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Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 145 records · Page 8Linked to original sources
PubMed1984

Kinetics of the allosteric transition of aspartate transcarbamylase. Chemical quench studies.

Using a chemical quench device, the rate of synthesis of carbamyl aspartate from the substrates aspartate and carbamyl phosphate was followed as a function of the time between mixing the enzyme with substrates and quenching with trichloroacetic acid. This function, which is linear at long times, shows (at 4 degrees C) a transient lag phase of product of roughly 10 ms. However, when the catalytic subunit (in which the enzymatic activity is desensitized) is used instead of the enzyme, the lag disappears. Therefore the lag seems to be associated with the control functions of the enzyme, i.e. to represent the allosteric transition involved in substrate-substrate (homotropic) co-operativity. Thus the relaxation time for the activation process is roughly 10 ms. The implications of these results are examined.

Allosteric Regulation↗
PubMed1984

Genetic complementation in somatic cell hybrids of cerebroside sulfatase activator deficiency and metachromatic leukodystrophy fibroblasts.

Several cases of metachromatic leukodystrophy (MLD) have been described with normal or near normal activities of arylsulfatase A (cerebroside sulfatase). However, the ability of intact cultured fibroblasts to hydrolyze cerebroside sulfate was impaired. Since the impairment was corrected by cerebroside sulfatase activator, a deficiency of activator was implied. In the absence of direct demonstration of deficiency, other types of evidence were needed to support the premise that the genetic defect was not associated with the arylsulfatase A locus as in classical MLD. Therefore, somatic cell hybrids of activator deficiency and MLD fibroblasts were analyzed. Complementation was indicated by enhanced hydrolysis of cerebroside sulfate, supporting the view that cerebroside sulfatase activator deficiency and MLD are nonallelic.

Cerebroside-Sulfatase↗
PubMed1984

Microheterogeneity of arylsulfatase a: Treatment with hydrolytic enzymes.

Cerebroside sulfatase also known as arylsulfatase A from human liver displays six microheteromer bands upon narrow pH range isoelectric focusing. Sialic acid residues only partially account for this enzyme multiplicity since neuraminidase treatment reduces the number of bands to three. Uptake studies with cultured fibroblasts strongly suggest arylsulfatase A has covalently bound mannose 6-phosphate residues. However, treatment with alkaline phosphatase and a battery of glycohydrolases failed to reduce the number of enzyme charge forms below three. These results imply that the neuraminidase-resistant charge microheterogeneity is not due to structures associated with the carbohydrate moiety of arylsulfatase A.

Cells, Cultured↗
PubMed1984

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion.

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.

Animals↗
PubMed1984

Acquisition of native structure from reduced Trimeresurus flavoviridis phospholipase A2.

Oxidation of reduced T. flavoviridis phospholipase A2 under suitable conditions resulted in recovery of its active, native structure. The oxidized product was eluted at the same positions as native phospholipase A2 on reserved phase column and DEAE-Toyopearl 650M column chromatographies. The native and regenerated proteins were also identical in mobility on polyacrylamide gel electrophoresis and in circular dichroism spectra. Des-octapeptide(1-8)-phospholipase A2 (L-fragment), which shows only greatly reduced activity, was reduced and oxidized but no effective reformation of the native structure was found, indicating that the entire sequence is required for efficient reorganization.

Animals↗
PubMed1984

Genotype assignments in a family with the pseudo arylsulfatase a deficiency trait without metachromatic leukodystrophy.

Two children in a family with five siblings were investigated because of low levels of fibroblast arylsulfatase A activity. Neither child had metachromatic leukodystrophy (MLD) and they were diagnosed as having benign pseudo arylsulfatase A deficiency trait (PD). Analysis of arylsulfatase A subunit profiles in fibroblasts provided data for genotype assignments for each family member. Father and mother were assigned an n/pd and n/mld phenotype, respectively. The low enzyme siblings were both assigned pd/mld; two of the three normal enzyme siblings were assigned an n/n phenotype and one an n/mld.

Adult↗
PubMed1984

Diagnosis of pseudo-arylsulfatase A deficiency with electrophoretic techniques.

Deficient arylsulfatase A activity in man has long been associated with the neurodegenerative disease, metachromatic leukodystrophy. However, similar deficiency has been noted in clinically normal individuals, and is referred to as the pseudoarylsulfatase A deficiency condition. Although direct quantitative analysis of arylsulfatase A activity failed to differentiate between these two conditions, analysis of residual arylsulfatase A activity with either Cellogel electrophoresis or isoelectric focusing in polyacrylamide gels now has been shown to distinguish between them unequivocally. With both techniques, cultured fibroblasts from patients with pseudo-arylsulfatase A deficiency showed faint but clear bands of arylsulfatase A activity. Under identical conditions, fibroblasts from patients with metachromatic leukodystrophy showed no trace of activity. These methods can be adapted easily for general laboratory analysis in cases when results from quantitative arylsulfatase A assays are noninformative.

Cerebroside-Sulfatase↗
PubMed1984

Compensatory development of immature LH cells after long-term gonadectomy: an immunocytochemical, electron microscopic and cell count study.

Following long-term castration of male rats (for 3, 6, 12 and 18 months), some populations of five gonadotroph types, (i.e., immature cell, types III, III/IV, IV and so-called signet-ring cell, appeared in the pituitary glands. Their ultrastructures were electron microscopically observed after immunohistochemical identification of them using anti-rat LH beta serum. In unoperated control rats, 60 days old, the predominant III/IV type was intermingled with some populations of III and IV types, but no immature LH cells or signet-ring cells were detected among them. In the present observation not all of the gonadotroph types turned into signet-ring cells. Immature small LH cells containing a few small secretory granules began to appear (2.0%) at 3 months, and had increased to a maximum (52.5%) at 18 months. The percentage of the signet-ring cells was high (24.1%) at 3 months, but reduced (2.4%) at 18 months. High populations of small, oval and immature LH cells are assumed to occur as a consequence of mitotic division of the most of the immature LH cells. This may compensate for the loss of signet-ring cells in order to maintain the high serum LH and FSH concentrations after long-term castration.

Animals↗
PubMed1984

A new sea urchin toxin and its effect on spontaneous transmitter release at frog neuromuscular junctions.

The authors extracted a dialyzable substance from sea urchin (Diadematidae) collected in Fiji, and investigated its effect on the frequency of miniature endplate potentials (MEPPs) in the sartorius muscle of the frog. This substance was shown to be different from previously known biologically active substances in many respects. The increase of MEPP frequency by the application of toxin was significant and dose-dependent. The frequency began increasing immediately after application and returned to normal after washing. This increasing action of toxin on MEPP frequency was strengthened by increased calcium concentration, but even in Ca-depleted solution toxin action was significant. Toxin action in Ca-free Mg2+ solution was found to be just as remarkable as in Ca2+ solution, and was dependent on magnesium concentration. Toxin action was also found to depend significantly on sodium concentration. The toxin did not change membrane potential of the muscle appreciably. From these results, the depolarization of the nerve terminal was not considered to be the main effect of toxin on MEPP frequency. The increasing action of toxin on MEPP frequency was interpreted as being the result of increased permeability of the nerve terminal to both divalent cations and Na+.

Animals↗
PubMed1984

Evaluation of a bait trap for Habu, the venomous snake, Trimeresurus flavoviridis.

The box trap for the venomous snake, Trimeresurus flavoviridis, 25 X 45 cm and 15 cm high, was made of a sheet of polypropylene board, and involved the use of a small rodent in a wire mesh cage as bait. The efficacy of this trap was evaluated in the field on Tokunoshima Island, one of the southern-most islands of Japan. The catch rate of the trap was expressed by the probability of snakes caught in trap-days, a product of the number of traps set and the period of set days. In one experiment, a result was successfully achieved by setting the boxes for almost 2,000 trap-days each for 2 different test materials in a limited geographical area. The one-way entrance made of flexible polyethylene sticks arranged conically allowed significantly more invasion of snakes than that of a swinging hinged door as shown by the catch rates of 12 snakes/1721 trap-days to 0/1682, respectively. Using traps with a conical entrance, the catch rate with the albino rat, Ratus norvegicus, was 6/2324 which was significantly higher than 0/2450 using rat feces as attractant. The catch rate with the field rat, Ratus rattus, at 31/2402 was significantly about 6 times higher than 5/2446 with the albino rat, whereas an albino rat was about 5 times more attractive than 2 albino mice at significantly different rates of 17/2455 and 3/2284, respectively. The box trap baited with a small rodent proved attractive to snakes and attractiveness differed with the species of bait animals.

Animals↗
PubMed1983

Small-angle X-ray scattering study of adenosine triphosphatase from thermophilic bacterium PS3.

Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.

Adenosine Diphosphate↗
PubMed1983

Pseudo arylsulfatase A deficiency: evidence for a structurally altered enzyme.

Analysis of arylsulfatase A from pseudo arylsulfatase A deficiency fibroblasts by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoradiochemical nitrocellulose blot radiography revealed two subunit bands which migrated faster than subunit bands of enzyme from normal fibroblasts. Immunoreactive material was present only at levels comparable to enzyme activity. These findings imply that arylsulfatase A in pseudodeficiency is structurally altered, but it is catalytically equivalent to normal arylsulfatase A. This altered enzyme must be the product of the pseudodeficiency gene since no immunoreactive product of the metachromatic leukodystrophy gene could be detected in metachromatic leukodystrophy cells by the procedure employed. It is not clear from the present data if the attenuated arylsulfatase A activity in pseudodeficiency results from a decreased rate of synthesis or an increased lability of the mutant enzyme.

Cerebroside-Sulfatase↗
PubMed1983

Prenatal diagnosis of pseudo arylsulphatase A deficiency.

Prenatal diagnosis was performed on a pregnancy at risk for metachromatic leukodystrophy (MLD) in a family with the pseudo arylsulphatase A deficiency trait. Extracts of cultured amniotic fluid cells were deficient in arylsulphatase A indicating that the fetus was either affected with MLD or had the benign pseudodeficiency trait. In the cerebroside sulphate loading test, the at risk cells hydrolysed sulphatide like control cultured amniotic fluid cells implying that the fetus had pseudodeficiency. The pregnancy was carried to term and a male child was delivered. Placenta, urine and fibroblasts had very low activities of arysulphatase A. However, no sulphatide could be detected in urine and growing fibroblasts responded normally in the cerebroside sulphate loading test, suggesting pseudodeficiency. At 29 months, the infant is healthy and shows no stigmata of MLD. The prediction based on the results of the cerebroside sulphate loading test on cultured amniotic fluid cells appeared to be borne out.

Amniotic Fluid↗
PubMed1983

Pituitary folliculo-stellate cells immunostained with S-100 protein antiserum in postnatal, castrated and thyroidectomized rats.

Pituitary folliculo-stellate cells and associated cells were studied immunohistochemically throughout the life of male and female rats. The marginal layer cells of the pars distalis and intermedia as well as the folliculo-stellate cells were immunostained with anti-S 100 protein serum. The immunostained folliculo-stellate cells in one section were identified as themselves by their ultrastructural properties on the adjacent section. Corticotrophs, thyrotrophs and prolactin cells, characterized by the stellate shape, were not immunostained with anti-S 100 protein serum. Reactivity for S-100 protein appeared on postnatal Day 6 in the marginal layer cells of the pars intermedia, and appeared on Day 10 in those cells of the pars distalis. No immunostained folliculo-stellate cells appeared before Day 6, but a few of them appeared on Day 10. Thereafter they increased in number, having more intense immunostaining with advancing age. In castrated rats, the immunostained folliculo-stellate cells appeared quite numerously, with branched cytoplasmic processes surrounding the gonadotrophs. In the thyroidectomized rats, however, folliculo-stellate cells lacked ramified cytoplasmic processes, and their topographic affinity for thyrotrophs was negligible.

Age Factors↗