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Biomedical subjects

R Sato

Publications and source records attributed to R Sato.

At least 127 records · Page 7Linked to original sources
PubMed1998

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides.

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the A rabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G-->A change at 291 in the first putative exon, resulting in a Val-->Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G-->A change at the equivalent position (5751) within exon 10.

Amino Acid Sequence↗
PubMed1998

Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase.

The expression of the human SREBP-2 gene is transcriptionally regulated in a cooperative manner by sterol regulatory element-binding proteins (SREBPs) and the general transcription factor NF-Y [Sato, R., Inoue, J., Kawabe, Y., Kodama, T., Takano, T., and Maeda, M. (1996) J. Biol. Chem. 271, 26461-26464]. To understand the sterol-dependent transcriptional regulation by these factors in detail, we have examined the regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and squalene synthase genes, whose promoters have multiple potential sterol regulatory elements (SRE, SREBP binding site) and NF-Y binding sites. The promoter of the human HMG CoA synthase gene was cloned, sequenced, and functionally characterized by means of reporter gene assays. The results indicate that an inverted CCAAT box, two SRE motifs and two Sp1 sites localized in a 90-bp region coordinately regulate the transcription. In the case of the human squalene synthase promoter, two SRE motifs and an inverted CCAAT box between the motifs localized in a 51-bp region are responsible for the sterol-regulated transcription of the gene. Gel mobility shift assay reveals that these two inverted CCAAT boxes are recognized by NF-Y. The involvement of multiple responsive elements in the transcription of HMG CoA synthase and squalene synthase seems to induce a higher level of sterol-dependent regulation (3.5 to 5. 8-fold) compared with that of the SREBP-2 promoter, which contains a single pair of SRE motif and CCAAT box (1.8 to 2.6-fold). Reporter gene assays using constructs containing various nucleotide spacing lengths between the SRE motif and the CCAAT box demonstrate that the 16 to 20-bp spacing range is required for maximal transcriptional regulation. These results agree with the findings that the distances between the two motifs in the known sterol responsive elements in several genes, including the human HMG CoA synthase and squalene synthase genes, are in this range.

Base Sequence↗
PubMed1998

Characterization of two Chinese hamster ovary cell lines expressing the COOH-terminal domains of sterol regulatory element-binding protein (SREBP)-1.

Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH2-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH2-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t1/2 = 4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t1/2 = 1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway.

Animals↗
PubMed1998

Structures of P-type transporting ATPases and chromosomal locations of their genes.

P-type ATPases (E1E2-ATPases) are primary active transporters which form phospho-intermediates during their catalytic cycle. They are classified into P1 to P4 based on the primary structure and potential transmembrane segments. Although the classic P-type ATPases are cation transporters, two new members have recently been found; one is a flippase catalyzing the flip-flop movement of aminophospholipids, but the substrate and function of the other one remain unknown. It would be interesting to determine whether the cations and aminophospholipids are transported by similar or different mechanisms. P-type ATPases are believed to have been derived from a common ancestor, and their genes are found to be distributed in various chromosomal loci. However, gene duplication events can be traced from the tandem arrangement of genes and their linkage map. Na+/K+- and H+/K+-ATPases have not only closely related a subunits but also similar beta subunits. Renal Na+/K+-ATPase has an additional subunit gamma. Similar small polypeptides (phospholemman, Mat-8 and CHIF), which induce Cl- and K+ currents, have been found. The idea of their functional and structural coupling with P-type ATPases, especially with H+/K+-ATPase, is intriguing. Each P-type ATPase must have specific domains or sequences for its intracellular trafficking (sorting, retention and recycling). Identification of such regions and studies on the molecules playing role in their recognition may facilitate the unveiling of various cellular processes regulated by P-type ATPases.

Adenosine Triphosphatases↗
PubMed1998

A specific and rapid method for determination of adenosine 3'-monophosphate (3'-AMP) content and 3'-AMP forming enzyme activity in rat liver mitochondria, using reversed-phase HPLC with fluorescence detection.

To study the physiological significance of adenosine 3'-monophosphate (3'-AMP), an intracellular P-site inhibitor of adenylate cyclase, in rat liver mitochondria, a specific, rapid and reliable assay method for determination of 3'-AMP and the activity of its forming enzyme is required. 3'-AMP in rat liver was determined to be ca. 23+/-7 nmol/g wet weight, but no 2-deoxy-3'-AMP, another P-site inhibitor of adenylate cyclase, was detected, even when using a reversed-phase HPLC column with a fluorescent-reaction, as established in this study. By using the optimized assay method developed here, 3'-AMP forming enzyme activity in rat crude mitochondrial extract was found to be enhanced by EDTA and inhibited by p-chloromercurybenzoate. The optimum pH was ca. 5.8 and no divalent cation was required for activity. From these results, 3'-AMP forming enzyme(s) in rat liver mitochondria could be classified as acid exoribonuclease, which mainly existed in an active form. The results obtained in this study will help to gain more insight into the physiological roles of 3'-AMP in living systems.

Adenosine Monophosphate↗
PubMed1998

Purine metabolism of uric acid urolithiasis induced in newborn piglets.

To clarify the relationship between uric acid urolithiasis and purine catabolites in newborn piglets, the incidence of uric acid urolithiasis and the plasma concentrations of xanthine, hypoxanthine, uric acid and allantoin were examined in 32 piglets. The newborn piglets were divided into two groups: normal (over 1.2 kg, n = 18, group N) and low body weight (below 0.9 kg, n = 14, group L). The animals in both groups were given water (non-nutrition, n = 11, treatment W), artificial milk (normal nutrition, n = 12, treatment M), or a combination of water and allopurinol (prophylactic treatment for the urolithiasis, n = 9, treatment A), during the first 60-hr of birth. At necropsy, the incidence of urolithiasis was higher in the piglets that received treatment W than those in the treatment M or A in both the N and L groups. In group L, the plasma xanthine, hypoxanthine and uric acid concentrations were markedly increased in the piglets that underwent treatment W compared with the treatment M. In both the N and L groups, the plasma allantoin concentration was higher in the treatment W piglets as compared with the treatment M piglets. These results suggested that the occurrence of uric acid urolithiasis in the newborn piglets is attributable to increased purine catabolites due to a starvational condition after birth.

Aging↗
PubMed1998

Hematuria in patients with renal hypouricemia.

The characteristics of urate metabolism in renal hypouricemic patients with hematuria were studied to clarify the risk factors for hematuria in patients with renal hypouricemia. In 16 Japanese patients with isolated renal hypouricemia, urate metabolism was measured using the urate clearance study and the subtype of renal hypouricemia [defective presecretory reabsorption (Pre), defective postsecretory reabsorption (Post), enhanced tubular secretion (Secretion) and defective presecretory and postsecretory reabsorption (Pre&Post)] were determined by the pharmacological tests. Hematuria was seen in 7 out of the 16 patients (44%), all of whom were females (58%). Serum urate and urinary urate concentrations were significantly higher in the group with hematuria (Sur = 1.76 +/- 0.31 mg/dl and Uur/Ucr = 0.75 +/- 0.12: p<0.05) than in the group without hematuria (Sur = 1.44 +/- 0.46 mg/dl and Uur/Ucr = 0.56 +/- 0.04), although there was no difference in the urate excretion rate between the two groups. Hematuria was more likely to be accompanied by Post (75%) and Secretion (75%), which showed significantly higher urinary urate concentration (Uur/Ucr = 0.75 +/- 0.1 and 0.69 +/- 0.13, respectively) than by Pre (25%) and Pre&Post (0%), which showed lower urinary urate concentration (0.61 +/- 0.06 and 0.62 +/- 0.05, respectively). The risk factors for hematuria in patients with renal hypouricemia are the elevation of urinary urate concentration and the subtypes of Post and Secretion.

Adult↗
PubMed1998

Excess urate excretion correlates with severely acidic urine in patients with renal hypouricemia.

We evaluated the renal handling of urate in 12 Japanese renal hypouricemia patients, and studied the relationship between the renal handling of urate and the urinary pH. The patients were classified into the 4 subtypes of renal hypouricemia: (defective presecretory reabsorption (Pre), defective postsecretory reabsorption (Post), enhanced tubular secretion (Secretion), and defective presecretory and postsecretory reabsorption (Pre&Post) as based on a pharmacological test. Seven patients showed acid urine with a urinary pH of less than 5.9, although this was not accompanied by any abnormality of blood pH, partial pressure of carbon dioxide (PaCO2), or blood HCO3-. The urinary pH in the morning significantly correlated with the ratio of urate clearance to creatinine clearance in the morning, whereas the urinary urate concentration in the morning did not correlate with the urinary pH in the morning. In the Pre&Post patients, the urate excretion was higher and the urinary pH was more acidic compared to the other subtypes. The administration of K+-, Na+-citrate significantly alkalized the urinary pH in the patients with renal hypouricemia. These results suggest that the acidic urine was significantly related to the Pre&Post subtype of renal hypouricemia with the higher urate excretion, and that this subtype might be a risk factor for complications in renal hypouricemia. The alkalization of urine might be a useful treatment for the complication of renal hypouricemia.

Adult↗
PubMed1998

Liposome encapsulated hemoglobin as a resuscitation fluid for hemorrhagic shock.

The effectiveness of the blood substitute neo red cells (NRC) on hemorrhagic shock was evaluated in dogs. Shock was induced by bloodletting, and NRC was infused immediately until the dogs recovered from shock; the process was repeated 3 times (mild shock), or NRC was infused after shock was maintained for 30 min, and the process was repeated 3 times (severe shock). In mild shock, NRC with low viscosity reduced the peripheral vascular resistance and increased the cardiac index. However, in severe shock, only a slight decrease in the peripheral vascular resistance and no increase in the cardiac index were observed. In severe shock, the total oxygen consumption increased, but red blood cells or plasma did not change the oxygen transport and did not meet the increased oxygen demand. However, the NRC increased the oxygen transport and supplied the increased oxygen demand. In mild shock on the other hand, no increase was observed in the total oxygen consumption or the oxygen transport.

Animals↗
PubMed1998

[Minimally invasive direct coronary artery bypass for ischemic heart disease associated with preoperative severe complications].

Our experiences of minimally invasive direct coronary artery bypass (MIDCAB) were reported with review of literatures. Patient #1 was a 69 years-old man with left main lesion associated with cerebral infarction and impaired renal function. Preoperative CT examination showed calcified ascending aorta. There was no significant lesions in the large right coronary artery and the circumflex was small. The patient underwent MIDCAB via minithoracotomy on the fourth intercostal space with left internal thoracic artery (ITA) to left anterior descending artery (LAD). The patient was weaned from ventilator 3 hours after the operation and his postoperative course was uneventful. Patient #2 was a 80 years-old man with acute myocardial infarction requiring intraaortic balloon pumping (IABP) due to significant stenosis in the right coronary artery and left main lesion associated with chronic hemodialysis due to renal failure. The right coronary artery was dilated by balloon angioplasty at the time of emergency coronary angiogram. However, the emergent operation was required because of the left main lesion. The circumflex was relatively small and because of severe complication, MIDCAB was selected to improve his condition. He underwent MIDCAB via minithoracotomy. The heart was enlarged because of congestive heart failure and left inferior epigastric artery was used to extend the length of the left ITA. The composite graft of ITA and IEA was anastomosed to the LAD under beating heart. The IABP was removed 8 hours after the operation and his postoperative course was uneventful. The MIDCAB for coronary artery bypass was first reported by Benetti et al in 1995 and the procedure seemed to be very effective for preventing postoperative complications in selected patients as seen in ours.

Aged↗
PubMed1997

GATA-6 DNA binding protein expressed in human gastric adenocarcinoma MKN45 cells.

A cDNA for the GATA-6 (GATA-GT1) DNA binding protein was cloned from a library of the human gastric adenocarcinoma cell line MKN45. The deduced amino acid sequence (449 residues) indicates that the primary structure of human GATA-6 is highly homologous to that of the rat protein. The potential phosphorylation site for protein kinases (A and C), and histidine and alanine clusters are conserved. Whereas the rat H+/K+-ATPase alpha and beta subunit genes have two and three GATA protein binding sites in their promoter regions, respectively, the human alpha subunit gene has only one binding site [Maeda, M., Kubo, K., Nishi, T. and Futai, M. (1996) J. Exp. Biol. 199, 513-520]. We cloned the 5'-upstream region of the human H+/K+-ATPase beta subunit gene by genome walking and found that it also has a single GATA protein binding site near the TATA box. The GATA sites of the human alpha and beta subunit genes are recognized by the zinc finger domain of human GATA-6. The conservation of the GATA protein binding sites suggests that they are important for the gene regulation of the human and rat H+/K+-ATPase.

Adenocarcinoma↗
PubMed1997

Lipopolysaccharide-binding proteins and their involvement in the bacterial clearance from the hemolymph of the silkworm Bombyx mori.

Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/PAGE. They bound to all E. coli R mutants (Ra, Rb1, Rc, Rd1 and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)-coated and lipid-A-coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS-binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.

Acute-Phase Proteins↗
PubMed1997

Aminopeptidase N from Bombyx mori as a candidate for the receptor of Bacillus thuringiensis Cry1Aa toxin.

Cry1Aa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that Cry1Aa toxin bound to a 120-kDa protein. A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor. The 120-kDa and 110-kDa Cry1Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography. Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cry1Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative Cry1Ac toxin receptor, reported in Manduca sexta and Heliothis virescens. On this basis, the 120-kDa Cry1Aa toxin-binding protein from B. mori was identified as a member of the aminopeptidase family.

Amino Acid Sequence↗
PubMed1997

Gastric GATA-6 DNA-binding protein: proteolysis induced by cAMP.

The rat gastric GATA DNA-binding protein, GATA-6 (GATA-GT1), was stably expressed in CHO-K1 cells. The GATA-6 protein was localized in the nucleus but not in the cytoplasm. Interestingly, when cells were treated with dibutyryl cAMP, the GATA-6 protein was specifically degraded. Such a phenomenon was not observed in the presence of 5'-AMP or dibutyryl cGMP. The cellular level of the GATA-6 protein was restored upon removal of dibutyryl cAMP. Degradation was also induced by cholera toxin, which increased the cellular cAMP concentration, and was inhibited by a protein kinase A inhibitor. However, activators of protein kinase C did not have any effect. The degradation was inhibited by proteasome inhibitors (PSI (benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinal) and MG115 (benzyloxycarbonyl-Leu-Leu-norvalinal)) but not by those of lysosomes and serine proteases. These results suggest that a kinase-mediated protein phosphorylation is the cellular signal for degradation of the GATA-6 protein. This finding constitutes a novel aspect of regulation by GATA DNA-binding proteins, which are essential for developmental processes and tissue-specific transcription.

Amino Acid Sequence↗