Search PubMed⌕ Search

Biomedical subjects

Y Nakaoka

Publications and source records attributed to Y Nakaoka.

At least 37 records · Page 2Linked to original sources

A calcium-activated, large conductance and non-selective cation channel in Paramecium cell.

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 microM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare.

Animals↗

A phenotypically normal liveborn male after prenatal diagnosis of trisomy 20 mosaicism.

We report on a case of prenatally diagnosed trisomy 20 mosaicism. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) using a chromosome 20 specific probe were performed on the lymphocytes and extra-embryonic tissues after birth. All of them revealed normal karyotypes. The baby is developing normally at the age of 2 years.

Adult↗

The photodynamic action of methylene blue on the ion channels of Paramecium causes cell damage.

The photodynamic effects of methylene blue (MB) on wild-type and mutant strains of Paramecium Were studied. From measurements of survival and cell motility under the continuous application of light in the presence of MB, the mutant strains remained alive for about three times longer than the wild-type strain. Although the resting potential of the mutant cells was similar to that of wild-type cells, the continuous photodynamic action shifted the membrane potentials of the mutant and wild-type cells to a depolarized level and a hyperpolarized level, respectively, from that before light application. Under voltage clamping, the mutant cells reduced not only the outward current elicited by the photodynamic action but also the outward tail current elicited by the preceding pulse of hyperpolarization. We conclude that the mutant strain is defective in the activation of Ca(2+)-dependent K+ channels. This defect might cause a reduction in the Ca2+ influx because of the suppression of the membrane hyperpolarization, which results in the elongation of survival time under the photodynamic action.

Animals↗

Possible mediation of G-proteins in cold-sensory transduction in

The possible involvement of G-proteins in cold-sensory transduction was examined using voltage-clamped Paramecium multimicronucleatum into which non-hydrolyzable guanosine nucleotide analogues had been applied intracellularly. Guanosine-5'-O-3-thiotriphosphate, guanosine-5'-O-2-thiodiphosphate and aluminium fluoride all reduced the transient inward current in response to cooling, suggesting the possibility that G-proteins mediate cold-sensory transduction. Internal application of a Ca2+ chelator, EGTA, also reduced the current response. In addition to their effect on reducing the cold-sensory response, application of these chemicals modulated both the resting potential and the membrane conductance. Possible correlations between G-protein activity and the regulation of intracellular Ca2+ levels are discussed.

Journal Article↗

Defect of cold-sensitive response in calmodulin mutants of Paramecium and the restoration by cyclic nucleotide.

Wild type and calmodulin mutants (cam) of Paramecium tetraurelia were examined for cold-sensitive responses. Among mutants tested, cam12 and cam13 mutants, which have substitutions in N-terminal lobe of calmodulin molecule, reduced both responses in the swimming and the membrane potential. Under voltage clamp conditions, the cooling stimulus to the wild type cell induced a transient inward current whose amplitude increased with the rate of temperature drop. The cam12 cell did not induce inward currents in response to cooling with a rate slower than -0.4 degree C/s. The reduced current response of cam12 mutant was restored by an external application of a phosphodiesterase inhibitor, theophylline. Also, an intracellular injection of hydrolysis-resistant cyclic nucleotides, either 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5'-cyclic monophosphate (8-Br-cGMP), restored the current response. Such restoration was accompanied by shifts of the resting potential to hyperpolarized levels and by an increase in the membrane conductance. The results suggest the possibility that calmodulin and cyclic nucleotide regulate K+ channels responsive to the cooling stimulus.

Animals↗

Cold-induced decrease of K+ conductance and its inhibition by a calmodulin antagonist, W-7, in Paramecium tetraurelia.

Under voltage clamp, Paramecium tetraurelia was used to examine the cold-induced inward current and its inhibition by a calmodulin antagonist, W-7 [N-(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide]. Cooling of the cell caused an inward current. The amplitude of the current was increased as the membrane potential was made more positive than the resting potential, and it was significantly blocked by using CsCl-filled electrodes and tetraethylammonium in the bath solution, suggesting that the current was accompanied mainly by a decrease in K+ conductance. The cold-induced inward current was reversibly inhibited by the external application of W-7 in a concentration-dependent manner. EGTA-microinjection into the cell also reduced the current. These results indicate that the decrease in K+ conductance induced by cooling is Ca(2+)-dependent and is inhibited by W-7.

Animals↗

[Trial of radiation and cisplatin, carboplatin combination chemotherapy for advanced cancer (W-platinum chemoradiotherapy].

Cisplatin was reported to be an effective radiation sensitizing agent. The effect was also reported to depend on dose intensity. But the incidence of complication was demonstrated in the relationship with the dose escalation curve of anti-cancer agent. The major side effect of CDDP was occasionally serious, as in renal toxicity or bone marrow suppression. W-Platinum is the trial of concurrent chemoradiotherapy. Cisplatin and its derivative, Carboplatin, were selected as effective radiation sensitizing agents and to obtain high-dose intensity and additive cytotoxicities by interaction between the two drugs. Concomitant administration of two platinum anti-cancer agents has the advantage of reduction of side effects compared with administration of single anti-cancer agents to the same degree. The first case was a recurrence of epipharyngeal cancer after 3 courses of chemotherapy, including CDDP or CBDCA. This case was suspected to be cancer-resistant to CDDP. The second case was post-operative residual lung cancer. The pre-operative diagnosis was stage III A. A poor prognosis was expected. This case was disease-free and alive for 1 year after W-Platinum administration. The most frequent complication was bone marrow suppression. Patients were rescued from bone marrow suppression with administration of G-CSF. Renal toxicity could be suppressed with sufficient hydration.

Adenocarcinoma↗

Cloning of a novel ubiquitin-conjugating enzyme (E2) gene from the ciliate Paramecium tetraurelia.

We isolated a 1.7 kb gene (UbcP1) for a ubiquitin-conjugating enzyme from a P. tetraurelia cDNA library and sequenced it. Its deduced polypeptide sequence consists of 425 amino acid residues (48 kDa). The UbcP1 protein contains novel N- and C-terminal extensions in addition to a UBC domain, and within the UBC domain it shares low identity with sequences of other known E2s. A constructed phylogenetic tree suggests that the UbcP1 protein may represent a member of a distinct subfamily of E2s. Southern blot analysis showed that the N-terminal extension of the UbcP1 is conserved in P. multimicronucleatum.

Amino Acid Sequence↗

Detection of aneuploidy in human spermatozoa using fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66%, and 0.61%, respectively. For XX- and YY-sperm the frequencies were 0.28% and 0.27%, respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.90% and 49.66%, consistent with an expected 1:1 ratio.

Aneuploidy↗

Cold-sensitive Ca2+ influx in Paramecium.

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.

Animals↗

Dual infection with attaching and effacing Escherichia coli and enterotoxigenic Escherichia coli in post-weaning pigs.

Post-weaning diarrhoea in pigs occurred on two farms in Hokkaido, Japan, in 1994. Four piglets aged 35 or 45 days were examined after death. At necropsy, ecchymotic haemorrhages were seen on the mucosal surface of the caecum and colon. Histopathologically, numerous Gram-negative bacilli adhered to the brush border of the small intestines, but the brush border itself was intact. Typical attaching and effacing (AE) lesions were seen in the caecum and colon. Immunohistochemically, the bacilli which adhered to the brush border gave positive results with antisera against serogroup O149 of Escherichia coli; the bacilli which caused the AE lesions, however, belonged to serogroup O45. It was concluded that the disease resulted from dual infection with attaching and effacing E. coli (AEEC) and enterotoxigenic E. coli (ETEC).

Animals↗

Gastric attaching and effacing Escherichia coli lesions in a puppy with naturally occurring enteric colibacillosis and concurrent canine distemper virus infection.

A puppy suffering from chronic diarrhea was humanely killed at 90 days of age. Numerous Gram-negative bacilli were found adhering to the surface of as well as within epithelial cells from the stomach to the colon. Canine distemper virus inclusions were in the epithelial cytoplasm of the esophageal, gastric, and intestinal mucosa. Typical attaching and effacing ultrastructural lesions were in the stomach, and some bacilli were in the cytoplasm of the epithelial cells. Escherichia coli, isolated from the contents of the small intestine, belonged to serotype 0118: NM and were negative for plasmid-encoded EPEC adherence factor (EAF) and positive for the E. coli attaching effacing (eae) gene. Immunohistologically, bacilli attached to the epithelium from the stomach to the colon were positive for antisera against E. coli 0118. E. coli 0118: NM inoculated into human tissue culture cells (HEp-2 cells) were attached to the surface of the cells and within the cytoplasm. This is the first report of attaching and effacing E. coli (AEEC) infection in the stomach of the dog.

Animals↗

Participation of GTP-binding protein in the photo-transduction of Paramecium bursaria.

Analysis of the flow of light information in Paramecium, especially the participation of GTP-binding protein, was carried out. Injection of GDP-beta-S into Paramecium cell abolished the light-induced inward current. The partially purified rhodopsin-like protein (RLP) that we obtained activated frog rod outer segments (ROS) GTPase. A GTP-binding protein, with Mw 57,000 was detected by immunostaining with anti-rat G alpha rabbit IgG. These results suggest that the light information flows from the light-captured RLP to the GTP-binding protein.

Animals↗

Tuberculous spondylitis (Pott's disease) with bilateral pleural effusion.

We report a case of tuberculous spondylitis (Pott's disease) with bilateral pleural effusion in a 25-year-old male patient. Left-pleural effusion was observed on admission. The initial diagnosis was tuberculous pleuritis. However, during anti-tuberculosis chemotherapy, back pain and right-pleural effusion appeared. by further examination, we diagnosed an active tuberculous spondylitis of the 11th and 12th thoracic vertebrae possibly by the dissemination of Mycobacterium tuberculosis through blood circulation or lymphatic circulation. In cases in which anti-tuberculosis chemotherapy is not so effective, the possibility of the rarely-appearing extra-pulmonary manifestation of tuberculous spondylitis must be considered.

Adult↗

[A case of superior vena cava syndrome treated with combination radiation and CRE (CBDCA and VP-16) therapy].

Carboplatin and etoposide were reported to be excellent radiation sensitizers. We encountered a patient with SVC syndrome due to lung cancer who was successfully treated by combination carboplatin, etoposide and hyperfractionation radiotherapy. A 72-year-old man was admitted to our hospital because of remarkable face edema. Computed tomography revealed a huge lung tumor and compressed SVC due to tumor growth. Acute tumor regression was essential for this case. We performed combination chemotherapy and radiation. The regime consisted of CBDCA 300 mg (day 1 1 hr drip infusion) and etoposide 50 mg/day for 21 days by oral administration. Two daily fractionations of 1.4 Gy were delivered 5 days-a-week, with a 4 h interval between fractions (total dose 49.8 Gy). Complete response of huge tumor was attained in this case. The major side effect associated with the therapy was myelosuppression. The patient's quality of life has been remarkably improved with this therapy.

Aged↗

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo.

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca(2+)-sensitive fluorescence measurements. Increases in [Ca2+]i were evoked by the bath application of acetylcholine (1 microM or higher). The rise in [Ca2+]i was due to the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response occurred even in a Ca(2+)-free medium. The Ca2+ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 microM abolished the response to 10 microM acetylcholine; muscarine and carbamylcholine (100 microM each) evoked Ca2+ rises. Increases in [Ca2+]i were also evoked by the bath application of ATP (10 microM or higher). The Ca2+ rise by ATP was evoked even in a Ca(2+)-free medium. Adenosine (500 microM) did not cause any Ca2+ response. Suramin and reactive blue 2 (200 microM each) completely blocked the Ca2+ response to 500 microM ATP. Uridine triphosphate (500 microM) caused comparable Ca2+ responses with those to 500 microM ATP. These results suggested the involvement of P2U purinoceptors. The potentiation of Ca2+ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 microM and 100 microM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have Ca2+ mobilizing systems activated by acetylcholine and ATP.

Acetylcholine↗

Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare.

The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR-amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex.

Animals↗

Correlation between circadian periods and cellular activities in Paramecium bursaria.

Paramecium bursaria shows a circadian rhythm of photoaccumulation: photoaccumulation is stronger during the day than at night. We obtained five strains of P. bursaria having different circadian periods under continuous light conditions, ranging from 20.9 to 27.9 h. Various physiological activities were compared in the cells of these strains. The periods of contractile vacuole contraction were in the range 10-15 s, which was almost proportional to the periods of the circadian rhythm in each strain. Swimming velocities were inversely proportional to the circadian period; i.e. swimming velocities were high in strains whose circadian periods were short. Resting membrane potential was more depolarized in strains with longer circadian periods. Finally, the membrane resistance of the resting state was reduced in proportion to the increase of the circadian period. Such correlation between the cellular properties and the circadian period suggests that the circadian clock mechanism is associated with various physiological activities of the cell.

Animals↗