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Biomedical subjects

M Kai

Publications and source records attributed to M Kai.

At least 19 recordsLinked to original sources
PubMed1995

Chaperone function of calreticulin when expressed in the endoplasmic reticulum as the membrane-anchored and soluble forms.

A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane-anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with other calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations.

Amino Acid Sequence↗
PubMed1995

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.

Adenosine Triphosphate↗
PubMed1995

Hypoxic helium breathing does not reduce alveolar-arterial PO2 difference in the horse.

In a previous study we evaluated the mechanism of alveolar-arterial PO2 (AaPO2) reduction when nitrogen is replaced with helium in normoxia (FIO2 = 0.21). The reduction in AaPO2 was not due to changes in VA/Q inequality, pulmonary O2 diffusing capacity, or cardiac output, but to more complete diffusion equilibration as a consequence of the higher ventilation and thus PAO2 (which reduced the average slope of the hemoglobin O2 dissociation curve (ODC), and thus enhanced diffusive equilibration). We hypothesized that hypoxic He/O2 breathing in contrast would not reduce the AaPO2 because PAO2 and PaO2, although higher with He than N2, would remain constrained to the linear region of the ODC. Breathing hypoxic gas mixtures did constrain the PAO2 to the linear region of the ODC, even when PAO2 was increased by He/O2 breathing. Thus, the average slope of the ODC did not change when He replaced N2 and this explains the lack of change in AaPO2, as hypothesized.

Animals↗
PubMed1995

Occupational exposure in pediatric cardiac catheterization.

Radiation doses to staff involved with pediatric cardiac catheterization were measured using thermoluminescent dosimeters in 18 procedures. The average doses to the lens and thyroid, and the effective dose per procedure to the main operating physicians was 88, 180, and 8 microSv, respectively; to assistant physicians, 23, 51, and 2 microSv; and to technicians, 23, 27, and 2 microSv. In some procedures, the dose to the technician's hand approached 1,500 microSv. The doses received by physicians were proportional to cineangiographic time but with no correlation with integrated currents. The number of procedures which may be performed in a year by individual staff members was estimated to be 430 and 2,780 procedures for physicians and assistants, respectively. It was suggested that the front of the neck is an adequate position for the dosimeter to measure doses during pediatric catheterization.

Adolescent↗
PubMed1995

Infrequent somatic mutation of the MTS1 gene in primary bladder carcinomas.

We examined a candidate tumor suppressor gene on chromosome 9p21, MTS1/CDK4I (multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor), which has been found to be mutated frequently in cell lines derived from bladder carcinomas, for somatic mutations in 39 primary bladder cancers by means of SSCP (single-stranded conformational polymorphism) and DNA sequencing. Mutations were detected in two of these carcinomas; one was a 61-base deletion and the other a 1-base deletion. In both cases the homologous allele was missing, indicating that "two-hit" mutation of the MTS1 gene had taken place in these tumors. The results indicated that inactivation of the MTS1 gene is likely to be a contributing factor in some, but not the majority of, bladder cancers.

Base Sequence↗
PubMed1995

Protein C deficiency and stroke in early life.

Plasma concentrations of protein C, protein S and antithrombin III were measured in 33 unselected children with a history of cryptogenic stroke (group 1), four children with previously ascertained low plasma concentrations of protein C following stroke (group 2) and 42 healthy children undergoing minor surgery (group 3). Protein S and antithrombin III were normal in all patients. Low concentrations of protein C were found in two patients in group 1 and in six healthy children in group 3. Low protein C concentrations returned to normal over many months in three of the four patients in group 2. Prophylactic antithrombotic therapy and/or termination of pregnancy had been carried out unneccessarily in two families in whom inherited protein C deficiency was not confirmed. The suggestion that heterozygous protein C deficiency contributes to the risk of arterial stroke was not supported by this study.

Adolescent↗
PubMed1995

Exercise induced ventilation/perfusion inequality in the horse.

Exercise in normal human subjects causes deterioration of matching of ventilation to blood flow in the lungs, but only in about 50% of those examined. A previous study (Wagner et al. 1989) of 5 horses showed no significant worsening of ventilation/blood flow (VA/Q) relationships during heavy exercise as determined by multiple inert gas elimination technique (MIGET). Because of the small number of horses in that study and the 50% human incidence of exercise induced VA/Q mismatch, we studied an additional 6 Thoroughbreds, comparing VA/Q relationships at the walk (1.4 m/s, 0 degrees incline) and during galloping (9.6 +/- 0.3 m/s, 7% incline). Such data were collected under 4 different conditions wherein inspired gas was 1) air, 2) 21% O2 in helium, 3) 15% O2 in N2 and 4) 15% O2 in helium. Each horse exercised 4 times (morning and afternoon of 2 days, with inspired gas conditions randomised). There was a small but significant increase in VA/Q mismatch (similar under all 4 conditions). The second moment of the VA/Q distribution (determined by the MIGET) increased significantly (P < 0.01) from 0.31 +/- 0.01 at the walk to 0.38 +/- 0.02 during gallop. This increase however is small: 0.38 is well within the range of this parameter for normal human subjects (where the 95% upper confidence limit is 0.60). This study shows that a small amount of exercise induced VA/Q mismatch can occur in the horse as in man, but the mechanism remains to be elucidated and its clinical significance remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Strand break formation in plasmid DNA irradiated in aqueous solution: effect of medium temperature and hydroxyl radical scavenger concentration.

Plasmid pBR322 DNA (4363 base pairs) in aerobic aqueous solution was irradiated with 60Co gamma-radiation. The change of diffusion coefficients (D) of chemical species, rate constants (k) of radical-DNA interaction and solubilities of O2 in water cannot be ignored when a temperature varies more than a few tens of centigrade. It is important to examine the variation of the yields of DNA strand breaks as a function of temperature in order to analyze the mechanisms of DNA strand breaks from the chemical point of view. Hence, we observed the change of the yield of strand breaks with temperatures between -20 and 42 degrees C by agarose gel electrophoresis. We also observed the change of the yield of strand breaks with the concentration of OH scavenger (Tris) from 1 mmol dm-3 to 100 mmol dm-3 and summarized it with previous experiments. This summarization indicated that the order of the lifetime of OH radical in cellular environment is several nanosecond. This value is consistent with the measurement of the lifetime of 8.7 nanosecond for OH radical in mammalian cell (Roots, R. and Okada, S. (1975) Radiat. Res. 64, 306-320).

Culture Media↗
PubMed1995

Deodorization of laboratory animal facilities by ozone.

Deodorizing effect of ozone was investigated comparing two types of compact ozonizing apparatus made on an experimental basis. The concentrations of ammonia and trimethylamine were examined as an indicator for deodorizing effect of ozone in animal rooms of rats and guinea pigs at laboratory animal facilities of three different universities. Both of the ozonizing apparatus were able to remove ammonia and trimethylamine in animal rooms, with no significant difference in the performance of the two apparatus.

Ammonia↗
PubMed1995

Synchronous circadian rhythms of mRNA levels and activities of cholesterol 7 alpha-hydroxylase in the rabbit and rat.

Cholesterol 7 alpha-hydroxylase, the key enzyme in a series of metabolic changes for the production of bile acids from cholesterol, shows circadian rhythms. The mechanism of the circadian rhythms is the subject of controversy; moreover, few pieces of information are presently available. Because the rabbit is a good animal model for the study of human cholesterol-bile acid metabolism, in the present study we isolated a complete cDNA encoding rabbit cholesterol 7 alpha-hydroxylase (3022 base pairs (bp) long and 1503 bp open-reading frame encoding 501 amino acid residues). Using this isolate rabbit cDNA and the rat cDNA as probes, the circadian rhythms of this enzyme were studied in normal male rabbits and rats. The animals were maintained on a 12-h light-dark cycle. Three subjects each were then killed every 3 h for 1 day. The livers were harvested, and the mRNA levels and the activities of the enzyme were measured. Both mRNA levels and activities of the enzyme showed remarkable circadian rhythms, with higher values during the dark phase and lower values reaching minimum during the light phase. The enzyme activities correlated well with the mRNA levels. There was no shift in the cycles of the enzyme activities and the mRNA levels. These findings suggest that pretranslational regulation is the most likely mechanism for the circadian rhythms of cholesterol 7 alpha-hydroxylase. Because the rhythms of mRNA levels and activities of this enzyme are synchronous, the turnover of the enzyme should be as fast as that of the mRNA, and we can hereafter measure the mRNA levels instead of the enzyme.

Amino Acid Sequence↗
PubMed1994

Fluorogenic reactions for biomedical chromatography.

A number of fluorogenic reactions, which have been used for HPLC detection systems by means of pre- and/or postcolumn derivatization, are surveyed with respect to both sensitivity and selectivity for the determination of biomedically important substances. For the derivatization of the substances, two types of fluorogenic reactions, fluorescence-generating and fluorescence-tagging, have been studied. The former are usable in most instances for both pre- and postcolumn derivatization methods, and the latter only for precolumn derivatization methods. HPLC methods utilizing the fluorogenic reactions allow analytes to be detected at picomole-subfemtomole levels. In the fluorescence-generating reactions, several fluorogenic reagents possessing two or more reactive sites in the molecule, which show molecular recognition for a variety of analytes, permit facile and reproducible detection in HPLC because there are fewer interferences from biological matrices.

Amines↗
PubMed1994

Antigenic variation and serotyping of HIV type 1 from four World Health Organization-sponsored HIV vaccine sites. WHO Network for HIV Isolation and Characterization.

Serologic reactivities of serum or plasma from 55 HIV-1 subjects in four countries--Brazil, Rwanda, Thailand, and Uganda--were examined by V3 peptide immunoassay. Forty-seven (85.5%) of the 55 specimens tested positive to the homologous peptide. A strong correlation between serotype (i.e., pattern of serologic reactivity with a panel of peptides) and genotype was not found. However, the V3 peptide immunoassays may be useful for epidemiologic studies to trace the distinctive HIV-1 strains from different geographic regions of the world. The serology data obtained may be useful for the development of effective V3-based vaccines.

AIDS Vaccines↗
PubMed1994

Mechanism of reduction in alveolar-arterial PO2 difference by helium breathing in the exercising horse.

Previous work has shown that replacing N2 in air with He at the same inspired O2 fraction reduces the exercise-induced alveolar-arterial PO2 difference (AaPO2) in horses but has provided no mechanism explaining this effect. We sought to distinguish among possible causes by using the multiple inert gas elimination technique. Six horses were studied on a high-speed treadmill while they breathed either ambient air or normoxic He-O2. O2 uptake reached 138.0 ml.min-1.kg-1 and was not affected by He-O2. Temperature-corrected arterial PO2 was 76.7 Torr (air) and 86.9 Torr (He-O2) (P < 0.01). Corresponding AaPO2 was 22.3 and 15.9 Torr, respectively (P < 0.01). Mean AaPO2 predicted from ventilation-perfusion inequality did not change with He-O2 (12.7 Torr with air and 11.9 Torr with He-O2). Mean arterial PCO2 was 50.1 Torr with air and 44.1 Torr with He-O2 (P < 0.01); minute ventilation and tidal volume were correspondingly higher by 140 l/min and 1.0 liter, respectively, with He-O2. Pulmonary O2 diffusing capacity, cardiac output, and all ventilation-perfusion dispersion indexes did not change with He-O2. Intrapulmonary shunt was insignificant. Higher ventilation with He-O2 explained only approximately 4 Torr of the 10-Torr rise observed in arterial PO2. The remainder (and the corresponding fall in AaPO2) was due to more complete diffusion equilibration as a consequence of the higher minute ventilation and thus alveolar PO2, which reduced the average slope of the O2 dissociation curve, thereby increasing the ratio of diffusive to perfusive conductance.

Animals↗
PubMed1994

Characteristics of vacuolating toxin produced by Helicobacter pylori.

OBJECTIVE: We sought a good indicator cell line to use in detecting the vacuolating toxin produced by Helicobacter pylori and in examining the characteristics of the toxin. DESIGN AND METHODS: Using five cell lines [baby hamster kidney (BHK-21), human amnion (FL), human uterine cancer (HeLa), rabbit kidney (RK-13) and African green monkey kidney (Vero)], cytotoxicity assays were performed with 33 H. pylori strains. To analyse the proteins produced by toxigenic and non-toxigenic strains, immunoblotting was done with the sera of patients with gastroduodenal diseases. RESULTS: When RK-13, FL, Vero, BHK-21 and HeLa cells were used for the assay, 73, 61, 27, 27 and 21% of strains, respectively, were toxigenic. The vacuolating toxin is sensitive to heat at 70 degrees C. By immunoblot analysis, under non-denaturing conditions, immune sera recognized proteins with molecular weights of 600,000 and 500,000 in the toxigenic but not in the non-toxigenic strains. CONCLUSION: H. pylori produces vacuolating toxin, which is a heat-sensitive protein at 70 degrees C and is easily detectable by cytotoxicity assay using the RK-13 cell line. Toxigenic H. pylori strains produce various proteins, including this toxin, which are recognized by sera from patients with gastroduodenal diseases.

Animals↗
PubMed1994

Monte Carlo simulation of DNA strand breaks induced by monoenergetic electrons using higher-order structure models of DNA.

A new theoretical model for estimating yield of initial DNA strand break induced by several monoenergetic electrons is presented. It is based on the Monte Carlo track structure simulation and on new DNA structure models (one turn of double-strand DNA, nucleosome, solenoid), and links physical and chemical stages of radiation action. Direct and indirect effects are strictly distinguished. Some results of calculations indicated: (1) the number of single strand breaks per nucleus (6 microns in diameter) per Gy in pure water was about 10 times that in a cell environment (OH radical life time is assumed to be 8.7 ns). This is due to the difference in the time-dependent variation in the total number of the OH radical; and (2) the contribution of indirect effects to total damage decreased as the order of the DNA target model structure used in the stimulation increased (e.g. a one-turn model of double-strand DNA, approximately 98.4%; but the 30-nm solenoid model, approximately 86.1%). This was due to the protective effect of histone protein against OH radical attack. Double-strand breaks were scored if two single-strand breaks were located on the same base pair. The present study indicated that the information from morphological and biochemical examinations of the cell environment must be considered more carefully with computer simulation.

Computer Graphics↗
PubMed1992

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Fractionation↗