Hepatic protein synthesis rate of biopsied liver tissue in the assessment of preservation solutions.
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Animals↗
Biomedical subjects
T Asano
Publications and source records attributed to T Asano.
Time-course analysis of the mechanical properties of the canine basilar artery subjected to experimental subarachnoid hemorrhage.
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Animals↗
[Urodynamic studies before and after gradual urethral dilatation with metal sounds for female urethral stricture].
We evaluated the urodynamics in women with urethral stricture after urethral dilatation. A total of 16 patients underwent treatment with gradual urethral dilatation at increasing intervals (2 weeks, 1 month, 3 months and 6 months). Their urethra was dilated up to 30 F. with metal sounds. Urethral calibration with bougie à boule, uroflowmetry and urethral pressure profile were performed before urethral dilatation and 1 week after the last dilatation. The urethral caliber was increased from 18.1 +/- 0.6 F. to 23.5 +/- 1.0 F. (P less than 0.005), average flow rate from 9.4 +/- 0.8 ml/sec to 11.2 +/- 1.1 ml/sec (P less than 0.025) and maximum urethral pressure with a full bladder decreased from 105.4 +/- 9.4 cmH2O to 87.5 +/- 10.5 cmH2O (P less than 0.05). With gradual urethral dilatation with metal sounds, (1) the urethra was significantly dilated, (2) maximum urethral pressure was significantly decreased and (3) average flow rate was significantly increased.
Adult↗
Differential localization of G-proteins Gi and Go in the accessory olfactory bulb of the rat.
To clarify the functional differences among G-proteins, we investigated the localization of Gi and Go in the olfactory bulb of rats by both immunohistochemical and immunochemical techniques, using purified antibodies specific to the alpha-subunits of Gi1 (Gi1 alpha), Gi2 (Gi2 alpha), and Go (Go alpha), respectively. We found that Gi2 alpha is localized exclusively in the accessory olfactory bulb, but it is present at only low levels in the main olfactory bulb. The unique pattern of immunoreactivity specific for Gi2 alpha and Go alpha within the glomeruli of the accessory olfactory bulb and the results of immunoassays indicate that the accessory olfactory bulb is divided into two parts: the anterior region is rich in Gi2, while the posterior region is rich in Go. These findings suggest that the accessory olfactory bulb has two different functions. In addition, we found that the concentration of Gi2 alpha in the accessory olfactory bulb increases during puberty and reaches the adult level at 12 weeks after birth, while that in the main olfactory bulb remains constant. By contrast, the concentrations of Go alpha in the accessory olfactory bulb and the main olfactory bulb increase with similar kinetics. These findings suggest that Gi2 is a key protein in signal transduction in the accessory olfactory bulb, and increases in its level seem to be related to sexual maturation.
Animals↗
[Evaluation of gastroenterological disease by using 18F-FDG PET--differential diagnosis of malignancy from benignity].
In this study, we used 18F-FDG PET to differentiate gastroenterological malignancy from benignity. We investigated 122 patients with gastroenterological disease before treatment (with lesions exceeding 2.0 cm in diameter only). 60 min after injection of FDG, although 16 cases out of 17 benign disease did not reveal accumulation higher than normal tissue, 105 cases of malignant tumor except some of hepatocellular carcinoma revealed high accumulation of FDG. FDG uptake was expressed as the Ci/Cp ratio, calculated from radioactivity of the tumor (Ci) and the plasma (Cp). 89 cases out of 90 which show more than 2.0 of Ci/Cp ratio were malignant tumor. On the other hand, 32 cases which show less than 2.0 contained 16 cases of benign disease and 15 of hepatocellular carcinoma. FDG PET is a useful tool for differential diagnosis of malignant tumor from benign disease except some of hepatocellular carcinoma.
Deoxyglucose↗
The role of N-glycosylation of GLUT1 for glucose transport activity.
To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.
Affinity Labels↗
HA1077, a novel calcium antagonistic antivasospasm drug, increases both cerebral blood flow and glucose metabolism in conscious rats.
The effects of a novel calcium antagonistic antivasospasm drug, HA1077, on local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCGU) were studied in 33 anatomically discrete regions of the brain in conscious rats, using the quantitative autoradiographic [14C]iodoantipyrine and [14C]2-deoxyglucose techniques. HA1077 was infused i.v. over a 30-min period (1 or 3 mg/kg). HA1077 significantly increased LCBF in 9 of 33 sites in rats given 1 mg/kg, and in 14 of 33 sites in rats given 3 mg/kg compared to the control group given vehicle. Significant increases in LCGU were also noted in 16 of 33 sites in rats given 3 mg/kg. HA1077 increased both cerebral blood flow and glucose metabolism in conscious rats.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli.
Human T-cell leukemia virus type I (HTLV-I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli. The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease. Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease. The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24. The protease activity was inhibited by pepstatin A. These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV-I virion.
Amino Acid Sequence↗
Expression of the GLUT1 glucose transporter increases thymidine uptake in Chinese hamster ovary cells at low glucose concentrations.
An increase in expression of the GLUT1 glucose transporter gene has been observed to be associated with an increase in glucose transport activity upon oncogenic transformation of the cells. Increased expression of this glucose transporter isoform has been also observed in fetal tissues. To investigate the consequences of this phenomenon on cellular metabolism and cell growth, an expression vector containing the GLUT1 glucose transporter complementary DNA was transfected into Chinese hamster ovary cells. Overexpression of this glucose transporter isoform resulted in an increase in not only glucose uptake and utilization but also thymidine uptake when cells were exposed to glucose-deficient conditions. This increase in glucose metabolism and DNA synthesis may play an important role on the growth and/or survival of cancer cells and fetal tissues.
3-O-Methylglucose↗
Sulfated glycolipids are the platelet autoantigens for human platelet-binding monoclonal anti-DNA autoantibodies.
The human monoclonal autoantibody HF2-1/17, produced by a human-human hybridoma derived from lymphocytes of a lupus patient with thrombocytopenia, reacts with single stranded DNA and platelets. To determine the chemical nature of the autoantigen against which this antibody is directed on platelets, this platelet antigen was purified by the lipid extraction of sonicated platelets, DEAE-Sephadex chromatography, and high performance liquid chromatography. The purified glycolipids, a trace component in platelets, demonstrated high reactivity with the HF2-1/17 antibody using a competition enzyme-linked immunosorbent assay system or immunostaining of thin layer chromatograms. The purified glycolipids co-migrated with bovine sulfatides by thin layer chromatography. The purified glycolipids contain sulfate and galactose but not sialic acid or phosphate. Fast atom bombardment-mass spectrometry revealed these sulfatides to be sulfated monohexyl ceramides. The dominant species has a molecular weight of 794 while a minor form has a molecular weight of 812 due to an extra hydroxyl group and loss of a double bond. These results indicate that the platelet autoantigen against which the human monoclonal anti-DNA antibody is directed represents a family of novel monogalactosyl sulfatides.
Antibodies, Monoclonal↗
High-performance liquid chromatographic analysis of fatty acid compositions of platelet phospholipids as their 2-nitrophenylhydrazides.
A novel high-performance liquid chromatographic method for biologically important fatty acids incorporated into platelet phospholipids in esterified form has been developed. 2-Nitrophenylhydrazine hydrochloride was used as a pre-column labelling agent to convert the saponified platelet phospholipids directly into corresponding fatty acid hydrazides, without a complicated isolation procedure. Isocratic separation was achieved within only 36 min for twenty-five saturated and mono- and polyunsaturated fatty acids (C8:0-C22:6), including cis and trans isomers, on a YMC-FA column. The analytical results showed good quantitative accuracy. Fatty acid compositions were determined in platelet phospholipids obtained from normal subjects and patients with diabetes mellitus. The method is simple, rapid and adequate for labelling esterified fatty acids in biological materials, and has several advantages with regard to resolution, analysis time and sensitivity over previously published methods.
Blood Platelets↗
Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells.
We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.
Adenosine Diphosphate↗
The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415.
GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of aspartic acid for asparagine 415, which is conserved among all facilitative glucose transporter isoforms. Although a significant amount of the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells by transfection with expression vector, almost no increase in glucose transport activity was observed. Analysis of glucose uptake with Lineweaver-Burk plot depicts that the mutation induced a marked decrease (more than 5-fold) in turnover number and a slight increase (1.5-fold) in Km compared with the wild-type GLUT1. Results obtained with cytochalasin B and ethylidene glucose suggested that the inner but not outer glucose binding site was modulated. These results suggest that asparagine 415 is located close to the inner glucose binding site and the putative inner gate of GLUT1 glucose transporter and that an ionic charge in this domain might play an important role in the rate of conformational change between an inward-facing form and an outward-facing form of glucose transporter.
Animals↗
Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter.
GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.
Affinity Labels↗
Regulation of transmembrane signal transduction of insulin-like growth factor II by competence type growth factors or viral ras p21.
In BALB/c 3T3 cells pretreated with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (primed-competent cells), insulin-like growth factors I and II (IGF-I and IGF-II) bind to their own receptors (IGF-IR and IGF-IIR) and stimulate calcium influx and DNA synthesis by a mechanism involving a 40-kDa pertussis toxin substrate. In contrast, these IGFs do not act on unprimed quiescent cells. In this study, the 40-kDa pertussis toxin substrate was identified as Gi-2 alpha using anti-G protein antibodies. We analyzed the quality of signal transduction from IGF-II to Gi-2 alpha. There was no difference in the amount of Gi-2 alpha between quiescent and primed-competent cells, and both of these cells had similar Kd values and numbers of IGF-II-binding sites. Whereas IGF-II did not alter pertussis toxin-catalyzed ADP-ribosylation of Gi-2 alpha in quiescent cells, IGF-II reduced the pertussis toxin substrate activity by 35-50% via the IGF-IIR in primed-competent cells. The action of IGF-II lasted for up to 3 h when IGF-II was present in the medium, and it disappeared when IGF-II was removed. These results suggest that the signaling pathway triggered by IGF-II is uncoupled between the IGF-IIR and Gi-2 alpha in quiescent cells and that PDGF and EGF restore the IGF-IIR-Gi-2 coupling. This study also indicates that low concentrations of IGF-I reduce the pertussis toxin substrate activity of Gi-2 alpha in primed-competent cells in a time course slower than that of IGF-II, but not at all in quiescent cells. However, both of these cells had similar Kd values and numbers of IGF-I binding sites. Therefore, the IGF-I signaling pathway may also be uncoupled between the IGF-IR and Gi-2 alpha in quiescent cells and restored by PDGF and EGF. In BALB/c 3T3 cells transfected with temperature-sensitive Kirsten sarcoma virus bearing the v-Ki-ras gene (ts cells), a 40-kDa pertussis toxin substrate was also identified as Gi-2 alpha. In nonpermissive ts cells, IGF-II was without effect on the pertussis toxin substrate activity of Gi-2 alpha or on calcium influx.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Defect in negative selection in lpr donor-derived T cells differentiating in non-lpr host thymus.
Transplantation of bone marrow cells of lpr/lpr mice into irradiated normal mice fails to develop massive lymphadenopathy or autoimmunity but causes severe graft-vs.-host-like syndrome. To elucidate an abnormality of lpr/lpr bone marrow-derived T cells, we transplanted bone marrow cells of Mlsb lpr/lpr mice into H-2-compatible Mlsa non-lpr mice. Although lpr/lpr T cell precursors repopulated the host thymus as well as +/+ cells, a proportion of CD4+CD8+ cells decreased, and that of both CD4- and CD8- single-positive cells increased compared with those of +/+ recipients. Notably, in MRL/lpr----AKR and C3H/lpr----AKR chimeras, CD4 single-positive thymocytes contained an increased number of V beta 6+ cells in spite of potentially deleting alleles of Mlsa, whereas V beta 6+ mature T cells were deleted in the MRL/+ ----AKR and C3H/+ ----AKR chimeras. There was no difference between MRL/+ ----AKR and MRL/lpr----AKR chimeras in their proportion of V beta 3+ cells because both host and donor strain lack the deleting alleles. Interleukin 2 receptor expression of mature T cells, in the thymus and lymph node, was obviously higher in the MRL/lpr----AKR chimeras, in particular in the "forbidden" V beta 6+ subset. Moreover, lpr donor-derived peripheral T cells showed vigorous anti-CD3 response. These results indicate that lpr-derived T cells escape not only tolerance-related clonal deletion but also some induction of unresponsiveness in the non-lpr thymus.
Animals↗
Altered expression of glucose transporter isoforms with aging in rats--selective decrease in GluT4 in the fat tissue and skeletal muscle.
To elucidate the cellular mechanisms of glucose intolerance associated with aging, both the protein and mRNA levels of glucose transporter isoforms were studied in the various tissues of young (7-week-old) and aged (20-month-old) rats. GluT4 (adipose/muscle-type glucose transporter) protein, which is specifically expressed in insulin-responsive tissues, was selectively decreased per milligram of cellular membrane protein in both the epididymal fat tissues and the gastrocnemius muscle of the aged rats compared with the young rats. When the changes in total cellular membranes per gram of tissue are taken into account, a further decrease in GluT4 protein per gram of tissue was observed in the tissues of the aged rats compared with the young rats. The decreased amount of GluT4 protein in the fat tissues of the aged rats is probably due to the decreased protein synthesis rather than the stability, since GluT4 mRNA/micrograms of cellular total RNA was also decreased. In contrast, GluT4 mRNA in the gastrocnemius muscle was rather increased and a ratio of GluT4 protein/GluT4 mRNA was decreased by 70% in the aged rats, suggesting that the translational efficiency and/or stability of GluT4 protein is decreased in the skeletal muscle of the aged rats compared with the young rats. GluT2 (liver-type glucose transporter) protein and mRNA in the liver were also decreased in the aged rats, while no apparent decrease in GluT1 (HepG2/brain-type glucose transporter) protein/mg of cellular membrane protein was observed in the skeletal muscle and fat tissues of the aged rats compared with the young rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose Tissue↗
The effects of HA1077 on the cerebral circulation after subarachnoid haemorrhage in dogs.
We examined the effects of the recently developed calcium antagonist HA1077 on cerebral haemodynamics during the chronic stage of the two-haemorrhage canine model system of vasospasm. Regional cerebral blood flow (rCBF), regional cerebral blood velocity and regional cerebral blood volume in the canine parietal cortex were measured by Laser-doppler flowmeter. On the 7th day after the initial injection of autogenous blood, subarachnoid haemorrhage (SAH) produced a significant decrease in rCBF (59% of control, p less than 0.05) and blood velocity (48% of control, p less than 0.05) with no remarkable change in blood volume (108% of control). Bolus intravenous administration of HA1077 (0.1-0.3 mg/kg) dose-dependently increased the rCBF and blood velocity, without significantly changing the blood volume on Day 7 after SAH. HA1077 improves haemodynamic function manifested by an increase in rCBF and velocity in this SAH model, and may be suitable for the treatment of vasospasm in patients with SAH.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗