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Biomedical subjects

M Irie

Publications and source records attributed to M Irie.

At least 37 records · Page 2Linked to original sources
PubMed2001

Enzymatic properties of sialic acid binding lectin from Rana catesbeiana modified with a water-soluble carbodiimide in the presence of various nucleophiles.

The anti-tumor activity of sialic acid binding lectin from Rana catesbeiana (cSBL) was increased by chemical modification with a water-soluble carbodiimide (EDC) in the presence of nucleophiles such as ethylenediamine and glycine methylester. Investigations on ribonuclease (RNase) activities of the modified cSBLs were conducted to elucidate the fundamental mechanisms underlying enhancement of the anti-tumor activity conferred by these modifications. The following three characteristics were observed with modification. (i) RNase activity of the modified cSBL was enhanced towards double stranded RNA and RNA-oligo dA hybrids. The activity increase was observed even under physiologic ionic strength conditions; (ii) RNase activity of the modified cSBL towards single stranded RNA and poly U decreased, while the activity towards poly C was unaffected; (iii) the base preference of the B2 base recognition site of modified cSBL decreased for guanine. On the contrary, the preference for cytosine and adenine increased. This result may explain why the RNase activity towards poly C was not affected by EDC-modification as mentioned above.

Amphibian Proteins↗
PubMed2001

Amino acid sequence and characterization of a rice bran ribonuclease.

A base-nonspecific and acid ribonuclease (RNase Os) belonging to the RNase T2 family was purified from rice bran to a homogeneous state by SDS-PAGE. The primary structure of RNase Os was determined by protein chemistry and molecular cloning. The RNase Os was a simple protein and consisted of 205 amino acid residues. Its molecular weight was 22578 and its amino acid sequence showed that it was most similar to barley RNase among the known RNase T2 family enzymes having 157 amino acid residues identical with barley RNase. However, its N-terminus was blocked by a gamma-pyroglutamyl residue. The optimal pH of RNase Os was around 5.5. The base preference at the B1 and B2 site of RNase Os was estimated from the rates of hydrolysis of 16 dinucleoside phosphates, to be guanine as the case of RNase LE from tomato. RNase Os was successfully expressed from yeast cells using the E. coli yeast expression vector pYE-RNAP.

Amino Acid Sequence↗
PubMed2001

Effect of modification of the carboxyl groups of the sialic acid binding lectin from bullfrog (Rana catesbeiana) oocyte on anti-tumor activity.

The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBL > glycine methylester-modified cSBL > taurine modified cSBL > or = native cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

Amino Acid Sequence↗
PubMed2001

Recognition and transport characteristics of nonpeptidic compounds by basolateral peptide transporter in Caco-2 cells.

Recent studies have revealed that diverse compounds lacking peptide bonds, such as valacyclovir and delta-aminolevulinic acid (delta-ALA), can be recognized by H+-coupled peptide transporters (PEPT1 and PEPT2). In the present study, recognition and transport characteristics of nonpeptidic compounds by the basolateral peptide transporter, which is distinct from PEPTs, were compared with those by PEPT1 using the human intestinal Caco-2 cells. [14C]Glycylsarcosine uptake via PEPT1 was inhibited by all nonpeptidic compounds tested. Similarly, most nonpeptidic compounds showed an inhibitory effect on [14C]glycylsarcosine uptake by the basolateral peptide transporter, although some kinds of nonpeptidic compounds, such as valine methyl ester, did not. Direct measurements of valacyclovir and delta-ALA transport revealed that both compounds were able to be transported by the basolateral peptide transporter. Because delta-ALA has been used recently in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy, the intestinal transport characteristics of delta-ALA were further examined. Inhibition studies and Eadie-Hofstee plot analysis suggested that delta-ALA transport across the brush-border and basolateral membranes of the intestine was mainly mediated by peptide transporters. In addition, the apical-to-basolateral transport of delta-ALA was greater than that of the opposite direction. These findings provide the first evidence that the intestinal basolateral peptide transporter can recognize and transport nonpeptidic compounds, and play a definitive role in the absorption of delta-ALA.

ATP-Binding Cassette Transporters↗
PubMed2001

Investigation and evaluation of the change in the outpatients flow before and after the move of the hospital.

In order to estimate the changes in the service for outpatients with hospital movement, patient flow investigation has been performed at both old and new hospitals. IC memory card was handed out to each outpatient, and timestamps and places were recorded on the card at the places that the patient have been by using card writers set at each department. By comparing with two results, old and new hospitals, patient-waiting time at payment counter was remarkably shortened because of the effect of order-entry system. However, there were few changes in stay time at clinical departments. From the results, it is found that optimization of booking system was required for improvement of patients flow at clinical departments.

Appointments and Schedules↗
PubMed2001

Effect of a novel insole on the subtalar joint of patients with medial compartment osteoarthritis of the knee.

OBJECTIVE: To assess the efficacy of a lateral wedge insole with elastic strapping of the subtalar joint for conservative treatment of osteoarthritis (OA) of the knee. METHODS: The efficacy of a novel insole with elastic subtalar strapping and a traditional shoe insert wedge insole was compared. Ninety female outpatients with OA of the knee were treated with wedge insoles for 8 weeks. Randomization was performed according to birth date. Standing radiographs with unilateral insole use were used to analyze the femorotibial and talar tilt angles for each patient with and without their respective insole. Visual analog scale (VAS) score for subjective knee pain at the final assessment was compared with that at baseline in both groups. RESULTS: Participants wearing the elastically strapped insole (n = 46) had significantly decreased femorotibial angle (p < 0.0001) and talar tilt angle (p = 0.005) and significantly improved VAS pain score (p = 0.045) in comparison with baseline assessments. These significant differences were not found in the group with the inserted insole (n = 44). CONCLUSION: The novel strapped insole leads to valgus angulation of the talus, resulting in correction of the femorotibial angle in patients with knee OA with varus deformity, and may have a therapeutic effect similar to that of high tibial osteotomy.

Aged↗
PubMed2000

Syntheses of optically active trifluoronorcoronamic acids.

Syntheses of optically active trifluoronorcoronamic acid 6 and its diastereomer 9 are described. Highly stereospecific and diastereoselective S(N)2 cyclization of gamma-cyanohydrins 3a and 3b gave cyclopropyl nitriles 4a and 4b. Hydrolysis of the cyano group and deprotection of the amino group of 4a provide trifluoronorcoronamic acid 6. Hofmann rearrangement of the amide which was generated by hydrolysis of the cyano group and oxidative cleavage of the aryl ring of 4b to provide trifluoro-allo-norcoronamic acid 9.

Amino Acids↗
PubMed2000

Classical conditioning of oxidative DNA damage in rats.

This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modification relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA significantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P=0.013). The rats that showed a painful response at the Fe-NTA administration had significantly higher values of 8-OH-dG than those without pain (P=0. 003). These results not only provide the first evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Crystal structure of a plant ribonuclease, RNase LE.

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.

Amino Acid Sequence↗
PubMed2000

Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase.

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.

Amino Acid Sequence↗
PubMed2000

Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2.

Peptide transporters PEPT1 and PEPT2 transport numerous compounds including small peptides, peptide-like drugs and nonpeptidic compounds such as valacyclovir. PEPT1 and PEPT2 show low and high affinity for most substrates, respectively, but beta-lactam antibiotics without an alpha-amino group are the only known substrates that prefer PEPT1 to PEPT2. The aim of this study was to compare the recognition and affinity of various substrates between rat PEPT1 and rat PEPT2, and to determine the structural requirements influencing the substrate affinity. [14C]Glycylsarcosine uptake by PEPT1- or PEPT2-expressing transfectant was inhibited by di- and tripeptides, but not by amino acids, tetrapeptides or most cyclic dipeptides. All dipeptides and tripeptides examined showed more potent inhibition of [14C]glycylsarcosine uptake via PEPT2 than via PEPT1, irrespective of their charge and structure. Modification of the alpha-amino group of dipeptides reduced their substrate affinity to both transporters, as compared to unmodified dipeptides, but these dipeptides still showed potent inhibitory effects on PEPT2. Among the nonpeptidic substrates tested, only the eight-amino-octanoic acid displayed stronger inhibition of [14C]glycylsarcosine uptake in PEPT1 than in PEPT2. These findings suggest that alpha- or beta-amino carbonyl function is the key structure responsible for the higher affinity for PEPT2 than for PEPT1.

Animals↗
PubMed2000

Relationship of two ribonucleases with molecular masses of 45 kDa and 37 kDa from the culture medium of Lentinus edodes.

Lentinus edodes (shiitake) produces three base non- specific and acid ribonucleases, RNase Le2, RNase Le37 and RNase Le45. The primary structures of the former two RNases, having molecular masses about 24 and 37 kDa, respectively, have been elucidated to be members of the RNase T2 family. The latter two are excreted from mycelia into the medium. In this report, we estimated the primary structure of RNase Le45 using the following experimental evidence. (i) The partial amino acid sequence of RNase Le45 determined that up to about 60% of total protein was identical with that of RNase Le37. (ii) The amino acid composition of RNase Le45 was identical to that of RNase Le37. (iii) The elution profiles on HPLC of lysylendopeptidase and Staphylococcus aureus V8 protease digests of RCM-RNase Le45 (reduced and S-carboxymethylated RNase Le45) were very similar to those of RNase Le37, except for the absence of C-terminus peptide which contained O-glycosylated peptides. However, RNase Le45 contained about 70 residues of mannose and 4 residues of hexosamine. These values were more than twice those of RNase Le37. (iv) RNase Le45 was immunologically indistinguishable from RNase Le37. (v) After treatment with both glycosidase EndoH and alpha-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis. However, one of the major bands with the highest mobility from RNase Le45 and Le37 showed the molecular mass of 29 kDa in common, which is slightly larger than that of RNase Le2 containing no carbohydrate. These results indicated that RNase Le45 is an enzyme which is a heavily glycosylated species of RNase Le37.

Amino Acid Sequence↗
PubMed2000

Enzymatic properties of phenylalanine101 mutant enzyme of ribonuclease rh from Rhizopus niveus.

To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2', 3'-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8% of the native enzyme, respectively, and those of the other mutants were less than 1% of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as pi/pi or CH/pi interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.

Base Sequence↗
PubMed2000

Amino acid sequence of an unique ribonuclease with a C-terminus rich in O-glycosylated serine and threonine from culture medium of Lentinus edodes.

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase LE37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.

Agaricales↗
PubMed2000

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Amino Acid Sequence↗
PubMed2000

Detection of exogenous growth hormone (GH) administration by monitoring ratio of 20kDa- and 22kDa-GH in serum and urine.

We previously demonstrated that individual subjects have fairly constant ratios of serum concentrations of 20 kDa- (20K) and 22 kDa-GH (22K). The aim of this study is to demonstrate the possibility of utilizing the changes in the ratio of 20K/22K for detecting the exogenous administration of 22K. A male patient with idiopathic dilated cardiomyopathy (age 51) received 22K (4U, s.c.) every other day. The concentrations of 20K and 22K in serum and urine were measured using enzyme-linked immunosorbent assays before and after administration. The administration of 22K increased total GH concentration, and markedly decreased the ratio of 20K/22K in serum, especially 2-10 h after the administration. From calculations, it became clear that the concentration of exogenous 22K reached a peak between 2-4 h after the administration and decreased to a negligible level after 24 h. The ratio of 20K/22K in the 0-24 h urine was 5 times lower than that in the 24-48 h urine. These data suggest that, by monitoring the ratio of 20K/22K in serum or urine, it is possible to determine whether or not GH has been externally administered and to calculate the serum GH that has been administered.

Cardiomyopathy, Dilated↗
PubMed2000

Serum concentrations of 20K human growth hormone in normal adults and patients with various endocrine disorders. Study Group of 20K hGH.

The 20K hGH isoform is produced by alternative splicing of GH mRNA, and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunoadsorbent assays (ELISAs) specific to 20K and 22K hGH. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (N=282) in normal women, significantly higher than in normal men (64 +/- 170 pg/mL, N=226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, N=176) and women (6.3 +/- 2.1%, N = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%, N=33) and in patients with anorexia nervosa (9.0 +/- 1.9, N=8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of the 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or chronic excess of 22K hGH altering the metabolic clearance rate of 20K hGH. The values in patients with adult growth hormone deficiency (GHD), hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests such as insulin tolerance test and GRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH.

Acromegaly↗