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Biomedical subjects

Y Akamatsu

Publications and source records attributed to Y Akamatsu.

At least 37 records · Page 2Linked to original sources

Cloning and expression of human liver rhodanese cDNA.

cDNA for the human rhodanese (thiosulfate; cyanide sulfurtransferase, EC 2.8.1.1) was cloned from a human fetal liver cDNA library. Sequencing of the cDNA revealed an open reading frame that encodes a 297-residue polypeptide with a calculated mass of 33,427 daltons. When the rhodanese cDNA was transiently expressed in Escherichia coli and Cos7 cells, the rhodanese activity increased 40-fold and 150-fold, respectively. Sequence homology analysis showed that the human rhodanese is 89.6% identical to bovine, 90.2% identical to rat, 91.2% identical to mouse and Chinese hamster, and 71.4% similar to avian counterparts, respectively, and that rhodanese was highly conserved across evolution.

Amino Acid Sequence↗

A simple screening for mutant DNA binding proteins: application to murine transcription factor PEBP2alpha subunit, a founding member of the Runt domain protein family.

Mouse transcription factor PEBP2 (polyomavirus enhancer-binding protein (2) is composed of two distinct subunits alpha and beta. The alpha subunit has an ability to bind the specific DNA sequences, which is enhanced by formation of a heterodimer with the beta subunit. The DNA binding and heterodimerization activities of the alpha subunit are both localized within a 128-amino-acid (aa) region termed as the Runt domain for its homology to the Drosophila segmentation gene runt. To characterize the molecular determinants for these activities, the Runt domain was randomly mutagenized and produced in E. coli as a secreted form. Using E. coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay. Nine randomly picked single-aa substitution mutants showed various functional alterations in DNA binding and heterodimerization either separately or simultaneously. This observation suggests that the structure of Runt domain is highly ordered and is quite sensitive to modulations in its primary structure. The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins.

Amino Acid Sequence↗

Changes in psychophysiological functions during night shift in nurses. Influence of changing from a full-day to a half-day work shift before night duty.

The effects of changing from a full-day to a half-day shift work before a night duty shift on physiological and psychological functions during the night shift were investigated in 12 healthy unmarried nurses working on the same ward of a university hospital. Three shift patterns, i.e., a day shift following a day shift, a night shift following a day shift, and a night shift following a half-day shift, were studied in terms of physical activity level, sympathetic and parasympathetic activity levels, cortisol, prolactin, NK cell activity, and changes in mood states. The change to the half-day shift increased the duration of sleep before night duty by about 86 min and brought wake-up times forward by about 1 h, resulting in increases in rest and time before work. In addition, the change was revealed to reduce the influence of reversed-phase circadian rhythms on autonomic nervous activity during the night shift. The score for sleepiness was significantly lower at 0500 hours following a half-day shift. There were some marginal but not significant differences in the scores reflecting the degree of vigor, tiredness and irritation during the night shift. Although the prolactin concentration was significantly decreased at the start of the night shift after the half-day shift, there was no difference in cortisol concentration or NK cell activity between the usual night shift after a day shift and the night shift after the half-day shift. The half-day shift was not observed to cause any marked change in the fixed biorhythms of these nurses. The cortisol and NK cell activity levels were low during the night shift, suggesting that the night shift itself is a high stress level, which is prejudicial to biodefense.

Adult↗

Liver transplantation for severe hypoxemia caused by patent ductus venosus.

The authors describe the case of a 9-year-old girl who underwent liver transplantation because she suffered from severe hypoxemia caused by patent ductus venosus (PDV). Generally, severe hypoxemia (PaO2 < 50 mm Hg in room air or < 300 mm Hg in pure oxygen) is not an indication for liver transplantation because the hypoxemia may not be improved, and may lead to a fatal outcome. PDV, which is associated with mild liver dysfunction, is not an indication for liver transplantation by itself. But in our patient, most of the mesenteric venous flow directly entered the systemic circulation through the PDV just like the portosystemic shunt, and this caused the pulmonary arteriovenous shunt and hypoxemia. Thus, the authors operated on the patient in an attempt to restore her pulmonary function. Nitric oxide (10 to 20 ppm) was added to the inhaled gas to dilate the functional pulmonary capillaries and to deliver sufficient oxygen after the transplantation. Although the patient suffered various complications after the operation, the final results were excellent.

Angiography, Digital Subtraction↗

Analysis of bilirubin fraction in the bile for early diagnosis of acute rejection in living related liver transplantation.

The diagnosis of acute rejection in liver transplantation usually needs hepatic biopsy, but hepatic biopsy sometimes involves severe complications. We analyzed biliary bilirubin fraction after living related liver transplantation by using high performance liquid chromatography (HPLC) and investigated availability for the early diagnosis of acute rejection retrospectively. Nine children with liver cirrhosis due to biliary atresia were included in this study, who underwent living related liver transplantation at The Second Department of Surgery, Tohoku University School of Medicine. Bile was collected daily from a biliary canulae inserted into the hepatic duct of the graft under aseptic and without exposure to the light. We measured the proportion of bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG) and unconjugated bilirubin (UCB) of bile pigments in the bile by HPLC. In three of four patients with acute rejection, BDG + BMG (= Bc) was above 85% and BDG/Bc ratio was below 0.6 at the time of hepatic biopsy. After rejection therapy, BDG/Bc ratio increased in their bile. The remaining one case with acute rejection as well as bile duct injury due to arterial thrombosis of S2, Bc was below 85%, and BDG/Bc ratio was below 0.6. In four of the other five patients who had several severe complications, i.e., arterial or portal vein thrombosis, bile stasis due to cholangitis and sepsis due to necrotizing myofascitis, Bc was below 85% and BDG/Bc ratio was below 0.6. We concluded that analysis of biliary bilirubin fraction after liver transplantation could be reliable as a noninvasive maker and valuable for the early diagnosis of acute rejection.

Acute Disease↗

Changes in osteosclerosis of the osteoarthritic knee after high tibial osteotomy.

Radiographically demonstrated osteosclerosis was quantitatively evaluated in knees with medial compartmental osteoarthritis by dual xray absorptiometry. Bone density was measured in various areas of 144 knees with medial compartmental osteoarthritis, 23 knees of which were treated with high tibial osteotomy. The bone mineral density of the medial femoral and tibial condyles was greater than that of the lateral femoral and tibial condyles in all knees with medial compartmental osteoarthritis. The ratios of bone mineral density of the medial condyles to that of the lateral condyles were found to increase significantly with the progression of osteoarthritis. There was a significant positive regression line correlation between those ratios and the increase of varus deformity as expressed by the standing femorotibial angle. The former were calculated to be 1.0 when the latter was nearly 170 degrees (10 degrees anatomic valgus angulation). The ratio of bone mineral density of the medial femoral condyles to the lateral femoral condyles decreased sharply in all 23 knees within 1 year after high tibial osteotomy.

Absorptiometry, Photon↗

[Recovery process from myocardial stunning after transient ischemia: assessment with pulsed wave Doppler transmitral flow pattern].

Sustained left ventricular systolic dysfunction after transient myocardial ischemia is well known as "myocardial stunning", but little is known about the recovery in left ventricular diastolic function. Changes in left ventricular systolic and diastolic performance following dobutamine-induced ischemia were investigated in 13 patients with chest pain syndrome and normal coronary arteries (control) and 34 patients with coronary artery disease. Two-dimensional echocardiography and pulsed wave Doppler transmitral flow velocity curves were recorded at baseline, after infusion of a peak dose of dobutamine and at 20 min and 2 hours after dobutamine infusion. In control subjects, left ventricular ejection fraction and the peak early diastolic filling velocity increased at the peak dose of dobutamine. At 20 min after the cessation of dobutamine infusion, these values were restored to the baseline levels. In patients with coronary artery disease, ejection fraction and peak velocity increased at the peak dose of dobutamine but decreased at 20 min after infusion compared with baseline values despite the restoration of heart rate and blood pressure. Although ejection fraction increased at 2 hours compared with 20 min after infusion, peak velocity did not increase. Left ventricular diastolic dysfunction may be sustained longer than systolic dysfunction after transient myocardial ischemia.

Aged↗

Functional dissection of the alpha and beta subunits of transcription factor PEBP2 and the redox susceptibility of its DNA binding activity.

The mouse transcription factor PEBP2 is a heterodimer of two subunits: a DNA binding subunit alpha and its partner subunit beta. The alpha subunit shares a region of high homology, termed the Runt domain, with the products of the Drosophila melanogaster segmentation gene runt and the human acute myeloid leukemia-related gene AML1. To study the molecular basis for the DNA binding and heterodimerization functions of this factor, we constructed series of deletions of the alpha and beta subunits and examined their activities by electrophoretic mobility shift and affinity column assays. The minimal functional region of the alpha subunit for DNA binding and dimerization was shown to coincide with the Runt domain. On the other hand, the region of the beta subunit required for heterodimerization was localized to the N-terminal 135 amino acids. Furthermore, it was found that the DNA binding activity of the Runt domain is regulated by a reduction/oxidization (redox) mechanism and that its reductively activated state, which is extremely labile, is stabilized by the beta subunit. These findings add a new layer to the mechanism and significance of the regulatory interplay between the two subunits of PEBP2.

Animals↗

Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells.

We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO-K1. The resultant transfectant, CDT-1, exhibited about 20-fold higher specific activity of the enzyme in the membrane fraction than CHO-K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross-reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT-1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42-kDa protein was eliminated from solubilized membrane proteins of CDT-1 cells. Both the enzyme activity and the 42-kDa protein of CHO-K1 cells were enriched in the mitochondria-associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.

Animals↗

Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries.

MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.

Amino Acid Sequence↗

Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells.

We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase. The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells. The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues. In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa. In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein. When CHO-K1 cells transfected with the cDNA clone are labelled with [35S]methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead. The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase. A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide. This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone. These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme. Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.

Amino Acid Sequence↗

A new method for recording mandibular position during nocturnal bruxism.

Experimental equipment for recording mandibular excursive position during nocturnal bruxism has been devised. The equipment consists of chromium-cobalt alloy cast frameworks for each upper and lower dental arch and two pairs of tiny magnetic sensors and magnets, capable of electromyographic (EMG) recordings of the bilateral masseter muscles and simultaneously recording mandibular position. The two subjects tested were confirmed to be bruxing during sleeping. Although EMG records for the two subjects showed a common EMG pattern as a bruxer, the sensor records revealed that one subject was a bilateral bruxer and the other bruxed almost unilaterally. The present study demonstrated that this newly devised equipment is practical for studying mandibular position during nocturnal bruxism.

Adult↗

Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes.

SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.

Acyltransferases↗

Essential residues in V(D)J recombination signals.

Recombination signal sequences for V(D)J joining consist of a conserved heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer of a constant length (12 bp or 23 bp). In the present study, we have analyzed various recombination signal mutations for their effects in V(D)J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gene as a reporter. This method allowed us to study recombination in a single copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations in the first three residues severely dropped the joining rates. Among them, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joining rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three consecutive A residues was necessary for efficient recombination. Furthermore, the nucleotides flanking the A-rich core needed to be other than A residues, probably marking the border of the A-stretch. This may be important when the recombinase measures the distance between the heptamer and the nonamer to satisfy the 12/23-bp spacer rule.

Animals↗

Alteration by transforming growth factor-beta 1 of asparagine-linked sugar chains in glucose transporter protein in Swiss 3T3 cells.

GLUT1 protein in Swiss 3T3 cells is a 55-kDa glycoprotein with an N-linked oligosaccharide chain. We previously showed that the 65-kDa GLUT1 protein with modulated glycosylation was induced by transforming growth factor-beta 1 (TGF-beta 1) in Swiss 3T3 cells. To further investigate the altered structures of these sugar chains, the membrane glycoproteins solubilized with Triton X-100 were fractionated by lectin-affinity chromatography. The 55-kDa GLUT1 in control and TGF-beta 1-treated cells showed partial binding to Datura stramonium agglutinin (DSA), whereas the 65-kDa GLUT1 exclusively bound to DSA- and wheat germ agglutinin (WGA)-agarose. The 65-kDa GLUT1 in TGF-beta 1-treated cells was sensitive to endo-beta-galactosidase, which cleaves unsubstituted polylactosamine chains. While the 55-kDa GLUT1 in control 3T3 cells was similarly digested by endo-beta-galactosidase, that in TGF-beta 1-treated cells was resistant to this enzyme. These results suggest that the N-linked oligosaccharides of GLUT1 in Swiss 3T3 cells were altered by TGF-beta 1 to forms with more branched and/or repeated polylactosamines as well as with some substitution in the polylactosamines, resulting in a larger GLUT1 molecule. These GLUT1 proteins were exclusively located at the plasma membrane and served as a glucose transporter. However, the affinity to 2-deoxyglucose was significantly increased by TGF-beta 1, associated with the altered glycosylation of GLUT1 protein.

3T3 Cells↗

CD14 is a pattern recognition receptor.

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Animals↗