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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 523 records · Page 29Linked to original sources
PubMed1993

Characterization of the membrane sensor PenJ for beta-lactam antibiotics from Bacillus licheniformis by amino acid substitution.

The PenJ protein of the penicillinase gene (penP) expression system from Bacillus licheniformis is an antirepressor and membrane receptor for beta-lactam antibiotics. A putative beta-lactam antibiotic binding site including Ser402 and Lys405, which are homologous to the conserved sequence for the beta-lactam binding site (Ser-X-X-Lys) is present. An amino acid substitution was introduced at Ser402 to Ala, removing the hydroxyl group of the serine. The mutant PenJ, S402A, was still functional. However, two other mutants, S402T (Ser402-->Thr) and K405A (Lys405-->Ala), were not functional. Thus, the hydroxyl group of Ser402 does not appear to be important for penicillin binding. Amino acid substitutions (K539R, D591N and K539R.G541V) were introduced in PenJ in the region of the putative phosphoryl binding domain. None of these mutant PenJ proteins was a functional antirepressor. These results suggested that the putative phosphoryl binding domain might be an important region for signal transduction.

Amino Acid Sequence↗
PubMed1993

Survival rate of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants.

The relationship between cryoprotectant and exposure time on the development of IVF-derived embryos in culture after freezing and thawing was examined. Good to excellent quality Day 7 to 8 blastocysts and expanded blastocysts were suspended in 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol, and 1.3 M ethylene glycol monomethyl ether (EME). In Experiment I, after 30 and 120 min of exposure, embryos were placed directly into culture medium and washed three times. Embryos were cocultured with cumulus cells in culture medium. After a 48-h culture, there were no significant differences in fully expanded, hatching, and hatched rates between the 30- and 120-min exposure to all four cryoprotectants except for the fully expanded rate with PG (55 vs 83%). In Experiment II, to compare the cryoprotective effects of the four cryoprotectants, embryos were exposed to each cryoprotectant for variable times from 10 to 120 min, seeded, and cooled to -30 degrees C at 0.3 degree C/min, plunged into liquid nitrogen for storage, and then thawed. Survival was measured by coculturing the embryos with cumulus cells in culture medium. There were no significant differences in development to hatched blastocysts of embryos exposed to either PG (27-42%) or EG (37-56%). The number of embryos developing after exposure to EME was similar to that in PG or EG when exposure time did not exceed 40 min (43-49%). Our findings suggest that the toxicity of different cryoprotectants is related to the exposure time and that PG and EG were relatively nontoxic as is exposure to EME for short periods.

Animals↗
PubMed1993

Survival rate of frozen-thawed bovine IVM/IVF embryos in relation to post-thaw exposure time in two cryoprotectants.

The relationship between post-thaw exposure time in cryoprotectants and the viability of in vitro fertilized (IVF)-derived bovine embryos after culture was examined. Good- and excellent-quality Day 7 to Day 8 blastocysts and expanded blastocysts were frozen by the nonstepwise method using 1.6 M propylene glycol (PG) or ethylene glycol (EG) as the cryoprotectant. After the straws were thawed, the embryos were kept in the cryoprotectants for 1, 10, or 30 min. Then the embryos were placed directly into culture medium and washed three times, followed by coculture with cumulus cells. After 48 h of culture, there were no significant differences in survival rate among the various exposure times in PG (79-81%) and EG (71-75%). After 72 h of culture, there were no significant differences among the various exposure times in the fully expanded (PG 76-81%; EG 71-72%), hatching (PG 69-74%; EG 58-71%), and hatched blastocyst rates (PG 60-71%; EG 47-65%). Our findings suggest that different post-thaw exposure times up to 30 min using PG or EG as the cryoprotectant are not harmful to IVF bovine embryos.

Animals↗
PubMed1993

Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition.

The chitin synthase 3 gene (CACHS3) has been cloned from Candida albicans. The yeast CAL1 gene encoding the chitin synthase 3 of Saccharomyces cerevisiae was used as a probe for the isolation of the gene from C. albicans. The CAL1 homolog was identified in Southern blots of C. albicans genomic DNA and cloned from a C. albicans genomic DNA library. The nucleotide sequences of two partial clones were determined and combined giving a total length of 4610 bp. A continuous open reading frame of 3525 bp encoding a predicted protein of 1175 amino acids and molecular mass of 131 850 daltons was identified. A comparison of the deduced amino acid sequences of CAL1 and the Candida chitin synthase 3 protein showed 59.3% identity. Southern blot analysis indicates that the CACHS3 gene is present in a single copy in the genome and maps to chromosome I. Northern blot analysis shows that expression of chitin synthase 3 gene is dramatically increased during the transition from the yeast to the hyphal form of C. albicans. This change in transcription level strongly suggests that CACHS3 may play a role in Candida morphogenesis.

Amino Acid Sequence↗
PubMed1993

Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa.

The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5'-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.

Amino Acid Sequence↗
PubMed1993

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries.

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.

Journal Article↗
PubMed1993

Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system.

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5 degrees C, 5% CO(2)) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0 degrees C, cooled from 0 degrees C to -6 degrees C at -1 degrees C/minute, seeded, held for 10 minutes, and cooled again at -0.3 degrees C or -0.5 degrees C/minute to -30 degrees C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30 degrees C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at -0.3 degrees C vs -0.5 degrees C/minute was not significantly different (P>0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of -0.3 degrees C/minute (64.6%, 31/48) than at -0.5 degrees C/minute (22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10/21); DEG (30%, 3/10); EG (74%, 20/27); and PG (40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.

Journal Article↗
PubMed1993

Extrastriate cortical neurons correlated with ocular convergence in the cat.

Responses of neurons in the lateral suprasylvian area to visual stimulation in association with ocular convergence were studied in eight alert cats trained to track a visual target moving in depth. Activities of 18 (3%) of 659 cells were related to ocular convergence. These 18 neurons were divided into two groups: activities of seven neurons (40%) correlated with peak velocities of convergence eye movement at both fast and slow target speeds (group I), while those of five neurons (30%) correlated with them only at faster target speed (group II). Activities of six other neurons correlated with peak velocity of ocular convergence at faster target speed but were not tested at slower speed. Activities of four group-I neurons (60%) did not or only weakly correlate with lens accommodation, while those of four group-II neurons (80%) correlated with peak velocity or amplitude of lens accommodation. It can therefore be concluded that the four group-I neurons are primarily related to ocular convergence. Other cells were either convergence-related, lens accommodation-related or both. It is suggested that these different types of neurons contribute in combination to optimal control of convergence eye movement.

Accommodation, Ocular↗
PubMed1993

The efficacy of radiation therapy for a malignant melanoma in the mucosa of the upper jaw: an analytic study.

An analysis has been made of the effect of radiation therapy in 28 patients with a malignant melanoma (Stage I: 18 cases; Stage II: 10 cases) in the mucosa of the upper jaw. Treatment had been provided by one of the following methods: intraoral mold (10 cases), interstitial brachytherapy (two cases), intraoral electron therapy (nine cases), or external irradiation (seven cases). The results have shown that the survival rate for all 28 patients was 25%, and that the survival rate for stage I patients treated by intraoral electron or brachytherapy was 47%. The primary tumor control rate in percentages was 79% (22/28) in all 28 radiotherapy patients; 92% (11/12) for tumors treated by a mold or an interstitial implant; 67% (6/9) for tumors treated by an intraoral cone; and 71% (5/7) for tumors treated by external irradiation with or without surgery. A neck metastasis that was found in 19 patients was treated by surgery, radiotherapy, and/or immunochemotherapy, and the result was successful in nine patients. The major factor in the failure of treatment was a distant, metastatic dissemination. This analysis revealed that radiotherapy has achieved similar or better results than surgery and may be advocated for the management of a localized malignant melanoma in the mucosa of the upper jaw.

Adult↗
PubMed1993

Purification of hemagglutinin from Haemophilus paragallinarum using monoclonal antibody.

The purification of hemagglutinin from Haemophilus paragallinarum was attempted using affinity chromatography with monoclonal antibody. The antigen eluted from the affinity column using potassium thiocyanate buffer agglutinated chicken erythrocytes. In immunoblotting of the eluted antigen, a single band with monoclonal antibody was found as well as the crude antigen. When the chickens were immunized with the eluted antigen, they produced the hemagglutination inhibition (HI) antibody, and they showed protection against challenged exposure with H. paragallinarum strain 221. These results indicated that the HA antigen of H. paragallinarum was a protective antigen.

Animals↗
PubMed1993

Role of Asp51 and Glu105 in the enzymatic activity of a ribonuclease from Rhizopus niveus.

The active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and Glu105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that Glu105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.

Amino Acid Sequence↗
PubMed1993

Accommodative and pupillary responses to sinusoidal target depth movement.

We evaluated the accommodative and pupillary responses to sinusoidal target depth movement in 10 normal subjects and one patient by analysing frequency responses and associated Bode plots. The phase of the pupillary response significantly lagged behind that of accommodative response, which suggests that they are controlled by separate central nervous systems. We measured the baseline shift of each response trace using a regression line and found that they were larger at higher stimulation frequencies. A patient with severe asthenopia showed a considerable baseline shift in accommodative response. These analyses may be useful for the quantitative assessment of dynamic accommodation and pupillary response.

Accommodation, Ocular↗
PubMed1993

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.

Amino Acid Sequence↗
PubMed1993

Cloning and sequencing of a dehalogenase gene encoding an enzyme with hydrolase activity involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme responsible for the conversion of the chemically unstable intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida PpY101LA into which the linA gene had been introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which could convert gamma-HCH to 2,5-DDOL, was subcloned, and subsequent deletion analyses revealed that a ca. 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the linB gene) of 885 bp within the region. The deduced amino acid sequence of LinB showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for LinB, suggesting that LinB may be a member of haloalkane dehalogenases with broad-range specificity for substrates.

Amino Acid Sequence↗
PubMed1993

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases.

We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA.

ATP-Binding Cassette Transporters↗
PubMed1993

Velocity profile of human horizontal saccades.

To assess saccadic trajectories, we evaluated the velocity profile using the 'skewness ratio' (the proportion of acceleration time to total saccadic duration) as a parameter. Horizontal cross-center saccades against target movements of 10 degrees, 20 degrees and 30 degrees were measured in 6 normal subjects using the magnetic search coil method. As the amplitude increased the period of the deceleration phase became prolonged: 10 degrees saccades were almost symmetrical and 30 degrees saccades were skewed to a ratio of about 0.3. A close relationship between duration and skewness ratio was found, and the skewness ratios of temporal saccades were smaller than nasal ones. This skewness ratio would be a useful parameter for assessing the velocity profile of human saccades.

Adult↗