Biomedical subjects

R Kitamura

Publications and source records attributed to R Kitamura.

At least 55 records · Page 3Linked to original sources

PubMed1993

Metabolic activation of aflatoxin B1 by human placental microsomes.

The metabolic activation of aflatoxin B1 by human placental microsomes was studied. Aflatoxin B1 showed relatively high mutagenic activity in Ames test when incubated with human placental microsomes. Addition of alpha-naphthoflavone or aminoglutethimide, known inhibitors of cytochrome P450 1A and P450 19, respectively, into the test system partially inhibited the mutagen-producing activity. It was suggested that the activation of aflatoxin B1 in human placental microsomes is mediated by at least these two forms of cytochrome P450.

Aflatoxin B1↗

PubMed1992

Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

Adult↗

PubMed1992

Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing.

A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10-fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1.

Adult↗

PubMed1992

Stable expression of monkey cytochrome P-450IA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B1.

A monkey cytochrome P-450IA1 cDNA (MKah1) was transfected into Chinese hamster CHL cells using a vector containing the SR alpha promoter, and sublines stably expressing P-450IA1 were established. The cells showed 25-fold higher sensitivity to the cytotoxic effect of aflatoxin B1 than the parental CHL cells. This hypersensitivity was almost completely suppressed by alpha-naphthoflavone, which is a known specific inhibitor of P-450IA. The cells expressing P-450IA1, but not CHL cells, showed a positive response to aflatoxin B1 in an assay for mutagenicity at the HGPRT locus.

Aflatoxin B1↗

PubMed1992

Pharmacological studies on 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one (T-614), a novel antiinflammatory agent. 3rd communication: the involvement of bradykinin in its analgesic actions.

In order to elucidate the analgesic mechanism of 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-on e (T-614), its effects on the kinin-forming system were examined both in vivo and in vitro. T-614, at doses more than 10 mg/kg p.o., exhibited a significant inhibitory effect on the increased levels of bradykinin released into the pouch fluid of kaolin-induced inflammation in rats. In the kaolin-induced writhing response in mice, which is shown to be mainly dependent on the action of bradykinin, T-614 reduced not only the writhing frequency but also the peritoneal levels of bradykinin in a dose-dependent manner. Whereas, in the zymosan-induced writhing response in which prostaglandin I2 (PGI2) is shown to be an important mediator, it did not exert an obvious inhibition on either writhing responses or peritoneal PGI2 levels at a highest dose of 100 mg/kg. T-614 did not inhibit the activities of serine proteases, such as trypsin, thrombin, kallikrein and plasmin. Furthermore, it did not affect the kinin-forming enzymes of rat plasma in vitro. The above results suggest that the analgesic effects of T-614 may be partly mediated by the inhibition of bradykinin release in the local inflamed tissue.

Analgesia↗

PubMed1992

Human fetal liver cytochrome P-450: capacity to form genotoxic metabolites.

Unlike most experimental animals, human fetal liver possesses forms of cytochrome P-450. Thus, the purpose of this study was to clarify the toxicological significance of these forms of cytochrome P-450 to understand possible roles of these cytochromes in producing genotoxic metabolites from promutagens. In fact, human fetal livers showed considerable capacity to activate aflatoxin B1 and IQ (2-amino-3-methylimidazo [4,5-f] quinoline). Three of four forms of cytochrome P-450, P-450HFLa-d, which we could purify from human fetal livers were capable of activating promutagens to mutagens. One of these three forms, namely P-450HFLa, catalyzed the metabolic activation of aflatoxin B1 and IQ. An expression plasmid containing HFL33 cDNA encoding P-450HFLa was constructed and the protein expressed in insect (Sf9) cells and in human cancer cells, MCF-7. Aflatoxin B1 was efficiently activated to a mutagen upon addition of the lysate of Sf9 cells to the incubation mixture for the assay. Transformants of MCF-7 cells expressing P-450IIIA7 (HFLa) showed higher sensitivity to aflatoxin B1 than the parental MCF-7 cells as detected by cytotoxicity.

Animals↗

PubMed1992

Examination for lipid peroxidation in liver microsomes of guinea pigs as a causal factor in the decrease in the content of cytochrome P-450 due to ascorbic acid deficiency.

The content of cytochrome P-450 in liver microsomes from guinea pigs was decreased by ascorbic acid-deficiency. Since ascorbic acid is an antioxidant in vivo, the possible involvement of lipid peroxidation in this phenomenon was investigated. In fact, the level of lipid peroxides in liver homogenates of guinea pigs was increased by ascorbic acid deficiency. The level was significantly decreased when the animals were given tocopherol acetate (25 mg/kg/day, s.c.) with an ascorbic acid-free diet. The activities of aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase, and the content of cytochrome P-450 spectrally determined did not restore the control level by the administration of tocopherol acetate to the ascorbic acid-deficient animals. Western blot analysis of liver microsomes with antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) showed that ascorbic acid-deficiency resulted in a decrease in the amount of cytochrome P-450 immunochemically related to P-450IA2, but not the amounts of the forms of cytochrome P-450 cross-reactive with antibodies to P-450IIB1 and P-450IIIA4. The reduced amounts of cytochrome P-450 cross-reactive with antibodies to rat P-450IA2 in liver microsomes of ascorbic acid-deficient animals remained unchanged even when lipid peroxidation was inhibited by tocopherol acetate, suggesting that there is a mechanism(s) other than lipid peroxidation involved in the reduction of amounts of cytochrome P-450 by ascorbic acid deficiency.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1991

Occurrence of autoimmune antibodies to liver microsomal proteins in association with fulminant hepatitis in the LEC strain of rats.

The Long Evans Cinnamon (LEC) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats. About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis; sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC rats.

Animals↗

PubMed1990

A search for the cortical gustatory area in the hamster.

The gustatory area was searched in the cerebral cortex of the hamster by means of a combined approach using electrophysiological, behavioral, and histological experiments. The chorda tympani (CT), which innervates taste buds on the anterior part of the tongue, projected to a confined area anterior to the middle cerebral artery and just dorsal to the rhinal fissure. The trigeminal component of the lingual nerve (LN) area was located anterodorsal to the CT area, and the glossopharyngeal nerve (GN), which innervates taste buds on the posterior part of the tongue, was posterior to the CT area. The center of the CT and GN areas belonged to the dorsal part of the dysgranular insular cortex, and the LN area was within the primary somatosensory granular cortex. Bilateral symmetrical ablations of the CT and GN areas abolished the conditioned taste aversion (to sodium saccharin) that had been acquired before ablations, indicating a role of these areas in some cognitive processes of taste perception. Injections of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) in the CT and GN areas, centered in the dysgranular insular cortex, revealed that this cortical region had major fiber connections with the contralateral homotypical cortical area, ipsilateral amygdala (central, lateral and basolateral nuclei), ipsilateral parvicellular part of the posteromedial ventral nucleus of the thalamus, bilateral parabrachial nucleus, contralateral nucleus of the solitary tract, raphe nuclei, and the locus ceruleus. Conversely, injections of WGA-HRP in these target areas showed anterograde and/or retrograde transport in the similar dysgranular insular cortex and additionally in the ventral part of the granular insular cortex. The present results suggest that the cortical gustatory area of the hamster is about 1.5 X 1.5 mm in size with the topographic organization between anterior and posterior parts of the tongue, and is located mainly in the dysgranular insular cortex around the middle cerebral artery.

Afferent Pathways↗

PubMed1990

Isolation of tumor-associated antigen from sera of bovine leukemia virus-infected cattle.

Tumor-associated antigens (TAA) expressed on the surface of enzootic bovine leukemia (EBL) cells were detected and separated from sera of bovine leukemia virus (BLV)-positive cattle using monoclonal antibody-conjugated immunoaffinity matrix. Eluted fraction from these sera showed 3 polypeptides with molecular weights of 70K, 52K, and 30K daltons, and these polypeptides reacted with a monoclonal antibody against TAA. However, only 70K peptide was isolated from culture supernatant of EBL B-cell line. We also tried to examine a reversed passive hemagglutination test to develop a rapid screening system of serum TAA level, but its sensitivity was below the level of detection when EBL sera was applied directly. This is the first report on the existence of tumor antigens in sera from leukemic cattle.

Animals↗

PubMed1989

Response properties of lateral hypothalamic neurons during ingestive behavior with special reference to licking of various taste solutions.

Activity of 58 single neurons in the lateral hypothalamic area (LHA) was recorded while Wistar male rats were drinking water and various taste solutions in a test box. A cue tone was presented before opening of a shutter for access to a drinking spout. Except 8 neurons which were non-responsive in the present experimental paradigm, 50 neurons were classified into 3 types according to their response properties: (1) 10 neurons changed their activity with arousal state or circadian rhythm, (2) 10 neurons responded to specific sensory stimuli, i.e. 2 were classified as taste-responsive neurons, which responded excitatory to sodium salts, 3 neurons responded to olfactory stimulation, 5 to somatosensory stimulation applied to the perioral region, and (3) the remaining 30 decreased their activity during licking of liquids regardless of their qualities. Besides this classification, activity of 28 of 58 LHA neurons was altered after onset of the cue tone (or before start of licking), i.e. 24 increased their activity (learned anticipatory response), and 3 modulated their tonic activity into burst discharges corresponding to sniffing, and 1 increased its activity in relation to stepping toward the drinking spout. These data suggest that about half of the LHA neurons increased their activity in anticipatory (searching or approaching) periods just before ingestion, and decreased activity in rewarding periods during ingestion of water or sapid solutions.

Action Potentials↗

PubMed1989

Sensory and motor responses of trigeminal and reticular neurons during ingestive behavior in rats.

Activities of 53 neurons in the brain stem were recorded with chronically implanted fine wires in freely eating and drinking rats. Twenty units were isolated from the trigeminal mesencephalic nucleus; 18 were spindle afferents and 2 periodontal afferents. The spindle units were classified into 4 types: 5 units showed rhythmical activity related only to the jaw opening phase during both licking and chewing, 8 units discharged at jaw opening phase during licking, but both at jaw opening and jaw closing phases during eating, 2 units increased phasic activity at jaw opening phase during licking, but increased tonically independent of jaw movements during eating, and the remaining 3 units responded only at jaw closing phase both in licking and eating behavior. Nine units were assumed to be alpha motoneurons isolated from the trigeminal motor nucleus; 2 innervating the temporalis muscle, 3 the masseter muscle, 2 the digastric muscle, and the remaining 2 presumably the pterygoid muscles. These units, with a mean tonic spontaneous rate of about 10 impulses/s, showed phase-related rhythmical burst activities during licking and eating. Four units were isolated from the trigeminal main sensory nucleus. Sixteen units were isolated from the medullary reticular formation and 1 from the pontine reticular formation. Of these 16 units, 3 from the parvocellular part of the medullary reticular formation were presumably premotor interneurons, and 3 units in the "intertrigeminal region", 2 units in the "juxtatrigeminal region", and 8 units in the "supratrigeminal region" were responsive to multiple sensory modalities. These results demonstrate that almost all the units sampled showed characteristic rhythmic activities associated with the position of the tongue or mandible during rhythmical jaw movements.

Action Potentials↗

PubMed1989

Taste responses of cortical neurons in freely ingesting rats.

1. Activities of 35 taste-responsive neurons in the cortical gustatory area were recorded with chronically implanted fine wires in freely ingesting Wistar rats. Quantitative analyses were performed on responses to distilled water, food solution, and four taste stimuli: sucrose, NaCl, HCl, and quinine hydrochloride. 2. Taste-responsive neurons were classified into type-1 and type-2 groups according to the response patterns to licking of the six taste stimuli. Type-1 neurons (n = 29) responded in excitatory or inhibitory directions to one or more of the taste stimuli. Type-2 neurons (n = 6) showed responses in different directions depending upon palatability of the liquids to rats: neurons showing excitatory (or inhibitory) responses to palatable stimuli exhibited inhibitory (or excitatory) responses to unpalatable stimuli. 3. Correlation coefficients of responses to pairs of stimuli across neurons suggested that palatable stimuli (water, food solution, sucrose, and NaCl) and unpalatable stimuli (HCl and quinine) elicited reciprocal (excitatory vs. inhibitory) responses in type-2 neurons, whereas type-1 neurons showed positively correlated responses to specific combinations of stimuli such as food solution and NaCl, sucrose and HCl, NaCl and quinine, and HCl and quinine. 4. A tendency toward equalization of effectiveness in eliciting responses among the four basic taste stimuli was detected on the cortex. The ratios of mean evoked responses in 29 type-1 neurons in comparison with spontaneous rate (4.4 spikes/s) were 1.7, 1.9, 1.8, and 1.9 for sucrose, NaCl, HCl, and quinine, respectively. 5. The breadth of responsiveness to the four basic taste stimuli was quantified by means of the entropy measure introduced by Smith and Travers (33). The mean entropy value was 0.540 for 29 type-1 neurons, which was similar to 0.588 previously reported for rat chorda tympani fibers, suggesting that breadth of tuning is not more narrowly tuned in a higher level of the gustatory system in the rat. 6. Convergent inputs of other sensory modalities were detected exclusively in type-1 neurons. Thirteen (45%) of 29 type-1 neurons also responded to cold and/or warm water, but none of 6 type-2 neurons responded to thermal stimuli. Two (7%) of 29 type-1 neurons responded to almond and acetic acid odors, but the 6 type-2 neurons did not. Two (13%) of 16 type-1 neurons responded to interperitoneal injection of LiCl, which is known to induce gastrointestinal disorders, with a latency of approximately 5 min, but 4 type-2 neurons tested were not responsive to this stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1989

[In vitro replication of plasmid pKYM].

The crude extract (fraction II) prepared from E. coli could replicate plasmid pKYM, only when the extract contained the rep protein which was produced by the plasmid and essential for its multiplication in vivo. The intermediate of replication was accumulated when a dideoxynucleotide triphosphate was added to the reaction mixture. By analyzing the intermediate, the initiation site of the deoxyribonucleic acid replication and the direction of replication could be determined. The replication initiated inside the ori region of pKYM and the direction was unidirectional. The analysis also suggested that the synthesis of the lagging strand stopped at almost the same site as the initiation site.

Adenosine Triphosphatases↗

PubMed1989

[Localization and gustatory responsiveness of cortical taste area in the hamster].

Mapping of evoked potentials on the cortical surface following electrical stimulation of the chorda tympani and the glossopharyngeal nerve, and anodal D.C. stimulation of the tongue indicated that the cortical taste area (CTA) was located in the dysgranular insular cortex just dorsal to the rhinal fissure near the middle cerebral artery in the hamster. Bilateral lesion of the CTA attenuated or disrupted the conditioned taste aversion that had been acquired preoperatively. This fact suggests that the CTA plays a role in some cognitive processes of taste. Experiments using anterograde and retrograde axonal transport of wheat-germ agglutinin conjugated horseradish peroxidase showed that the CTA received inputs from the contralateral CTA, amygdala, thalamic taste area and the pontine taste area, and that neurons in the CTA (chiefly in layer V) sent axons to the contralateral CTA, amygdala, and the thalamic, pontine and bulbar taste areas. Responses of 87 CTA neurons to the four basic taste stimuli were recorded in urethane-anesthetized hamsters. Majority of the CTA neurons (85%) existed in the dysgranular insular cortex. Neurons responding best to sucrose tended to be located rostrally, those to NaCl caudally, and those to HCl were distributed evenly in the CTA. Judging from the breadth of responses to the four tastes and across-neuron correlation coefficients, responsiveness of neurons in layer V is more narrowly tuned than in layers II-IV and VI.

Animals↗

PubMed1988

Taste effects of 'umami' substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques.

Behavioral and electrophysiological experiments were performed to examine whether or not the taste of 'umami' substances such as monosodium glutamate (MSG), disodium 5'-inosinate (IMP), and disodium 5'-guanilate (GMP) is really unique in hamsters. When the animals were conditioned to avoid ingestion of MSG (or IMP) or their mixture by pairing its ingestion with an i.p. injection of LiCl, suppression of drinking generalized to IMP (or MSG), GMP, NaCl, and other sodium salts. Suppression of drinking after conditioning to NaCl generalized to MSG, IMP, GMP, and inorganic sodium salts. These learned aversions to umami substances and sodium salts were abolished by bilateral deafferentation of the chorda tympani, but were not affected by destruction of the bilateral glossopharyngeal nerves. The integrated whole-nerve responses of the chorda tympani to MSG, IMP, and NaCl were similar to each other, consisting of the initial dynamic phase and the following tonic phase. Synergism of chorda tympani responses to a mixture of MSG and IMP was not observed. Across-fiber response patterns of the chorda tympani for MSG, IMP, or their mixture were very similar to that for NaCl. Even the high concentrations of umami substances (0.3 M MSG, 0.3 M IMP, and the mixture) did not elicit any detectable responses in the glossopharyngeal nerve. These results suggest that the taste of umami substances is not unique in the hamster, but is similar to that of sodium salts, and is mediated exclusively via the chorda tympani.

Animals↗

PubMed1988

Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene.

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.

Animals↗

PubMed1988

Sensory inputs from the oral region to the cerebral cortex in behaving rats: an analysis of unit responses in cortical somatosensory and taste areas during ingestive behavior.

1. The responses of 90 cortical neurons in the somatosensory and gustatory areas were recorded with chronically implanted fine wires in freely moving Wistar rats. The responses were analyzed mainly while the animals were freely licking solutions and eating dry pellets. Cortical neurons were classified into several groups according to their response properties. 2. "Mechanosensitive" neurons (n = 20) showed rhythmic phasic activity in different phases of the licking cycle, depending on the location of their receptive field in the peripheral orofacial region. 3. "Movement-related" neurons (n = 27) changed their activities tonically during licking, chewing, or grooming behavior. The responses were either excitatory or inhibitory. Receptive fields and adequate stimuli could not be identified. These neurons might receive somatosensory (except light tactile) inputs from wide or deep areas of intra- or perioral regions, or might be related to orofacial active movement. 4. "Taste" neurons (n = 35) increased or decreased their discharge rates during licking of particular taste solutions. Some taste neurons received convergence from somatosensory inputs. 5. "Temperature" neurons (n = 2) responded exclusively to water of temperatures lower or higher than room temperature. The responses were opposite in direction between cold and warm stimuli. 6. "Anticipation" neurons (n = 4) increased their impulse discharges before the start of licking in the situation in which the animal expected access to the drinking tube. 7. "Attention" neurons (n = 2) responded to arousal stimulation such as sound, a flash of light, and body touch. These neurons showed only a slightly increasing response during ingestive behavior. 8. The locations of 56 of 90 units were histologically identified. Mechanosensitive neurons were located in the appropriate parts of the somatotopic pattern within the primary somatic sensory area in the granular cortex. Taste neurons were found evenly in the dysgranular cortex and the agranular insular cortex. Other types of neurons were located mainly in the dysgranular cortex between the granular cortex and agranular insular cortex, and some were intermingled with taste neurons in the agranular insular cortex. 9. The present study has shown that cortical neurons in the orolingual somatosensory and taste areas have different response characteristics related to each aspect of ingestive behavior.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗