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Biomedical subjects

N Kamo

Publications and source records attributed to N Kamo.

At least 55 records · Page 3Linked to original sources
PubMed1996

[Anesthesia for cesarean section: drug transfer to fetus during anesthesia].

One case of prenatally diagnosed congenital diaphragmatic hernia was reviewed in terms of the anesthetic managements. Concentrations of diazepam, pancuronium and fentanyl were measured in maternal, fetal and umbilical serum. As expectedly, the transition of diazepam through the placenta was large and the concentration of diazepam in the fetal serum was equal to that of maternal serum, but only a small amount of pancuronium was transferred.

Adult↗
PubMed1995

The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane.

We measured the flash-induced absorption anisotropies of mutant bacteriorhodopsin (bR), D96N, in the purple membrane suspension. The measured anisotropy decay at 410 nm differed from that at 570 nm. These wavelength-dependent anisotropies show that the motion of absorption dipole of non-excited bR is faster than that of M-intermediate. The motion of non-excited bR is considered as the rotational motion of whole protein in the purple membrane. This fact suggests that the photo-excitation induces the conformational change of the protein and/or the inter-protein interaction within the membrane, which prevents the motion of M-intermediate.

Anisotropy↗
PubMed1995

Nucleosides and nucleotides. 141. Chemical stability of a new antitumor nucleoside, 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine in alkaline medium: formation of 2'-C-cyano-2'-deoxy-1-beta-D-ribo-pentofuranosylcytosine and its antitumor activity.

We have designed 2'-C-cyano-2'-deoxy-1-beta-D-arabino- pentofuranosylcytosine (CNDAC) as a potential mechanism-based DNA-strand-breaking nucleoside, which showed potent tumor cell growth inhibitory activity against various human tumor cell lines in vitro and in vivo. When measuring the pKa of the 2' alpha-proton of CNDAC, we found that CNDAC epimerized to 2'-C-cyano-2'-deoxy-1-beta-D-ribo-pentofuranosylcytosine (CNDC) with concomitant degradation of both CNDAC and CNDC to cytosine and 1,4-anhydro-2-C-cyano-2-deoxy-D-erythro-pent-1- enitol. Kinetic analysis of these reactions showed that abstraction of the acidic 2'-proton of CNDAC and CNDC initiated the reactions, which quickly reached an equilibrium. In the equilibrium, a concentration ratio of CNDAC and CNDC was about 3:5. Concomitant degradation of these nucleosides was found to be rather slow. Deuterium incorporation experiments with CNDAC in a D2O buffer suggested the mechanism of the beta-elimination reactions is an E1cB type. These epimerization and degradation reactions were found even in neutral conditions (pH 7.5) and also occurred in RPMI 1640 cell culture medium. The discovery of which nucleoside possesses the predominate tumor cell growth inhibitory activity was important. While both nucleosides showed potent tumor cell growth inhibitory activity against three human tumor cell lines (colon carcinoma WiDr, small cell lung carcinoma SBC-5, and stomach carcinoma MKN-74 cells) in 48 h of incubation, in 20 min of incubation, CNDAC was 11-50 times more effective than CNDC. In vivo antileukemic activity of these nucleosides against a mouse P388 model, CNDAC was obviously superior to CNDC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1995

Reason for the lack of light-dark adaptation in pharaonis phoborhodopsin: reconstitution with 13-cis-retinal.

The reconstitution of pharaonis phoborhodopsin was performed by incubation of its opsin with 13-cis-retinal. Spectrum change was very slow, and two phases of the change were observed: the first and second phases are due to the transient formation of 13-cis pigment and spontaneous isomerization to all-trans-retinal, respectively. Slow binding supports an idea that the retinal binding pocket of ppR is highly restricted. Being bent in the configuration, 13-cis-retinal cannot be accommodated in the pocket due to the steric hindrance. This is a possible reason for the lack of light-dark adaptation.

Archaeal Proteins↗
PubMed1995

Conductance change in phospholipid bilayer membrane by an electroneutral ionophore, monensin.

Monensin is a polyether antibiotic ionophore and is considered an electroneutral Na/H antiporter. Its addition, however, increased the conductance of phospholipid bilayer membrane, and this increase was observed only when the medium contained Na+. Analysis of the current-voltage curve suggested that the increase was due to the formation and the translocation of an univalently charged species. The conductance at zero external voltage was proportional to the second power of monensin concentration and increased with the decrease in pH of the medium. Modified monensin whose terminal carboxyl was esterified showed much larger increase (ca. 100 times) in conductance than intact monensin. We concluded that the complex between the dimer of protonated monensin and Na+ contributed to the electrogenic transport of monensin. This complex bears a +1 charge, which is consistent with the analysis of current-voltage curves. Contrary to the conductance, the Na+ transfer rate of liposomal membrane measured with 23Na-NMR was proportional to the monensin concentration, meaning that the electrogenic component contributes little to the total monensin-mediated Na+ transport in the present system. It should be noted that this electrogenic component may change the membrane potential.

Electric Conductivity↗
PubMed1995

Ultraviolet action spectra for peroxide generation in human and pig epidermal keratinocytes loaded with dihydrorhodamine 123.

We developed a new and simple method for measuring peroxides in a single living cell, and the generation of peroxides upon ultraviolet (UV) irradiation was measured in human and pig epidermal keratinocytes. The method was based on the fact that the non-fluorescent dye, dihydrorhodamine 123, reacts in the presence of peroxides, such as H2O2, and changes into the fluorescent rhodamine 123, and hence the fluorescence intensity is proportional to the amount of reacted peroxide. The epidermal keratinocytes were loaded with the dihydrorhodamine under a fluorescence microscope and exposed to UV radiation. Taking C as the content of peroxides generated within the cell and I as the increase in fluence (radiation intensity x time = photons/cm2), the following empirical relationship was established: C = Cs (1-exp(-kI)), where Cs is the content of peroxides at the saturation state, and k is a kinetic parameter. The dependence of the two parameters on wavelength in the range 280-400 nm was studied. In human keratinocytes Cs had a peak at 310 nm and a small peak (shoulder) at 380 nm, while k increased gradually toward shorter wavelengths. In pig keratinocytes, on the other hand, k had a peak around 380 nm and a shoulder at 330 nm, while Cs remained unchanged. Aminotriazole, an inhibitor of catalase, and low temperatures increased the stationary levels of peroxide generation in pig keratinocytes upon UV irradiation, indicating that the reaction used for measuring intracellular peroxides is competitive with the intrinsic reactions in scavenging peroxides.

Amitrole↗
PubMed1994

Activation energy for permeation of phosphonium cations through phospholipid bilayer membrane.

The conductance caused by translocation of various phosphonium cations across phospholipid (from soybean) bilayer membrane was measured. Phosphonium cations used were tetraphenylphosphonium (TPP+) and triphenylalkylphosphonium cations formulated as (Phe)3-P(+)-(CH2)nCH3 (n = 0-5). The conductance was dependent on voltage applied externally to the membrane in accordance with a theory developed by previous authors. Using the theory, values of beta K(i) were determined, where beta and k(i) are a linear partition coefficient and a rate constant of transmembrane ion transport, respectively. Since beta k(i) depended on the phosphonium ion concentration, values extrapolated to infinite dilution, (beta k(i))0, were determined. Temperature dependence of (beta k(i))0 allowed us to estimate the activation energy of transport, Ea. For TPP+ thermodynamic values obtained were consistent with values calculated by Flewelling and Hubbell [(1986) Biophys. J. 49, 541-552]. When (Phe)3-P(+)-(CH2)nCH3 (n = 0-5) were used, E(a) depended on the odd or even of n. This "odd and even" pattern was observed in a variety of phenomena such as solubility in water, equivalent ionic conductivity in water, and 31P NMR chemical shift.

Biological Transport↗
PubMed1994

Shape of the chromophore binding site in pharaonis phoborhodopsin from a study using retinal analogs.

To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, alpha-retinal, cyclohexylretinal and 5-isopropyl-alpha-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.

Archaeal Proteins↗
PubMed1994

Generation of active oxygens, cell deformation and membrane potential changes upon UV-B irradiation in human blood cells.

The generation of peroxides (presumably hydrogen peroxide) by UV-B irradiation of human blood cells was detected. Non-fluorescent dihydrorhodamine 123 (DHR) is oxidized to fluorescent rhodamine 123 (R123) by H2O2 or peroxides with a stoichiometry of 1:1 in the presence of exogeneous peroxidase, and the fluorescence of R123 within the cells was measured using flow-cytometry. UV irradiation gave rise to changes in the cellular volume and the membrane potential, whose extent and direction were dependent on the type of blood cells. The production of peroxides (H2O2) in polymorphonuclear leukocytes is the largest among blood cells at the lower dose (< 0.1 J/cm2), and the production decreases with an increase in the dose, while the production in platelets is the smallest at the lower dose, but above 0.4 J/cm2 it increases suddenly so that at the higher dose (1.2 J/cm2) it amounts to 3.3 x 10(-16) mol/cell. For monocytes and lymphocytes, the production increases gradually with the increase in the dose.

Blood Cells↗
PubMed1993

Quartz crystal microbalance for the detection of microgram quantities of human serum albumin: relationship between the frequency change and the mass of protein adsorbed.

We have developed a piezoelectric immunosensor for the detection of microalbumin. Human serum albumin (HSA) in the range 0.1-100 micrograms mL-1 could be detected using a flow cell; the immunosensor is sensitive enough to monitor levels of albuminuria. The immunosensor did not respond to bovine serum albumin, only to HSA, implying that the specificity for HSA was high. We investigated the relationship between the frequency change (delta F) and adsorption per unit area of piezoelectrically active quartz crystal (delta M). delta M was estimated with radioisotope-labeled anti-HSA or HSA. When anti-HSA was adsorbed onto the surface of the crystal or HSA was bound to anti-HSA supported by the crystal, values of magnitude of delta F/delta M were larger than the value predicted from theory (Sauerbrey's equation). Furthermore, magnitude of delta F/delta M for HSA was larger than that for anti-HSA.

Adsorption↗
PubMed1993

Membrane transport of tetraphenylphosphonium and its homologues through the planar phospholipid bilayer: concentration dependence and mutually competitive inhibition in membrane passive transport.

The concentration dependence and mutually competitive inhibition in the membrane transport of the lipophilic cations tetraphenylphosphonium (TPP+) and its homologues were studied by use of a planar phospholipid bilayer. The current-voltage characteristics were analyzed by use of a model developed by Ketterer et al. (J. Membr. Biol. 1971, 5, 225-245). The conductance at 0 mV [G(0)], which represents the transport ability, increased with an increase in concentration, and the G(0) values revealed saturation at higher concentrations. The relationship between G(0) and concentration was well fitted to a Michealis-Menten-type equation with a saturable component (maximum conductance [G(0)max] and concentration yielding one-half of G(0)max[Km]). The G(0)max and Km values were calculated to be 4.1 x 10(-8)-242 x 10(-8) S/cm2 and 13-489 microM, respectively, depending on the lipophilic cations used. The G(0) values normalized by the corresponding G(0)max values were plotted against the concentrations normalized by the corresponding Km values. Normalized curves for TPP+ homologues were all superimposed on a single curve. The mechanism for the saturation of G(0) values may involve the adsorption of TPP+ homologues to a planar phospholipid bilayer, because the Km values calculated in the present study were comparable to those for adsorption to phosphatidylcholine liposomes. We further determined the mutual inhibition of membrane transport by these homologues. Tetraphenylmethylphosphonium at 1 mM, a concentration threefold higher than the Km value for this compound, reduced the G(0) for other TPP+ homologues by 38-55%, whereas reduction by 10 mM tetraethylammonium was minimal. The type of inhibition was classified as mutually competitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗
PubMed1992

Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation, tetraphenylphosphonium: correction of probe intracellular binding and mitochondrial accumulation.

The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.

Amphotericin B↗
PubMed1992

Detection of antistreptolysin O antibody: application of an initial rate method of latex piezoelectric immunoassay.

Latex plezoelectric immunoassay (LPEIA) is a new latex immunoassay using a plezoelectric quartz crystal (Kurosawa et al. Chem. Pharm. Bull. 1990, 38, 1117). This assay requires no immobilization of antigen or antibody on an electrode surface of a plezoelectric crystal, while the immobilization is indispensable for ordinary immunoassays using a plezoelectric crystal as a microbalance. The present paper improves a previous method (end-point analysis) by introducing the initial rate method using a batch cell; reduction of assay volume (1.2 mL) and shortening of assay time (2-3 min) were achieved. This assay was applied to the detection of antistreptolysin O antibody (ASO) in serum. The frequency change was proportional to the ASO concentration up to 1040 IU mL-1, and the method has good sensitivity for actual clinical application. The volume of serum required for the assay was 0.02 mL. Twenty-four clinical specimens were analyzed with this LPEIA, and the values obtained were compared with those obtained with a turbidimetric latex agglutination method. The correlation coefficient between these values was 0.950 (P < 0.01).

Agglutination↗
PubMed1992

In archaebacteria, there is a doxorubicin efflux pump similar to mammalian P-glycoprotein.

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca(2+)-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.

Archaeal Proteins↗
PubMed1992

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.

Archaeal Proteins↗
PubMed1992

Plasma-polymerized allylamine film used as a new solid phase in immunoradiometric assay (IRMA): effect of antibody (F(ab')2 fragment) concentration on dose response in two-site IRMA.

Allylamine (ALAM) film was plasma-polymerized on a flat glass (referred to as ALAM(GLA): GLA refers to a flat glass plate) for use as a solid phase in two-site immunoradiometric assay (two-site IRMA). Adsorption of F(ab')2 anti-human immunoglobulin G [formula: see text] to ALAM(GLA) was larger than adsorption on a polyvinyl chloride plate (referred to as PVC). Contrary to the expectation that the dose response for human IgG (hIgG) on ALAM (GLA) was better than that on PVC, the dose responses on both solid phases were the same. This phenomenon was independent of molecular size of the antigen (Ag) (hIgG or Fc fragment of hIg G (hIgG-Fc)) and also the reaction with protein A (pA). Because direct measurements of binding with 125I-labeled hIgG (hIgG*) or hIgG-Fc (hIgG-Fc*) showed no difference between ALAM(GLA) and PVC, the phenomenon was not due to the second step in the system of two-site IRMA (an Ag--Ab reaction (Ab refers to antibody)). These results indicated that the phenomenon was due to the first step (the adsorption of [formula: see text] to a solid phase). When the concentration of [formula: see text] immobilized on the solid phases was lowered, a significant increase in the dose response was observed for ALAM(GLA).

Adsorption↗