Biomedical subjects
T Endo
Publications and source records attributed to T Endo.
Electrocardiographic changes induced by diesel exhaust particles (DEP) in guinea pigs.
We have previously reported that diesel exhaust particles (DEP) caused a negative inotropic effect that was followed by cardiac arrest in the isolated atrial preparation of guinea pigs. The purpose of this study was to examine the systemic effects of DEP on electrocardiographic (ECG) changes using guinea-pigs. We found that intravenously administered dimethyl sulfoxide (DMSO) extract of DEP solution induced arrhythmias and deaths via complete atrioventricular (AV) block in guinea pigs. The LD of DEP solution was 132.0 +/- 7.2 mg/kg. The coefficient of variance (CV) of LD measured by the modified Hatcher-Magnus method was relatively small (5.5%). Fractions of DEP extracted by hexane, ethanol or methanol, 4-hydroxyphthalic acid 2-methyl ester, a compound isolated from methanol extract of DEP did not induce significant ECG changes in guinea pigs. As compared with fresh DEP solution, the DMSO/DEP solution used in the present study induced similar cardiac toxicity after being stored in a freezer at 4 degrees C for 3 days. These results suggest that stable and water-soluble fractions of DEP may be responsible for cardiotoxicity.
Decreased DCC mRNA expression in human gastric cancers is clinicopathologically significant.
Loss of heterozygosity (LOH) or decreased expression of the deleted in colorectal cancer (DCC) gene, a candidate tumor suppressor gene, has been suggested to correlate with progression in several types of tumors. In human gastric cancers, although DCC LOH has been reported to be frequent in expanding-type tumors, the relation of decreased DCC mRNA expression with clinicopathological features, including metastatic events, remains to be investigated in detail. Therefore, expression levels of DCC mRNA were examined in 52 advanced gastric cancers by the reverse transcription-polymerase chain reaction method combined with Southern blot analysis. Overall, decreased DCC mRNA expression was observed in 27 of the tumors (52%). According to Ming's [Cancer 39, 2475-2485 (1977)] classification, the expanding type showed a significantly higher incidence (71%) than the infiltrative type (17%) (p = 0.0002). Immunohistochemical analysis demonstrated the presence of DCC expression in cancer but not non-cancerous cells in infiltrative-type lesions. An additional follow-up study of tumor recurrence in 42 patients undergoing curative resection implicated decreased DCC expression in metachronous liver metastasis. The results indicate that decreased DCC mRNA expression is closely associated not only with the expanding-type phenotype but also with liver metastasis of gastric cancers. Assessment of this parameter in primary lesions may thus find predictive application.
Antiemetic effects of sendide, a peptide tachykinin NK1 receptor antagonist, in the ferret.
The antiemetic activity of sendide, a new peptide tachykinin NK1 receptor antagonist, against cisplatin-induced emesis was investigated using ferrets. The frequency of cisplatin (10 mg/kg, i.p.)-induced retching (104.6 +/- 14.3/6 h) and vomiting (19.0 +/- 3.0/6 h) was significantly reduced by pretreatment with sendide (3.0 mg/kg, s.c.) (14.0 +/- 8.1/6 h and 1.8 +/- 1.2/6 h, respectively). Intravenous bolus injection of substance P (1-10 microg/kg) or 5-hydroxytryptamine (5-HT) (10-50 microg/kg) produced a dose-dependent increase in the abdominal afferent vagus nerve activity. The change from pre-injection level in the afferent nerve activity induced by substance P (1 microg/kg, i.v.) (453.7 +/- 51.5%) was significantly reduced by pretreatment with either sendide (100 microg/kg, i.v.) (276.1 +/- 50.1%, P < 0.05) or granisetron, a 5-HT3 receptor antagonist (1 mg/kg, i.v.) (146.3 +/- 14.0%, P < 0.01). The amount of 5-HT released into the solution during a 1-h exposure to 2-methyl-5-HT (10(-6) M), a 5-HT3 receptor agonist, was significantly increased (317.9 +/- 46.7%, P < 0.05) compared with that of the control tissues (160.4 +/- 8.1%). The 2-methyl-5-HT-induced 5-HT release was significantly inhibited by administration of sendide (10(-6) M) (174.0 +/- 21.6%, P < 0.05) or granisetron (10(-6) M) (186.6 +/- 27.3%, P < 0.05). Since sendide does not penetrate the central nervous system, these results suggest that the antiemetic effects of sendide are due to the inhibition of NK1 and 5-HT3 receptors on the emetic peripheral detector sites.
Identification of regions in which positive selection may operate in S-RNase of Rosaceae: implication for S-allele-specific recognition sites in S-RNase.
A stylar S-RNase is associated with gametophytic self-incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S-RNase is responsible for S-allele-specific recognition in the self-incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (dS) and non-synonymous (dN) substitutions in rosaceous S-RNases detected four regions with an excess of dN over dS in which positive selection may operate (PS regions). The topology of the secondary structure of the S-RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S-RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S-RNases the PS regions also form two sites and are candidates for the recognition sites for S-allele-specific discrimination.
Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan.
alpha-Dystroglycan, which is a cell surface component of dystroglycan complex, is known to bind laminin in basal lamina of muscle cells and Schwann cells. We found previously that a novel O-glycan, Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man, is the major oligosaccharide in bovine peripheral nerve alpha-dystroglycan, and that this structure might mediate the binding of laminin. In order to determine whether this structure is specific for peripheral nerve alpha-dystroglycan or present on different forms of alpha-dystroglycan, we analyzed the structures of the sialylated O-glycans of rabbit skeletal muscle alpha-dystroglycan. Their structures were elucidated to be a mixture of a core 1 O-glycan and the same O-mannosyl glycan that we found in bovine peripheral nerve. These results indicate that alpha-dystroglycan in different species and tissues share a common structure of its major O-linked acidic carbohydrate, suggesting its relevance to the basic functional role of alpha-dystroglycan.
T-cell receptor variable gene analysis of renal allograft-infiltrating cells in biopsy specimens using a nonradioisotopic micromethod.
BACKGROUND: A sensitive micromethod for T-cell receptor (TCR) analysis is needed for clonality analysis of renal allograft-infiltrating T cells (RAITs) obtained by needle biopsy. METHODS: TCR cDNA was amplified by the anchored polymerase chain reaction and was hybridized with 28 different TCR beta variable (TCRBV) genes fixed on nylon membranes, and the percentage of each TCRBV gene was measured spectrophotometrically. RESULTS: The specificity and linearity of the hybridization technique and the constancy of the TCRBV percentages over a wide range of sample amounts were demonstrated by control experiments. Analysis of RAITs of biopsy specimens from four patients showed broad or skewed TCRBV usage, indicating the presence of polyclonal and oligoclonal RAIT populations, respectively. In one patient who received OKT3 immunosuppressive treatment, the TCRBV skewness was dramatically reduced after the treatment. CONCLUSION: We have established a powerful method for analyzing RAIT clonality, which is especially useful for monitoring RAIT dynamics after immunosuppression therapy.
Bidirectional transport of cadmium across apical membrane of renal epithelial cell lines via H+-antiporter and inorganic anion exchanger.
The purpose of this study was to investigate whether cadmium (Cd) efflux across the apical membrane of renal epithelial cells is mediated via a H+-antiport system, and to confirm the re-uptake of Cd from the apical membrane via an inorganic anion exchanger. LLC-PK1 and OK cell monolayers cultured on permeable membranes were incubated with 1 microM CdCl2 added to the basolateral medium, and the transport of Cd from the basolateral to apical medium and the accumulation of Cd in the monolayers were measured. Cd transport was increased by lowering the pH of the apical medium and was accompanied by a decrease in Cd accumulation. Coincubation with N'-methylnicotinamide or cisplatin, which act as substrates of the H+-antiport systems, decreased Cd transport and increased Cd accumulation in a concentration-dependent manner. To confirm the re-uptake of Cd, LLC-PK1 cell monolayers were pretreated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a specific inhibitor of an inorganic anion exchanger, before incubation with CdCl2. Pretreatment with DIDS significantly increased Cd transport and decreased Cd accumulation at an apical medium pH of 7.4, but not at pH 5.5. These results suggest bidirectional transport of Cd across the apical membrane of renal epithelial cells via a H+-antiport system (efflux) and an inorganic anion exchanger (influx), depending on the pH of the apical side.
Loss of Hsp70-Hsp40 chaperone activity causes abnormal nuclear distribution and aberrant microtubule formation in M-phase of Saccharomyces cerevisiae.
The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes. To elucidate the function of the yeast cytosolic hsp70 Ssa1p in vivo, we characterized a Saccharomyces cerevisiae ssa1 temperature-sensitive mutant (ssa1-134). After shifting to the restrictive temperature (37 degreesC), ssa1-134 mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2 N DNA content. We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed. Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJ homologue in yeast) was much weaker compared with wild-type Ssa1p. These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase. In support of this idea, a ydj1 null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than ssa1-134. Furthermore, both ssa1-134 and ydj1 null mutant cells exhibited greater sensitivity to anti-microtubule drugs. Finally, the observation that SSA1 and YDJ1 interact genetically with a gamma-tubulin, TUB4, supports the idea that they play a role in the regulation of microtubule formation.
pH-dependent transport of cadmium in rat renal brush border membrane vesicles: cadmium efflux via H+-antiport.
The effects of pH on the Cd efflux across the renal brush border membrane were investigated using isolated rat renal brush border membrane vesicles (BBMV). BBMV equilibrated at pH 5.5 or 7.5 (pHin) were mixed with an experimental buffer at pH 5.5 or 7.5 (pHout) containing CdCl2. After the incubation with CdCl2, BBMV were washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. The initial accumulation of Cd in BBMV incubated for 1, 3 or 5 min at pHin = 5.5 and pHout = 7.5 (outwardly directed H+-gradient) was significantly higher than that at pHin = pHout = 7.5, but the equilibrated Cd accumulation incubated for over 15 min was almost the same. FCCP, a protonophore, diminished the increasing effect of the H+-gradient on the initial Cd accumulation, whereas valinomycine, a K+ ionophore, did not. Kinetic analysis of the Cd accumulation incubated with different concentrations of CdCl2 suggests that Cd is taken up into BBMV via the unsaturable component and saturable component and the outwardly directed H+-gradient increases the Vmax value of the saturable component. The BBMV were incubated with CdCl2 at different temperatures under the outwardly directed H+-gradient, and the increase in Cd accumulation due to the gradient was analyzed by an Arrhenius plot. The plot of increased Cd accumulation was nonlinear, suggesting the involvement of a carrier-mediated process. These findings suggest that the outwardly directed H+-gradient increases the Cd efflux across the renal brush border membrane via H+-antiport system.
The visuo-motor pathway in the local circuit of the rat superior colliculus.
Intrinsic circuit of the superior colliculus (SC), in particular the pathway from the optic tract (OT) to neurons in the intermediate layer (SGI), was investigated by whole-cell patch-clamp recording in slice preparations obtained from 17- to 24-d-old rats. Stimulation of the OT induced monosynaptic EPSPs in neurons in the superficial gray layer (SGS) and the optic layer (SO), and disynaptic or polysynaptic EPSPs in a majority of SGI neurons. Stimulation of the SGS induced monosynaptic or oligosynaptic EPSPs in the SGI neurons. Both the monosynaptic EPSPs induced in the SGS/SO neurons by stimulation of the OT and those induced in the SGI neurons by stimulation of the SGS were mediated by AMPA- and NMDA-type glutamate receptors. Thus, we have clarified the existence of the glutamatergic excitatory pathway from the OT to the SGI neurons via SGS and SO neurons. The EPSPs in the SGI neurons induced by stimulation of the OT or SGS were remarkably enhanced by bicuculline, suggesting that the signal transmission in this pathway is under strong suppression by the GABAergic system.
Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I.
To evaluate the physiological significance of cyclic electron flow around photosystem (PS) I, we used a reverse genetic approach to focus on 11 chloroplast genes that encode homologs of mitochondrial complex I subunits (ndhA-K). Since their discovery, the exact function of the respiratory components in plant chloroplasts has been a matter of discussion. We disrupted one of these genes (ndhB) in tobacco by chloroplast transformation. Analysis of the transient increase in chlorophyll fluorescence after actinic light illumination and the redox kinetics of P700 (reaction center chlorophylls of PS I) suggest that the cyclic electron flow around PS I is impaired in the ndhB-deficient transformants. Transformants grew normally in a greenhouse, suggesting that the cyclic electron flow around PS I mediated by ndh gene products is dispensable in tobacco under mild environmental conditions.
Intramammary Extension of Breast Cancer: Diagnosis with MR Mammography.
Purpose: Intramammary extension of breast cancer was investigated using contrast-enhanced magnetic resonance (MR) imaging by comparing MR findings with the histopathology of specimens used for pretreatment planning. Materials and Methods: Thirty-nine breast cancers were examined. The dynamic protocol was performed with a 2- or 3-dimensional fast spin echo sequence (1.5 T), once before and 2-5 times immediately after Gd-DTPA administration with an original fat suppression technique. The findings were correlated with the histopathology. Results: All main tumors showed early enhancement. All with direct invasion histopathologically showed spicular enhancement. The four patients who had separate nodular enhancement showed satellite lesions histopathologically. Three-typed MR findings were found to be indicative of an intraductal component. Conclusion: Various forms of breast cancer were reliably demonstrated using MR imaging. MR imaging could be useful for the pretreatment planning for patients with breast cancer.
Characterization of temperature rise of the brain and the rectum following intracerebroventricular administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate in rats.
Intracerebroventricular administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or kainate caused a rise of the temperature of the brain and the rectum in urethane-anesthetized rats. An AMPA-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), significantly suppressed the AMPA- and kainate-induced rises of brain and rectal temperatures. An N-methyl-d-aspartate receptor antagonist, MK-801, also suppressed the rises of the brain and rectal temperatures induced by AMPA or kainate, but the profiles of the suppressive effects of MK-801 were different between rats treated with AMPA and kainate. An antipyretic agent, indomethacin, completely suppressed the AMPA-induced rises of brain and rectal temperatures. Although indomethacin completely suppressed the kainate-induced rise of the rectal temperature as well, the brain temperature was still raised. These findings suggest that distinct mechanisms may be involved in the temperature rise of the brain and the rectum mediated through AMPA and kainate receptor stimulation.
Corrigendum to: 'Evolutionary significance of intra-genome duplications on human chromosomes'
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Multiple digit formation in Xenopus limb bud recombinants.
We prepared recombinant limb buds of Xenopus tadpoles by grafting a mesenchyme mass of the hindlimb bud. The Xenopus recombinant limb buds with dissociated and reaggregated mesenchyme developed more than 30 digits with cartilage segmentation, while those with undissociated mesenchyme developed a limb with normal cartilage pattern. Before the formation of multiple digits, a patchy expression pattern of fgf-8, an AER marker, was observed in the distal region of recombinant limb buds. shh, a ZPA (zone of polarizing activity) marker, was expressed broadly in the distal region of recombinants. Recombinant limb buds with the reaggregated mesenchyme of anterior halves formed anterior digits with claws, and those with the mesenchyme of posterior halves formed posterior digits without claws. The temporal and spatial changes in the potency of multiple digit formation are discussed with reference to the regenerative capacity of Xenopus limb buds.
Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas.
Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue.
Reinvestigation of the functions of the hydrophobic segment of Jem1p, a yeast endoplasmic reticulum membrane protein mediating nuclear fusion.
Jem1p is a DnaJ-like protein of the yeast ER membrane, which is required for nuclear membrane fusion during mating. We have determined the position of translation initiation codon for the JEM1 gene. Translation of Jem1p starts from the second ATG codon of the previously assumed JEM1 open reading frame, leading to the synthesis of a precursor protein of 645 amino acids long. The translated Jem1p precursor contains an N-terminal hydrophobic sequence that functions as a signal sequence and is removed upon import into the ER lumen. Jem1p is peripherally associated with the ER membrane probably through protein-protein interactions.