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H Kitasato

Publications and source records attributed to H Kitasato.

70 records · Page 4Linked to original sources
PubMed1980

The interaction between the effects of insulin and ouabain on the activity of Na transport system in frog skeletal muscle.

The effects of insulin and ouabain on 22Na efflux and net Na loss were examined in frog sartorius muscles. After removal of ouabain, 22Na efflux which had been inhibited by ouabain remained at the inhibited level for more than 2 hr. In ouabain-free solution, insulin stimulated the Na efflux from ouabain-preincubated muscles. The stimulating effect of insulin on 22Na efflux was completely blocked by the presence of ouabain. Insulin shifted the Na efflux vs. intracellular Na concentration relationship toward the region of lower Na concentrations, and made the slope of log (Na efflux) vs. log [Na]1 relationship less step. On the other hand, ouabain shifted the Na efflux vs. intracellular Na concentration relationship toward the region of higher Na concentration, and made the slope of the log (Na efflux) vs. log [Na]1 relationship steeper. The relationship between insulin-stimulated Na efflux and intracellular Na concentration obtained from ouabain-preincubated muscle was located in the region of higher Na concentrations than that obtained from control muscle. These findings suggest that insulin increases an apparent affinity of binding sites for Na ions, and the possibility that insulin increases the Vmax of Na transport system may be excluded. In contrast with the effect of insulin, ouabain may be considered to decrease the apparent affinity of binding site for Na ions.

Animals↗
PubMed1980

Effects of ouabain on Na efflux in high internal Na and insulin-preincubated muscles.

To gain an insight into the mechanism of stimulation by insulin of Na efflux, the effect of high concentrations of internal Na on the inhibition by ouabain of Na efflux was compared to the inhibition by ouabain of insulin-stimulated Na efflux. The rate coefficient of 22Na efflux from "high-Na" muscle exposed to ouabain was lower than that from "low-Na" muscle exposed to ouabain. Similarly, the rate of net Na loss from the "high-Na" muscle which had been exposed to ouabain was lower than that frm the "low-Na" muscle. These findings indicate that Na transport units whose internal Na-binding sites have been occupied by Na is susceptible to ouabain. The insulin-stimulated Na efflux was inhibited by ouabain to a larger extent than was the Na efflux from the control muscle, although the final level of the rate coefficient of 22Na efflux from the muscle exposed to both insulin and ouabain was not always lower than that from the muscle exposed only to ouabain. The similarity between the effects of ouabain in "high-Na" muscle and in insulin-preincubated muscle suggests that insulin increases the fraction of Na transport units whose internal Na-binding sites have been occupied by Na.

Animals↗
PubMed1980

Apparent affinity changes induced by insulin of Na-K transport system in frog skeletal muscle.

The dependency of Na efflux on intracellular Na concentration was examined on sartorius muscles of Rana catesbeiana. In normal Ringer, the rate coefficient of 22Na efflux is nearly proportional to the internal Na concentration, i.e., Hill's coefficient in the dependency of Na efflux on internal Na concentration was around two. As long as insulin is present in the bathing solution, the rate coefficient of 22Na efflux retains the stimulated level. Insulin caused a leftward shift of the relationship between Na efflux and the logarithm of internal Na concentration, and concomitantly decreased Hill's coefficient. A model of the coupling of Na transport and hydrolysis of ATP was proposed. The theoretical relation derived from this model accounted for these findings quite satisfactorily. According to this model, insulin shifts as a cofactor the equilibrium between dephosphorylated and phosphorylated states of carriers, whose Na binding sites are not occupied by Na, toward the phosphorylated state.

Animals↗
PubMed1973

K permeability of Nitella clavata in the depolarized state.

Membrane current responses to sudden potential changes were recorded in solutions of various [K](o) on 52 internodal cells of Nitella clavata. The membrane current after sudden depolarization had a component sensitive to [K](o) which increased with time from 0.3 to 2.0 s and remained steady thereafter. This late current became zero at values of E and [K](o) which suggests that the current was nearly all carried by K(+). The potassium conductivity represented by this current increased with depolarization, with a half-maximum value at about -70 mV, and saturation at about -30 to -20 mV. The potassium conductance also increased with increasing [K](o), but less rapidly than predicted for constant potassium permeability. This failure of the conductance to increase with [K](o) was relatively the same at all membrane potentials and may be explained by a model with a finite number of channels. No attempt was made to model the dependence of g(K) on time after depolarization or on membrane potential. However, the finding that the membrane potential did not affect the way in which the permeability depended on [K](o) suggests that the membrane potential change does not affect the affinity of the sites, and that the increase in g(K) with time after depolarization is brought about by an increase in the number of channels with such sites.

Calcium↗
PubMed1968

The influence of H+ on the membrane potential and ion fluxes of Nitella.

The resting membrane potential of the Nitella cell is relatively insensitive to [K](o), but behaves like a hydrogen electrode. K(+) and Cl(-) effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P(K) and P(Cl) are independent of pH but are a function of membrane potential. Slope ion conductances, G(K), G(Cl), and G(Na) were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H(+) was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K(+) equilibrium potential. In the range of external pH 5 to 6, H(+) chord conductance was substantially equal to the membrane conductance. However, the [H](i) measured by various methods was not such as would be predicted from the [H](o) and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H(+) extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H(+) equilibrium potential is always maintained notwithstanding a continuous H(+) inward current which should result from the potential difference.

Action Potentials↗
PubMedDate not supplied

Regulation of fluid flow through corneal stroma in the bullfrog.

Regulation of fluid flow through corneal stroma was investigated in the bullfrog. Corneal specimens were mounted by clamping their limbal sclera between the two chambers of a Ussing-type chamber. The epithelial surface was covered with Ringer's solution, while the endothelial surface was superfused with Ringer's solution at various pressures ranging from 0-60 mm Hg. At 0 mm Hg, the cornea swelled, while at 10 mm Hg the corneal thickness remained unchanged. Further elevation of the hydrostatic pressure of the endothelial superfusion solution caused a decrease in corneal thickness, suggesting that the hydrostatic pressure in the in vivo frog corneal stroma is about 10 mm Hg. At 10 mm Hg of endothelial superfusion pressure, piercing the epithelial cell layer with a 30-gauge needle caused only slight corneal swelling. Removing glucose from the epithelial perfusion solution induced a slowly progressing increase in corneal thickness. Iodoacetate did not interfere with the swelling of the pierced cornea after the removal of glucose from the epithelial perfusion solution. To examine the possibility that the interstitial fluid flows across the stroma-scleral boundary, corneal specimens having unclamped sclera were incubated in Ringer's solution containing 3 mmol/L dextran of various molecular weights ranging from 8800-162,000, and the volume of the preparation was monitored by sequential measurement of the weight. In the presence of dextran with a molecular weight higher than 70,000, the corneal volume decreased at the beginning of incubation, and after reaching the minimal volume it slowly increased, indicating that the stroma-scleral boundary is permeable to dextran of even a molecular weight of 162,000, although dextran molecules diffuse much more slowly than water, and the concentration of unfilterable solutes in the stroma is lower than 3 mmol/L. In experiments using the Ussing-type chamber at 10 mm Hg of endothelial superfusion pressure, a decrease of NaCl in the superfusing solution to 1/2 caused rapid corneal swelling followed by slow recovery. Adding NaCl to the 1/2 NaCl Ringer's solution caused a further corneal thinning in a concentration-dependent manner. The same extent of decrease in corneal thickness as induced by adding NaCl was achieved by the same concentration of glucose as of NaCl, implying that the value of the reflection coefficient of the endothelial cell layer to either Na+ or Cl- is about half that of glucose. Our results show that even a small difference in the concentration of low molecular weight solutes (e.g., Na+ and Cl-) exerts a force that draws water from the cornea.

Animals↗
PubMedDate not supplied

DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.

The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.

Animals↗
PubMedDate not supplied

Tumorigenicity of the E6 and E6-E7 gene constructions derived from human papillomavirus type 33.

Human papillomavirus type 33 (HPV33) belongs to the group of HPV types frequently found in severe cervical dysplasias and carcinomas. By analogy with HPV types 16 and 18 selectively expressing E6 and E7 genes in malignant tissues, we studied the HPV33 E6 and E7 open reading frames in various configurations with upstream promoter and noncoding region (NCR) known to contain a particular 78-bp tandem repeat. HPV DNA fragments were cloned into expression vectors between the SV40 or the mouse metallothionein I promoter and the neo gene, transfected into NIH3T3 cells, selected by neo resistance, and inoculated into nude mice. In these bioassays, a weak transforming activity was detected for E6 open reading frame, and could be significantly enhanced either by the NCR or the E7 open reading frame. No tumorigenicity could be detected for E7 alone or in configuration with the upstream NCR. Further analyses of tumor cells showed that HPV33-derived genes were not sufficient to induce an anchorage-independent phenotype and, interstingly, there was no requirement for virus-transfected tumor cells to retain HPV sequences during tumor progression. We concluded that the transformation function of HPV33 resides in E6 gene as assayed by tumorigenicity. An enhancer of the E6 promoter is located in the NCR. On the other hand, in the absence of the NCR, E6 tumorigenicity may be augmented by the E7.

Animals↗
PubMedDate not supplied

Properties of Syrian hamster cells transformed by human papillomavirus type 16.

Adult Syrian hamster kidney cells were transfected with a mixture of plasmids containing human papillomavirus type 16 (HPV16) E6/E7 open reading frames (ORFs), activated Ha-ras gene and neomycin resistance gene. From these cultures two lines were isolated which were oncogenic for newborn and 5-day-old but not for 3-week-old hamsters. Sublines oncogenic for 3-7-week-old hamsters were derived from tumours formed in animals inoculated within 5 days of birth. The cells contained HPV 16 DNA in an integrated form and HPV16 transcripts. The transcript patterns in low and high oncogenicity sublines were different. Very few tumour-bearing animals possessed antibodies reactive with E6- and E7-derived synthetic peptides. On the other hand a majority of these animals gave a positive reaction in the lymphoproliferation assay with either E6 and E7 peptides or extracts from the transformed cells.

Animals↗