Biomedical subjects

N Kamo

Publications and source records attributed to N Kamo.

At least 37 records · Page 2Linked to original sources

PubMed1999

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-beta-glucoside.

The structural stability of bacteriorhodopsin (bR) solubilized by octyl-beta-glucoside was studied by measuring the denaturation kinetics under visible light irradiation and in the dark. The denaturation of bR solubilized by 50 mM octyl-beta-glucoside was very slow at room temperature when it was left in the dark. However, its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation and in the dark could be well described by a single decay constant. The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light irradiation was 12.5 kcal/mol, which was much smaller than the corresponding value in the dark, 26.2 kcal/mol. These results strongly suggest that some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D crystalline state. Comparing the denaturation behavior in the 2D crystalline state and that in the octyl-beta-glucoside-solubilized state, our findings suggest that protein-protein interaction contributes to the stability of this protein.

Bacteriorhodopsins↗

PubMed1999

Photoinactivation of vesicular stomatitis virus with fullerene conjugated with methoxy polyethylene glycol amine.

Fullerene is known to effectively produce mainly singlet oxygen by absorbing light energy, and therefore may be useful as a virucidal photosensitizer. This study was designed to investigate the virucidal activity of a water-soluble fullerene derivative, which is conjugated with methoxy polyethylene glycol amine to enhance its water solubility. Vesicular stomatitis virus (VSV) was inactivated upon illumination with this water-soluble fullerene, in a concentration- or dose-dependent manner. The titer of VSV was reduced by >5log 10 in the presence of 10 mg/ml (400 microM) fullerene derivative with 120 J/cm2 white light irradiation. VSV inactivation was inhibited by oxygen removal or by the addition of sodium azide, a known singlet oxygen scavenger. The substitution of H2O by D2O, which is known to prolong the lifetime of singlet oxygen, promoted the virucidal activity. These results indicate that singlet oxygen may play a major role in VSV photoinactivation by the water-soluble fullerene derivative. The concentration needed for virus inactivation is higher than that of other sensitizers such as methylene blue.

Antiviral Agents↗

PubMed1998

Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin.

Natronobacterium pharaonis has retinal proteins, one of which is pharaonis phoborhodopsin, abbreviated as ppR (or called pharaonis sensory rhodopsin II, psR-II). This pigment protein functions as a photoreceptor of the negative phototaxis of this bacterium. On photoexcitation ppR undergoes photocycling; the photoexcited state relaxes in the dark and returns to the original state via several intermediates. The photocycle of ppR resembles that of bR except in wavelengths and rate. The cycle of bR is completed in 10 ms while that of ppR takes seconds. The Arrhenius analysis of M-intermediate (ppR(M)) decay which is rate-limiting revealed that the slow decay is due to the large negative activation entropy of ppR. The addition of azide increases the decay rate 300-fold (at pH 7); Arrhenius analysis revealed decreases in the activation energy (activation enthalpy) and a further decrease in the activation entropy.

Journal Article↗

PubMed1998

V108M mutant of pharaonis phoborhodopsin: substitution caused no absorption change but affected its M-state.

Crystallographic data reveal that Met-118 in bacteriorhodopsin (bR) contacts directly with the C9 methyl group of retinal, and Khorana et al. [J. Biol. Chem. 268, 20305-20311 (1993)] suggest that this contact may regulate the absorption maximum (lambdamax). We have replaced the amino acid (Val-108) corresponding to Met-118 of bR by methionine in pharaonis phoborhodopsin (ppR), whose lambdamax is ca. 500 nm, while those of other bacterial rhodopsins such as bR, halorhodopsin, and sensory rhodopsin are red-shifted by 60-90 nm. By flash-photolysis measurement, we could not recognize a large spectral red-shift of the V108M mutant. On the other hand, the decay of ppRM (M-intermediate) of the mutant was approximately three times as fast as that of wild-type, and an M-like intermediate (M') whose lambdamax is blue-shifted by 60 nm from that of M became appreciable. The replacement abolished the shoulder of the ppRM spectrum. From these findings, we infer that the distance between the retinal and the 108-position in ppR is relatively long, and that in the M-state this distance is shortened.

Amino Acid Sequence↗

PubMed1998

Control of the unidirectional topological orientation of a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c reconstituted into proteoliposomes.

Control of the unidirectional topological orientation was achieved for a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c (RC/cyt c) reconstituted into proteoliposomes. Using the method of Ueno et al. [Ueno et al. (1995) Mater. Sci. Eng. C3, 1-6], we prepared RC/cyt c by conjugating cyt c to the H-subunit of RC of Rhodobacter sphaeroides R-26 using a bifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), as previously reported. The freeze-thaw method was used to incorporate RC/cyt c into liposomes that contained dipalmitoyl-L-alpha-phosphatidylcholine and dipalmitoyl-L-alpha-phosphatidylglycerol (1:9). The topological orientation of RC/cyt c in the proteoliposomes was determined using three methods: (i) release of the cyt c moiety from the proteoliposomes by cleaving the disulfide bond in the linker residue, (ii) electron transfer from free cyt c outside the proteoliposomes to the RC moiety, and (iii) photo-induced membrane potential of RC- and RC/cyt c-reconstituted proteoliposomes. The results indicated that about 90% of the RC/cyt c in proteoliposomes was oriented with the H-subunit exposed on the outside of the liposomes, whereas only about 60% of the RC in proteoliposomes had this orientation. Thus, we successfully controlled the unidirectional topological orientation of the RC moiety in liposomes using the RC/cyt c complex.

Animals↗

PubMed1998

Virus inactivation in superoxide dismutase preparations by ultraviolet light irradiation.

Viral inactivation in superoxide dismutase (SOD) derived from human red cells was carried out by ultraviolet light C (UVC) irradiation. With 400 J/m2 UVC irradiation, the titer of canine parvovirus (CPV, a nonenveloped virus), M13 bacteriophage (M13, a nonenveloped phage) and vesicular stomatitis virus (VSV, an enveloped virus), which were spiked into SOD solution, were reduced by > 4.6 log10 (detection limit), 7.0 log10 and 6.2 log10, respectively. The SOD activity was maintained and the band pattern of SOD on an electrophoresis gel was not changed even by 1000 J/m2 UVC irradiation. These results indicate that UVC irradiation is a promising method for the inactivation of both enveloped and nonenveloped viruses in SOD preparations while maintaining its activity.

Animals↗

PubMed1997

Functional expression of pharaonis phoborhodopsin in Escherichia coli.

Pharaonis phoborhodopsin, the photoreceptor of the negative phototaxis of archaebacterial Natronobacterium pharaonis, was functionally expressed in the heterologous system of Escherichia coli. Flash-photolysis on a millisecond time scale indicated that the photochemical properties of ppR expressed in E. coli were the same as those of the native ppR in N. pharaonis. We concluded that the integral membrane protein ppR is correctly folded in vivo in the eubacterial E. coli membrane.

Archaeal Proteins↗

PubMed1997

Adenosine 5'-triphosphate binding to bovine serum albumin.

Binding of ATP to bovine serum albumin was shown by ultrafiltration and NMR. The binding was pH dependent. Scatchard analysis revealed that at pH 5.4, 6.4 and 7.4, dissociation constant Kd was 13, 40 and 120 microM, respectively, and no binding was observed at pH 8.4. The binding stoichiometry was 1:1 for all pH. Dimer of BSA did not bind ATP. From chemical shifts of 31P-NMR, Kd was estimated to be 15 microM at pH 5.4, which is very close to that determined by ultrafiltration. While adenosine did not interfere with the binding. GTP, dCTP, ADP, UTP, AMP, phosphate and pyrophosphate were competitive inhibitors and their inhibition constants Ki were 25, 32, 36, 50, 130, 1000 and 186 microM, respectively. Fatty acids such as lauric acid and palmitic acid did not interfere with the binding. Warfarin was a non-competitive inhibitor. Cl- competitively inhibited the binding, and the inhibition constant was 20 mM. The dissociation constants of the Cl- binding were reported to be 0.42 mM for the first binding site, 10-5 mM for the second and 303-143 mM for the third [G. Scatchard, W.T. Yap, J. Am. Chem. Soc., 86 (1964) 3434; G. Scatchard et al., J. Am. Chem. Soc. 79 (1957) 12]. This suggests that the ATP binding site may be the second Cl- binding site.

Adenosine Triphosphate↗

PubMed1997

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol.

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.

Bacteriorhodopsins↗

PubMed1997

Drug extrusion in Corynebacterium glutamicum.

We selected a mutant of Corynebacterium glutamicum, EBR, which can grow in a medium containing cytotoxic ethidium bromide (EtBr) at a high concentration of 100 microM. The resistance to EtBr in the mutant was reversed by 2 microM reserpine, a potent inhibitor of mammalian p-glycoprotein and bacterial multidrug resistance (MDR) transporter, whereas reserpine alone had a minimal effect on cell growth. The mutant showed a much higher efflux rate of EtBr than wild-type cells, and the efflux was completely inhibited by 2 microM reserpine. In addition to reserpine, structurally unrelated chemicals such as quinidine, trifluorperazine, tetraphenylarsonium chloride, chlorpromazine and quinine inhibit the EtBr efflux, revealing that the putative efflux system(s) can recognize a variety of chemicals. The efflux activity was correlated with the membrane potential but not the intracellular ATP contents. We, therefore, concluded that the EtBr resistance may be involved by proton-motive-force driven multidrug efflux system(s).

Adenosine Triphosphate↗

PubMed1997

Culture in the presence of sugars increases activity of multi-drug efflux transporter on Haloferax volcanii.

We found that when a growth medium contained glucose, wild-type cells of Haloferax volcanii were able to grow even in the presence of doxorubicin (DOX), an anti-cancer reagent, whereas they usually cannot grow in its presence. The reason was that cells grown in the presence of glucose (glucose-grown cells) showed high multi-drug efflux activity even though the growth medium contained no DOX or substrates of the transporter. This transporter was ATP-driven and the elevation of efflux activity was not due to an increase in intracellular ATP contents. The activity was increased not only by glucose but also by sugars that could be metabolized.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Factors affecting M13 bacteriophage inactivation by methylene blue photosensitization.

We have investigated the factors that affect the virucidal activity of methylene blue (MB) photosensitization. The M13 bacteriophage was more rapidly inactivated at higher temperatures (6 degrees C < 24 degrees C < 38 degrees C). Rate constants for inactivation were 0.072, 0.139 and 0.260 (log10 inactivation)/ (J/cm2) at 6 degrees C, 24 degrees C and 38 degrees C, respectively. On the other hand, dye penetration into virus particles, which was monitored by the fluorescence of YOYO-1, was unchanged with incubation temperature. These data suggest that temperature dependency of M13 inactivation was due to factors other than dye permeability. The pH of the virus suspension also affected the rate of M13 inactivation by MB. The M13 bacteriophage was inactivated faster in basic suspensions and slower in acidic suspensions compared with neutral buffers. These results suggest that temperature and pH are factors that influence the extent of MB photosensitization, and hence, the control of these factors will be necessary for MB phototreatment of plasma products in transfusion medicine.

Antiviral Agents↗

PubMed1997

Photoinactivation of virus infectivity by hypocrellin A.

We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or beta-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Animals↗

PubMed1997

Potential involvement of both type I and type II mechanisms in M13 virus inactivation by methylene blue photosensitization.

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D2O), azide ion (N3-) and oxygen-dependent studies. Inactivation of M13 bacteriophage and singlet oxygen (1O2) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D2O and inhibited by N3-, suggesting that 1O2 participates in M13 inactivation by MB photosensitization. However, N3- did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52-67% of M13 inactivation observed in the presence of oxygen. These results suggest that 1O2-mediated (Type II) and sensitizer-mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.

Antiviral Agents↗

PubMed1996

Novel bacterial rhodopsins from Haloarcula vallismortis.

New bacterial rhodopsins of the cruxrhodopsin (cR) tribe were identified in a type strain Haloarcula vallismortis. The genes encoding a bacteriorhodopsin-like ion pump (named cR-3), a halorhodopsin-like ion pump (chR-3) and a sensor rhodopsin (csR-3) were cloned and sequenced. Together with the data for vsRII (Seidel et al., Proc. Natl. Acad. Sci. USA 92, 3036-3040 (1995); cpR-3 in our notation), the primary structures of a set of four rhodopsins are now all known only in this species. They are separated by almost the same distances in homology, suggesting that they have derived from a single ancestral rhodopsin. The degree of conservation in the amino acid sequence of each helix showed that helices C and G are relatively well conserved in all rhodopsins, whereas helices DEF are conserved especially in sensor rhodopsin-I, possibly because these helices are needed for interaction with the transducer protein (Htr).

Amino Acid Sequence↗

PubMed1996

Rhodamine 123 efflux transporter in Haloferax volcanii is induced when cultured under 'metabolic stress' by amino acids: the efflux system resembles that in a doxorubicin-resistant mutant.

In this paper, we report that an archaebacterium, Haloferax volcanii, cultured in medium containing a large excess of amino acids showed very low levels of rhodamine 123 (RH123), which is a potent substrate for P-glycoprotein and the bacterial multidrug efflux transporter. This low level involved the active efflux of RH123 from the cells. The level of intracellular RH123 was increased and the efflux inhibited by the Ca2+-channel antagonist verapamil and also by various anti-cancer drugs. The efflux transporter was suggested to be ATP-driven. We have previously selected a mutant of H. volcanii with resistance to doxorubicin, by repeatedly culturing cells in 1.5 microM doxorubicin [Miyauchi, Komatsubara and Kamo (1992) Biochim. Biophys. Acta 1110, 144-150]. The acquisition of resistance to doxorubicin involves the active expulsion of lipophilic drugs such as RH123 and doxorubicin. It is notable that the drug spectrum and ATP-dependency of the amino acid-induced efflux transporter resemble those of the efflux transporter induced by doxorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement.

Localization of chondroitin sulphates in periodontal ligaments (PDL) of rat molar roots during physiological and experimental tooth movement were analysed immunohistochemically with the use of monoclonal antibodies, 3B3 and 2B6, specific to chondroitin 6-sulphate (CH-6S) and chondroitin 4-sulphate/dermatan sulfate (CH-4S/DS), respectively. The maxillary first molars of experimental animals were forced to move laterally with a 10 g weight by U-shaped wires for 3 and 7 d. In control animals, 3B3 epitope was seen in the PDL near to the bone surface facing the distal half of roots, which corresponded to the compressive side during physiological tooth movement. Immunoreactivity for 2B6 was weak or negative in the PDL. Both epitopes were present at osteoid, precementum, lacunae and canaliculli of osteocytes and cementocytes. In 3-d-treated animals, the early stage of hyalinization characterized with visible cells and fibres was observed in the PDL at the buccal side of the mesial root, which showed intense immunoreactivity for 3B3. Further 3B3 positive area seen in control animals changed its position from the distal to the buccal side of the PDL. Immunoreactivity for 2B6 did not change in the PDL of 3-d-treated animals. In 7-d-treated animals, the typical hyalinization characterized with no visible cells and fibres was seen in the PDL at the buccal sides of both mesial and disto-buccal roots, where both epitopes were present at the peripheral part of the tissue. Observation of serial sections suggested that the 3B3-positive area was present at the peripheral part of the 2B6-positive area. The present results suggest that the expression of CH-6S is related to the compressive force in non-hyalinized and hyalinized PDL, whereas that of CH-4S/DS is not influenced by the mechanical stress.

Alveolar Process↗

PubMed1996

Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane.

We measured the absorption anisotropies of bacteriorhodopsin (bR) within a purple membrane suspension after photo-excitation in the millisecond time range. The purple membranes used were isolated from Halobacterium salinarium grown at three different culture temperatures, 37.0, 43.0 and 47.5 degrees C. For the membranes from the 37.0 degrees C culture, the observed anisotropies at wavelengths of 410, 570 and 680 nm showed almost the same slow decay. The slow decaying of the anisotropies originated from the rotation of the membrane itself. Using the membranes from the 43.0 and 47.5 degrees C culture, however, we found that the anisotropy change varied at each wavelength measured. In these cases, it is shown from detailed data analysis that 1) the rotational motion of photo-intermediates within the membrane is more restricted than that of non-excited bR and 2) the distorted arrangements of the proteins within the membrane remain, even after photo-intermediates return to ground-state bR. This restricted motion is probably caused by the conformational changes in photo-intermediates, which prevent the rotation of the monomer protein and/or lead photo-intermediates to bind with neighboring proteins.

Bacteriorhodopsins↗