Prevention of ischemic sinusoidal lining cell injury during liver transplantation in pigs using an artificial heart and lung.
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Animals↗
Biomedical subjects
K Fujimori
Publications and source records attributed to K Fujimori.
ATP-activated single-channel currents recorded from cell-free patches of pheochromocytoma PC12 cells.
Single-channel recordings were made using cell-free membrane patches (outside-out configuration) isolated from pheochromocytoma PC12 cells. ATP (50 microM) activated single channel currents in the isolated patches and the currents inactivated with a half-decay time of about 5s. The single channel conductance was about 13 pS in external solution with 140 mM Na. The amplitudes of the single-channel currents were decreased when external Ca was increased from 1.8 to 16.2 mM, suggesting that Ca blocks ion permeation through the channel. These properties of single-channel currents may underlie those of the macroscopic current.
Adenosine Triphosphate↗
Neurokinin A suppresses a voltage-gated K+ current in smooth muscle cells from rat vas deferens.
The effects of neurokinin A (NKA) on freshly isolated smooth muscle cells from the rat vas deferens were investigated electrophysiologically. NKA (10(-7) M) prolonged the duration of action potentials without affecting resting potentials. Under voltage-clamp conditions, NKA (10(-7) M) suppressed an outward K+ current activated by depolarizing voltage steps. Substance P (SP, 10(-7) M) did not affect the K+ current, suggesting that NKA is more potent in suppressing the K+ current than SP is, as is also true for the contractions of smooth muscles of the rat vas deferens. The suppression of the K+ conductance now reported may contribute to NKA-induced smooth muscle contraction.
Action Potentials↗
Probing the calcium-induced conformational transition of troponin C with site-directed mutants.
The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.
Animals↗
Difference between substance P- and acetylcholine-induced currents in mammalian smooth muscle cells.
The effects of substance P (SP) and acetylcholine (ACh) on membrane currents were compared using freshly isolated smooth muscle cells from guinea-pig ileum. Both SP (100 nM) and ACh (10 microM) induced inward currents at negative holding potentials. The ACh-induced current, but not the SP-induced current, showed relaxation upon voltage-stepping. The SP-induced current was increased by hyperpolarization more negative than -50 mV whereas the ACh-induced current was decreased by hyperpolarization. The results suggest that the SP-induced inward current is elicited via an ionic pathway different from that involved with ACh-activated channels.
Acetylcholine↗
Lysosomal glycogen accumulation in rat liver and its in vivo kinetics after a single intraperitoneal injection of acarbose, an alpha-glucosidase inhibitor.
A single intraperitoneal injection of acarbose (400 mg/kg) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with alpha-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (beta NAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2.5 mg/g liver within 2 days, and then decreased gradually at a rate of 0.4 mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1.5 mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid alpha-glucosidase. Therefore, assuming that the liver breaks down about 40 mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.
Acarbose↗
Reversible and selective antagonism by suramin of ATP-activated inward current in PC12 phaeochromocytoma cells.
1. The effects of suramin on an adenosine 5'-triphosphate (ATP)-activated inward current were investigated in PC12 phaeochromocytoma cells with whole-cell voltage-clamp techniques. 2. Suramin (30 to 300 microM) inhibited the ATP-activated current in a dose-dependent manner. The inhibitory effects were reversible and competitive. 3. Suramin also suppressed the current activated by adenosine 5'-O-(3-thiotriphosphate) but did not affect the current activated by nicotine. Suramin did not affect the suppression of a K current induced by methacholine. 4. The results suggest that suramin antagonizes the ATP-receptor-operated membrane current reversibly and selectively.
Adenosine Triphosphate↗
Chlordecone inhibits three types of ion channels in a neural cell line.
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Adenosine Triphosphate↗
An ATP-activated conductance in pheochromocytoma cells and its suppression by extracellular calcium.
1. ATP-activated inward current in PC12 pheochromocytoma cells was characterized using the whole-cell voltage-clamp technique. 2. ATP (100 microM) applied extracellular elicited an inward rectifying current with a reversal potential of about +7 mV. The current was desensitized in seconds in spite of continued presence of ATP. 3. A comparison was made of the ability of ATP and its analogues. The order of potency in activating the inward current was ATP greater than ATP gamma S greater than ADP; AMP, adenosine and alpha, beta-methylene ATP were inactive at concentrations up to mM. 4. The ATP-activated current was also observed when external Na+ and Ca2+ were replaced with K+, TEA, Tris or glucosamine. The order of ion selectivity was Na+ greater than K+ greater than TEA not equal to Tris greater than glucosamine. 5. The ATP-activated current was also recorded in extracellular solutions containing Ca2+ as the only external cation. The amplitude increased as the concentration of Ca2+ was increased in the range between 1.8 and 16.2 mM. However, the current amplitude decreased at higher Ca2+ concentrations and the current was not recorded in 110 mM-Ca2+ solution. 6. In the presence of 140 mM-Na+ in the external solution, the current amplitude also decreased as the external Ca2+ concentration was increased (from 1.8 to 16.2 mM). 7. The results indicate that Ca2+ as well as monovalent cations permeate through the ATP-sensitive pathway and that Ca2+ blocks ion permeation, including its own permeation through the pathway. This regulation by extracellular Ca2+ is different to the ATP-activated current in smooth muscle cells.
Adenosine Triphosphate↗
A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier.
For successful use of radiolabeled monoclonal antibodies (MAbs) for diagnosis and therapy, it is helpful to understand both global and microscopic aspects of antibody biodistribution. In this study, antibody distribution in a tumor is simulated by splicing together information on global pharmacokinetics: transport across the capillary wall, diffusive penetration through the tumor interstitial space, and antigen-antibody interaction. The geometry simulated corresponds to spherical nodules of densely packed tumor cells. This modeling analysis demonstrates that: 1) antigen-antibody binding in tumors can retard antibody percolation; 2) high antibody affinity at a given dose tends to decrease antibody percolation because there are fewer free antibody molecules. The result is a more heterogeneous distribution; 3) the average antibody concentration in the tumor does not increase linearly with affinity; and 4) increasing antibody dose leads to better percolation and more uniform distribution. This mathematical model and the general principles developed here can be applied as well to other biologic ligands.
Antibodies, Monoclonal↗
Extracellular adenosine 5'-triphosphate-evoked norepinephrine secretion not relating to voltage-gated Ca channels in pheochromocytoma PC12 cells.
Characteristics of extracellular ATP-stimulated norepinephrine (NE) secretion were investigated by the batch method in relation to membrane current measured with the whole cell voltage-clamp technique. Extracellular ATP stimulated [3H]NE secretion from PC12 cells in a concentration range from 10 microM to 1 mM. The maximal effect of ATP was about 5 times larger than that of nicotine. ATP-stimulated secretion was extracellular Ca-dependent, but it was not inhibited by the Ca-channel blockers, cadmium (300 microM) or nicardipine (10 microM). Extracellular ATP activated an inward current in PC12 cells and the peak amplitude of the current was about 5 times larger than that activated by nicotine. These findings suggest that extracellular ATP activates receptor-operated channels and causes NE secretion.
Adenosine Triphosphate↗
Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab')2, and Fab in tumors.
In order to understand the pharmacology of monoclonal antibodies and their conjugates, one must consider both global and microscopic aspects of antibody distribution. Here we present an analysis of antibody distribution in tumors based on the following factors: (a) molecular weight and valence of the antibody; (b) global pharmacokinetic profile following i.v. bolus injection; (c) penetration through the vascular wall; (d) diffusive and convective transport through interstitial space in the tumor; (e) antigen-antibody interaction; (f) antibody metabolism. Partial differential equations were developed to incorporate these factors and then solved numerically using parameter values from animal experiments, from clinical protocols at our institution, from studies of antibody binding characteristics in vitro, and from the literature. Salient findings from this model are that (a) antigen-antibody interaction in the tumor can retard antibody percolation away from blood capillaries, thus constituting a "binding site barrier"; (b) high antibody affinity tends to decrease antibody penetration and result in a more heterogeneous distribution; (c) high molecular weight [IgG greater than F(ab')2 greater than Fab] slows percolation and results in less uniform spatial distribution; (d) the average antibody concentration in the tumor does not increase linearly with antibody dose; (e) raising the rate of antibody metabolism results in low concentration and poor percolation; (f) perhaps most interesting, there is predicted to be a range of antibody dose and affinity within which the specificity ratio and average concentration could be kept high while limiting the heterogeneity of distribution. PERC, the computer program package developed for these analyses, provides a convenient and flexible way to assess the impact of global and microscopic parameters on the distribution of immunoglobulin in tumors. For calculations presented here, the input data were obtained from experimental sources, and qualitative features of the output proved consistent with the few interpretable observations available. However, detailed validation would require much more data than are currently at hand. The mathematical findings should therefore be considered as aids to concept development and as a set of null hypotheses with which to guide experimentation. Experiments and simulations will continue in tandem. It should be noted that the PERC package (and also the general principles delineated here) can be applied as well to biological ligands other than antibodies.
Antibody Affinity↗
NaCl-aided Hoechst 33258 staining method for DNA quantification and its application.
We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.
Animals↗
An experimental study on the effects of elastase, bleomycin and infection on the growth and tensile strength of elastic tissue in rabbit fetal membranes.
The effects of bleomycin, elastase and infection on the tensile strength of rabbit fetal membranes and the structure of the elastic tissue of rabbit amnion were studied. The experimental subjects were rabbits to which bleomycin (total dose, 20 mg) or elastase (total dose, 60 mg) was administered from 18 days of pregnancy. In addition to the fetal membranes from these treated animals, fetal membranes with amniotic infection induced by injection of Escherichia coli into the amniotic fluid (AF) of rabbits at 22 or 23 days of pregnancy were examined, and those from rabbits administered physiological saline solution were used as controls. The results obtained were as follows. Determination of tensile strength of the fetal membranes revealed that the infected membranes had minimum rupture pressure (18.67 +/- 6.30 mm Hg), followed by the elastase administration group (46.67 +/- 3.51 mm Hg). The value in the bleomycin administration group (99.67 +/- 12.89 mm Hg) was significantly higher than that in the control group (61.80 +/- 6.30 mm Hg). Histological changes in the fibroblast layer of these fetal membranes included elastic fibrosis due to excessive collagen production, which was mostly manifested as fibrosis densa, in the bleomycin administration group. In the elastase administration group, the elastic fibers were eliminated or became minute, as in the infection groups. The above results showed the adverse effects of elastase and bleomycin and the destructive effect of infection on the growth and properties of elastic tissue in the amnion, suggesting their pathophysiological importance.
Animals↗
Existence of muscarinic suppression of a K current in PC-12 pheochromocytoma cells.
Muscarinic influence on membrane currents of PC-12 pheochromocytoma cells were investigated with whole cell voltage-clamp methods. An outward K current was observed when depolarizing voltage steps were applied to the cells. Methacholine (MCh, 300 microM), a selective agonist for muscarinic receptors, partially suppressed the K current, and the suppression was enhanced by removal of external Ca. The effect of MCh was antagonized by a low dose (100 nM) of atropine. Nicotine (10 microM) induced an inward current in these cells but did not affect the K current activated by depolarizing voltage steps. A Ba current flowing through voltage-gated Ca channels was not changed by MCh. The results indicate the existence of a MCh-sensitive K current in PC-12 cells and suggest that the membrane currents of these cells are modulated by cholinergic agents through muscarinic mechanisms in addition to well-known nicotinic mechanisms.
Atropine↗
[Effects of extract from a herbal drug, cnidium rhizome (senkyu), on contraction, heart rates and membrane potentials of isolated guinea pig atria].
Effects of extract from a herb, Cnidium rhizome (Senkyu), on isolated guinea pig atria were investigated pharmacologically and electrophysiologically. The methanol extract from Cnidium rhizome decreased the contraction and slightly increased the heart rates of the isolated atria. Extracts from five other herbs, such as Japanese anglica root (Toki), Peony root (Shakuyaku), Moutan bark (Botanpi), Glycyrrhiza (Kanzo), Bupleurum (Saiko), affected neither the contraction nor the heart rates. The methanol extract from Cnidium rhizome was fractionated with chloroform and water fractions. The chloroform fraction exerted potent negative inotropic and chronotropic effects in isolated atria. The contraction was attenuated by two major components in the chloroform fraction, ligustilide and senkyunolide, but the heart rates were scarcely affected by these components. The chloroform fraction induced changes in resting potentials and configurations of normal action potentials recorded in the isolated left atria: the resting potentials were depolarized, and the upstroke velocity of the action potentials decreased. Neither ligustilide nor senkyunolide exerted such effects. The upstroke velocity of action potentials recorded in partially depolarized atria was reduced by the chloroform fraction as well as ligustilide and senkyunolide. The mechanisms underlying the effects of the extract from Cnidium rhizome were discussed.
Animals↗
Quantitative determination of follicle size distribution in the guinea pig ovary after hemicastration and PMSG treatment.
In order to investigate the action point of intraphysiological or supraphysiological elevation of FSH during the preovulatory period on follicular development, adult guinea pigs underwent unilateral ovariectomy on days 10, 12 and 14 of the estrous cycle (N = 6 each group). Thereafter, guinea pigs were injected twice daily with either vehicle or pregnant mare's serum gonadotropin (PMS). After 2 days, the remaining ovaries were removed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 microns) and stained with Azan. All follicles greater than 70 microns were classified by size and atretic stage. The follicular size distribution was not affected by hemicastration at day 10, although the ratio of atretic follicles (greater than 400 microns) decreased from 51% to 32% (P less than 0.01). Hemicastration at day 12 increased the largest nonatretic population (70-99 microns group) from 17% to 26%, and the ratio of atretic follicles (greater than 400 microns) decreased from 35% to 23%. The peak size distribution of follicles was shifted from 70-99 microns to 200-299 microns by PMS, and follicles 600-899 microns in size contained an increased percentage of atresia, which resulted in the bimodal distribution of viable follicles greater than 400 microns. These data suggest that 2 day hemicastration promotes an influx of primordial follicles into growing follicles and suppresses the atretic process by a different mechanism depending on the date of hemicastration in the estrous cycle. Conversely, hemicastration + PMS accelerated viable follicle growth to increase the percentage of atresia.
Animals↗
[Fundamental studies on bearing stress of basal seat under denture. 1. Difference height of ridge and load].
The purpose of this report is to investigate influence of difference in shape of ridge (height) and load (point, direction and strength) on bearing stress of basal seat. The stress against various loads (200-2,000 gf) onto artificial tooth (maxillaly first molar) were measured by using dentures, buried stress sensor, upon simulations of maxillaly edentulous posterior ridges with three types in height (9-15 mm). The summary of results are shown below: 1. When vertical loads onto mesial pit were applied, stress in working was most at ridge. The stronger load was, the more increased stress, and the shorter height of ridge was, the more decreased stress. Stress in balancing was most at palate. The stronger load was and the shorter height was, the more increased stress. 2. When oblique loads onto buccal cusp were applied 30 degrees to vertical, stress in working was most at palate. The stronger loads was, the more increased stress. In balancing, the stronger load was and the shorter height was, the more happened negative atmospheric pressure and breakaway of denture. 3. When oblique loads onto lingual cusp were applied 30 degrees to vertical, stress were most at buccal in working and at palate in balancing. At both areas, the stronger load was and the shorter height was, the more increased stress.
Alveolar Process↗