed1983

Spontaneous subperiosteal hematoma of the orbit.

Spontaneous subperiosteal hematoma of the orbit is extremely rare. This is a case of an 81-year-old woman. The presenting symptom was protrusion of the right eye with severe intraorbital pain. Computed tomography scanning showed a high-density area in the upper part of the right orbit, which, at operation, was found to be a subperiosteal hematoma of the orbit.

Aged↗
PubMed1983

[Multiple malignant intracerebral schwannomas in von Recklinghausen's disease--report of a case].

Intracerebral schwannoma is very rare, and only nine cases of solitary intracerebral schwannoma have been reported since the first description by Gibson et al, in 1966. This report is the first case of multiple intracerebral schwannomas in Japan. Intracranial extracerebral schwannomas in von Recklinghausen's disease are sometimes encountered, but intracerebral ones have not been discovered. A case of multiple intracerebral schwannomas associated with von Recklinghausen's disease is reported in a 23-year-old male. He had been healthy until January 1979 when he was pointed out an abnormal shadow on chest roentgenogram. Although thoracic tumor revealed schwannoma in histological examination of surgical specimen, it transformed to malignant one during next 15 months. In July 1980, he complained of severe headache, vertigo, nausea and vomiting, and bilateral papilledema was proved. He was admitted to our clinic on August 8, 1980. Two large intracerebellar and four intracerebral tumors were revealed by computed tomography (CT). Angiography demonstrated avascular masses. The cerebellar tumors were totally extirpated on August 12, 1980. Histological diagnosis of the removed tumors approved malignant schwannoma, but their correlation to the chest tumor was unclear. Follow-up CT scans demonstrated that the supratentorial tumors increased in size during about three months. The chest tumor also increases in size. He died on November 12, 1980, but autopsy was not done. Pathogenesis of intracerebral schwannomas in this case is controversial, and we suggest that it is (1) multiple schwannomas in von Recklinghausen's disease with malignant transformation, or (2) metastasis from the chest tumor. But the conclusion remains unclear.

Adult↗