Biomedical subjects

M Sugawara

Publications and source records attributed to M Sugawara.

At least 55 records · Page 3Linked to original sources

PubMed2000

Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle.

To date, two different transporters that are capable of transporting alpha-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the system A-specific substrate. This new transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 amino acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the transport of alpha-[(14)C](methylamino)isobutyric acid and [(3)H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. The amino acid-induced current is Na(+)-dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K(0. 5) for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2+/-0.1 mM and that the Na(+):alanine stoichiometry is 1:1.

Alanine↗

PubMed2000

A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates.

OBJECTIVE: To determine the cause and pathogenic mechanisms of a 21-year-old patient's cardioskeletal myopathy. The patient's muscle atrophy and weakness began in distal parts of limbs; cardiac and facial muscles were later involved. BACKGROUND: Desmin myopathy is a skeletal myopathy often associated with cardiomyopathy, caused by mutations in the desmin gene and characterized by desmin accumulation in affected muscle fibers, a leading marker of myofibrillar myopathies. Two kinds of deletions and seven missense mutations in the desmin gene have been identified. METHODS: Clinical examination, electron microscopy of muscle tissue, two-dimensional gel electrophoresis, DNA sequencing, restriction enzyme analysis, and gene transfection were performed. RESULTS: Electron microscopy showed disruption of sarcomeres at Z discs and electron-dense aggregates in biopsied skeletal and heart muscle. Two-dimensional gel electrophoresis of the patient's skeletal muscle proteins showed massive accumulation of desmin. The authors identified a novel desmin mutation, L385P in one allele in the carboxyl end of the rod domain 2B in the patient's leukocytes and skeletal muscle; neither parent had the mutation. Serologic study and DNA markers confirmed the de novo mutation. A peptide harboring desmin rod domains 2A and 2B with L385P tagged with green fluorescent protein induced cytoplasmic aggregates, nuclear DNA condensation, and cell death. CONCLUSIONS: A novel de novo mutation, L385P, causes desmin myopathy. An expression study indicated the toxic effect of the L385P mutation.

Adult↗

PubMed2000

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter.

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.

Amino Acid Transport Systems, Neutral↗

PubMed2000

Evaluation and comparison of ion permeation and agonist selectivities for N-methyl-d-aspartate receptor channels with different subunit compositions in bilayer lipid membranes based on integrated single-channel currents.

To quantify the ion-permeation ability of the recombinant epsilon1-4/zeta1 channel activated by agonists, the magnitude of agonist-induced integrated single-channel currents for the epsilon2-4/zeta1 N-methyl-d-aspartate (NMDA) channels in bilayer lipid membranes (BLMs) was evaluated electrochemically based on the single-channel recordings. The recombinant epsilon2-4/zeta1 channels were purified from Chinese hamster ovary cells expressing each channel and incorporated in BLMs formed by the tip-dip method. Three typical agonists, l-glutamate, NMDA, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (l-CCG-IV), were investigated at a concentration of 50 microM. The magnitude of l-glutamate-induced integrated current was found to depend on the epsilon-subunit composition and to increase in the order of epsilon2/zeta1 > epsilon1/zeta1 approximately epsilon4/zeta1 > epsilon3/zeta1, which differs from that of the reported binding affinities (EC(50)) between l-glutamate and each channel type. On the other hand, the magnitude of the integrated currents induced by NMDA and l-CCG-IV did not vary among the four channel types. The order of agonist selectivity toward the epsilon2-4/zeta1 channels in terms of the magnitude of the integrated current was l-glutamate > l-CCG-IV approximately NMDA for the epsilon2/zeta1 channel, l-CCG-IV > NMDA > l-glutamate for the epsilon3/zeta1 channel, and l-CCG-IV approximately l-glutamate > NMDA for the epsilon4/zeta1 channel, suggesting that the agonist selectivity also depends on the epsilon-subunit composition. The present study shows that each epsilon1-4/zeta1 channel has its own ability of ion permeation, i.e., its own signal transduction ability, which is not parallel to its binding ability.

Animals↗

PubMed2000

Primary structure, functional characteristics and tissue expression pattern of human ATA2, a subtype of amino acid transport system A.

We report here on the primary structure and functional characteristics of the protein responsible for the system A amino acid transport activity that is known to be expressed in most human tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from the human hepatoma cell line HepG2. Human ATA2 (hATA2) consists of 506 amino acids and exhibits a high degree of homology to rat ATA2. hATA2-specific mRNA is ubiquitously expressed in human tissues. When expressed in mammalian cells, hATA2 mediates Na+-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li+-intolerant. The Na+:amino acid stoichiometry is 1:1.

Amino Acid Sequence↗

PubMed2000

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta.

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.

Amino Acid Sequence↗

PubMed2000

Massive accumulation of M and H subunits of neurofilament proteins in spinal motor neurons of neurofilament deficient Japanese quail, Quv.

Quiver (Quv) is a non-sense mutation of neurofilament protein L subunit (NF-L) that causes neurofilament deficiency with preserved microtubules in Japanese quail. Anti-NF-M and anti-NF-H mAbs stained cell bodies of motor neurons in Quv embryo spinal cords much more intense than those in control spinal cords. Volume of motor neurons in Quv spinal cords increased to 2.3 times of control motor neurons. Immunoblot of Quv spinal cords revealed a relative increase in non- and hypo-phosphorylated NF-M and NF-H, and a decrease in the total amount of NFs. Quv sciatic nerves showed faintly reacted phosphorylated NF-M and NF-H. These results suggest that deficiency of assembled neurofilament results in decreased axonal transport of NFs and accumulation of NFs in cell bodies of spinal motor neurons.

Animals↗

PubMed2000

cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1.

We describe in this report the cDNA structure, functional characteristics, genomic organization, and promoter analysis of the mouse H(+)-coupled low-affinity peptide transporter PEPT1. The mouse PEPT1 cDNA cloned from a kidney cDNA library is approximately 3.1 kb long and encodes a protein of 709 amino acids. When expressed heterologously in mammalian cells and in Xenopus laevis oocytes, mouse PEPT1 mediates H(+)-coupled electrogenic transport of the dipeptide glycylsarcosine. The mouse pept1 gene, cloned from a genomic DNA library in bacterial artificial chromosome, is approximately 38 kb long and consists of 23 exons and 22 introns. 5'-Rapid amplification of cDNA ends with poly(A)(+) RNA from mouse intestine has identified the transcription start site that lies 31 bp upstream of the translation start site. The promoter region upstream of the transcription start site does not contain the TATA box but possesses three GC boxes which are the binding sites for the transcription activator SP1. Functional analysis of the promoter region using the luciferase reporter assay in Caco-2 cells (a human intestinal cell line that express PEPT1 constitutively) and five different 5'-deletion fragments of the promoter has shown that essential promoter/enhancer elements are present within 1140 bp upstream of the transcription start site.

Amino Acid Sequence↗

PubMed2000

Overexpression of mRNAs of TGFbeta-1 and related genes in fibroblasts of Werner syndrome patients.

We analyzed mRNAs that were up- or down-regulated in fibroblasts from Werner syndrome (WS) patients compared with those from normal individuals. The mRNAs from normal and WS cells were first screened by differential display, and those mRNAs that were apparently up- or down-regulated were selected except for mRNAs related to extra-cellular matrix (ECM) proteins that are already known to be up-regulated in WS fibroblasts. Then, the expression levels of these mRNAs were semiquantified by northern blot analysis, and six up-regulated and two down-regulated mRNAs were identified in WS cell lines. Among the six up-regulated mRNAs were three mRNAs that coded TGFbeta-1 and two proteins, their expressions of which were increased by TGFbeta-1. These results together with the fact that TGFbeta-1 up-regulates the expression of ECM proteins strongly suggest that TGFbeta-1 has a key role in accelerated cellular senescence of fibroblasts of WS patients.

Base Sequence↗

PubMed2000

Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A.

We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.

Amino Acid Sequence↗

PubMed2000

Evaluation of beta- and gamma-effects of group 14 elements using intramolecular competition

To evaluate beta-effects and gamma-effects of group 14 elements, we have devised a system in which the intramolecular competition between gamma-elimination of tin and beta-elimination of silicon, germanium, and tin can be examined. Thus, the reactions of alpha-acetoxy(arylmethyl)stannanes with allylmetals (metal = Si, Ge, Sn) in the presence of BF(3).OEt(2) were carried out. The reactions seem to proceed by the initial formation of an alpha-stannyl-substituted carbocation, which adds to an allylmetal to give the carbocation that is beta to the metal and gamma to tin. The beta-elimination of the metal gives the corresponding allylated product, and the gamma-elimination of tin gives the cyclopropane derivative. In the case of allylsilane, the cyclopropane derivative was formed as a major product, whereas in the case of allylgermane the allylated product was formed predominantly. In the case of the allystannane the allylated product was formed exclusively. These results indicate that the gamma-elimination of tin is faster than the beta-elimination of silicon, but slower than the beta-elimination of germanium and tin. The theoretical studies using ab initio molecular orbital calculations of the carbocation intermediates are consistent with the experimental results. The effect of substituents on silicon was also studied. The introduction of sterically demanding substituents on silicon disfavored the beta-elimination of silicon probably because of the retardation of nucleophilic attack on silicon to cleave the carbon-silicon bond.

Journal Article↗

PubMed2000

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter.

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.

Acetylcarnitine↗

PubMed2000

Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2.

In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.

Animals↗

PubMed2000

Simultaneous measurements of temperature and pH in vivo using NMR in conjunction with TmDOTP5-.

NMR techniques for temperature and pH measurements have attracted increasing interest in recent years, motivated in part by the growing importance of medical hyperthermia for the treatment of cancer. The chemical shifts of thulium 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetrakis(methylene phosphonate) (TmDOTP5-) have been studied as a function of temperature and pH. The results demonstrate that TmDOTP5- resonance shifts are highly sensitive to temperature (approximately 1.0 ppm/degrees C) and pH (approximately 3.2 ppm/pH unit) at clinically relevant field strengths. A new method is presented which utilizes two magnetically non-equivalent protons in TmDOTP5- for simultaneous NMR measurements of both temperature and pH. The difference in the chemical shift values of pairs of 1H resonances provides a temperature sensitivity of about 1.6 ppm/ degrees C. The technique is demonstrated in live rats undergoing ultrasound-induced hyperthermia therapy.

Animals↗

PubMed2000

Relationship between the pressure and diameter of the carotid artery in humans.

The purpose of this study was to examine the assumption of similarity between pressure and diameter-change waveforms in humans. We measured carotid arterial pressure and diameter change, simultaneously, in six patients with heart disease. In all patients, the carotid arterial pressure-diameter relationship could, in practice, be regarded as being linear.

Adult↗

PubMed2000

Genetic mapping of the rat mutation creeping and evaluation of its positional candidate gene reelin.

We have previously described a rat autosomal recessive mutation, creeping (cre), causing severe ataxia and disarrangement of neuronal cells in the central nervous system. The mutant strain has recently been successfully inbred, named Komeda Zucker creeping (KZC) rat. In the present study, we have performed a genetic analysis of the creeping mutation, and mapped it to rat Chromosome (Chr) 4. Comparative mapping, together with the similarity of the phenotype, suggested that the creeping mutation is homologous to the mouse reeler mutation. In fact, reelin expression was markedly reduced in the homozygous mutant (cre/cre) animals compared with the normal littermates. Thus, the KZC rat should become a useful biological model with a novel mutation in the reelin gene.

Animals↗

PubMed2000

Does repeated balloon inflation during coronary angioplasty induce ischemic tolerance? Analysis based on regional work.

It has been reported that repeated brief balloon inflation during coronary angioplasty (PTCA) alleviates myocardial dysfunction. However, it has also been reported that PTCA does not induce ischemic tolerance. Six patients with stable angina pectoris were recruited for this study. They were scheduled for PTCA to a significant stenosis of the proximal left anterior descending artery (LAD). All patients had single-vessel coronary artery disease without angiographic evidence of collateral circulation and with normal wall motion. After the stenosis of LAD was dilated by a 30-s inflation, 60 s of balloon inflation was performed five times at 60-s intervals. Left ventricular regional work was determined in the first and fifth inflations, and the data were compared. Regional work of the interventricular septum decreased immediately after the balloon inflation (the first inflation: 5.3 +/- 1.0 --> 0.6 +/- 0.2 mJ/cm3; fifth inflation; 5.3 +/- 1.0 --> 0.6 +/- 0.3 mJ/cm3) and no statistically significant differences were found between the first and fifth inflations. After balloon deflation, the time required for the recovery of regional work was 30s in the fifth inflation, compared with 40 s in the first inflation (at 30 s after deflation, first inflation: 3.6 +/- 1.3 mJ/cm3; fifth inflation: 5.2 +/- 1.2 mJ/cm3). Although repeated balloon inflation did not change the amount of reduction in regional work, it improved the postischemic recovery of regional work. These results suggest ischemic tolerance.

Aged↗

PubMed2000

Evolving changes in Doppler mitral flow velocity pattern in rats with hypertensive hypertrophy.

OBJECTIVES: The aim of our study was to explore evolving changes in a mitral flow velocity pattern (MFVP) and its hemodynamic and pathological correlates in hypertensive rats in an isolated diastolic heart failure model. BACKGROUND: Development of left ventricular (LV) hypertrophy and concomitant diastolic dysfunction cause heart failure in hypertensive hearts even with normal systolic function; however, associated evolving change in MFVP is still unclear. METHODS: Mitral flow velocity pattern was recorded every 2 weeks from 7 to 19 weeks in six hypertensive rats. Hemodynamic and pathological correlates of Doppler mitral flow indexes were examined as an additional part of the study using the hypertensive rats at the age of 13 weeks (compensatory stage, n = 7) and at 19 weeks (heart failure stage, n = 8). RESULTS: Initial development of pressure overload LV hypertrophy resulted in a decrease in early diastolic filling wave (E), a reciprocal increase in the filling wave due to atrial contraction (A) and prolongation of deceleration time of E wave (relaxation abnormality pattern). These changes were associated with an increase in tau, an index of LV relaxation, but without a change in LV end-diastolic pressure. Transition to congestive heart failure caused an increase in E, a decrease in A and shortening of deceleration time. These changes were not associated with further increase in tau but with elevation of LV end-diastolic pressure, reflecting marked LV hypertrophy and myocardial fibrosis. CONCLUSIONS: Development of pressure overload LV hypertrophy is associated with evolving changes in MFVP from normal to relaxation abnormality pattern and, in turn, to pseudonormalized to restrictive pattern. Analysis of MFVP may be useful to follow not only functional but also constitutional changes of the myocardium in hypertensive hearts.

Animals↗