Biomedical subjects

A Takeuchi

Publications and source records attributed to A Takeuchi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Clinical and pathologic features of focal segmental glomerulosclerosis with mitochondrial tRNALeu(UUR) gene mutation.

BACKGROUND: Several families have been described in which an A to G transition mutation at position 3243 (A3243G) of the mitochondrial DNA (mtDNA) is associated with focal and segmental glomerulosclerosis (FSGS). However, the prevalence, clinical features, and pathophysiology of FSGS carrying mtDNA mutations are largely undefined. METHODS: Among 11 biopsy-proven primary FSGS patients of unknown etiology, we examined seven FSGS patients to determine whether any of the clinical and pathological features of FSGS were associated with an A3243G mtDNA mutation. In four subjects in whom the A3243G mtDNA mutation was discovered in blood leukocytes, as well as in urine sediments, we retrospectively reviewed the medical records and re-evaluated the renal biopsy specimen using light and electron microscopy. We further screened the patient's family members for the presence and degree of heteroplasmy for this mtDNA mutation and obtained medical histories that were consistent with mitochondrial cytopathy. RESULTS: The four individuals identified with the A3243G mtDNA mutation were female. Proteinuria was diagnosed in these individuals during a routine annual health checkup in their teenage years. None of the patients showed any symptoms related to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode, whereas diabetes mellitus in two of the patients and a hearing disturbance in one patient became manifest within a 3- to 13-year follow-up period. Strict maternal transmitted inheritance was confirmed by pedigree studies in all of these patients. Steroid therapy was ineffective in all four patients. In two of these patients, renal function declined slowly to end-stage renal failure. Histologic examination of biopsy specimens revealed that glomeruli were not hypertrophied, while electron microscopic examination identified severely damaged, multinucleated podocytes containing extremely dysmorphic abnormal mitochondria in all patients. CONCLUSIONS: FSGS may belong to the spectrum of renal involvement in A3243G mtDNA mutation in humans. Severely injured podocytic changes containing abnormal mitochondria may explain the pathogenesis of FSGS in association with the A3243G mtDNA mutation.

DNA, Mitochondrial↗

PubMed2001

Role of kidney-specific organic anion transporters in the urinary excretion of methotrexate.

BACKGROUND: High-dose folinic acid is used to accelerate methotrexate elimination to avoid renal toxicity of the drug. The present study was carried out to examine the role of the renal organic anion transporters OAT-K1 and OAT-K2 in the urinary excretion of methotrexate, especially in the methotrexate-folinic acid rescue therapy. METHODS: Madin-Darby canine kidney cells stably expressing OAT-K1 and OAT-K2 were used for the in vitro transport study; 5/6 nephrectomized rats were used to detect changes in mRNA expression levels of OAT-K1 and OAT-K2 and to evaluate methotrexate pharmacokinetics under conditions of renal insufficiency. RESULTS: Methotrexate efflux mediated by these transporters in stable transfectants was stimulated in the presence of extracellular folic acid and folinic acid, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate. The mRNA expression levels of OAT-K1 and OAT-K2 were markedly diminished after 5/6 nephrectomy, but those of multidrug resistance associated protein 2, which could transport methotrexate, were maintained. Renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats compared with that in sham-operated rats. Additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats but not in 5/6 nephrectomized rats. CONCLUSIONS: The decreased expressions of OAT-K1 and OAT-K2 may be attributable to the longer exposure to methotrexate and ineffective folinic acid rescue. In terms of contributing to patient safety, renal clearance of methotrexate, especially folinic acid-stimulated tubular secretion of the drug via these transporters, would be a key factor in methotrexate therapy.

Animals↗

PubMed2001

Transfusion transmitted virus infection in patients on maintenance haemodialysis and in hospital workers.

A newly discovered DNA virus, transfusion transmitted virus (TTV), was isolated from a post-transfusional hepatitis patient in Japan. A high prevalence (32-46%) of TTV infections in patients receiving maintenance haemodialysis (HD) has been reported but the occupational risk of TTV on HD units has not yet been determined. We determined the prevalence of TTV in workers in the same HD unit and the risk factors for TTV infection in HD patients, using logistic regression analysis. The prevalence of TTV DNA was 59.6% in 198 HD patients, significantly higher than that in the HD unit (13 of 39, 33.3%;P= 0.002) and non-HD healthcare workers (20 of 75, 26.7%; P= 0.001). A logistic regression analysis showed that male gender and negative test results for hepatitis G virus RNA were risk factors for TTV infection, but prior blood transfusion and duration of HD were not. Stepwise selection of multiple regression analysis showed that the presence of hepatitis C virus RNA was the only significant predictor for high serum ALT activity, and that the presence of TTV DNA was not. These results indicate that TTV is one of the prevalent human viruses transmissible either parenterally or nonparenterally in HD patients, but the occupational risk of TTV infection in HD unit workers is as low as in other healthcare workers. The pathogenic effects of TTV on the liver appear to be limited.

Adult↗

PubMed2001

Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat.

We previously found hydroperoxide-responsive proteins (HPRPs), which are comprised of peroxiredoxin I (Prx I), Prx II, Prx III, Prx VI, HSP27, G3PDH and two unidentified proteins (HPRP-2' and HPRP-5'), in human umbilical vein endothelial cells. It was demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) that most HPRPs are converted into variants with lower pI upon exposure to hydroperoxides. In this study, we examined the HPRP response on 2D gels upon exposure of human endothelial cells (ECV304) to paraquat (PQ2+), which generates reactive oxygen species (ROS) within cells. PQ2+ exerted cytotoxic effects in a dose-(10 microM-10 mM) and time-(24-168 h) dependent manner. Two-dimensional PAGE analysis revealed that HPRP-2', and oxidized forms of Prx I, Prx II and Prx III were clearly increased upon exposure of cells to sublethal levels of PQ2+. Microsequence analysis revealed that both HPRP-2 and -2' were identical with human DJ-1. Moreover immunoblot analysis confirmed the increase of oxidized forms of Prx II, Prx III and DJ-1 in response to sublethal levels of PQ2+. PQ2+ treatment failed to increase fluorescence intensity derived from DCF, which is believed to be an indicator for intracellular levels of hydroperoxide. Although pentachlorophenol (PCP), an uncoupler of the mitochondrial respiratory chain, clearly elevated the fluorescence, PCP had no effect on HPRP response. These observations indicated that DCF-derived fluorescence is not correlated with HPRP response. We consider that the response of Prxs and DJ-1 on 2D gels could reflect endogenous production of ROS in PQ(2+)-treated cells, and might be a sensitive indicator of oxidative stress status.

Animals↗

PubMed2001

Milk basic protein promotes bone formation and suppresses bone resorption in healthy adult men.

Milk contains several components effective for bone health. In the previous in vitro and in vivo studies, we have shown that milk whey protein, especially its basic protein fraction (milk basic protein [MBP]), promoted bone formation and suppressed bone resorption. This present study examines the effect of MBP on the biochemical markers of bone metabolism in healthy adult men. Experimental beverages containing MBP (300 mg of MBP a day) were given to 30 normal healthy adult men for 16 days. The serum osteocalcin concentration had increased significantly after 16 days of ingesting the experimental beverage containing MBP. Urinary cross-linked N-teleopeptides of type-I collagen (NTx) excretion had decreased significantly after 16 days of ingesting MBP. The urinary NTx excretion was related to the serum osteocalcin concentration after 16 days of ingestion. These results suggest that MBP promoted bone formation and suppressed bone resorption, while maintaining the balance of bone remodeling.

Adult↗

PubMed2001

Evaluation of diagnostic availability of continuous ANP assay and LA/AO ratio in left heart insufficient dogs.

Left atrial/aorta ratio (LA/AO) by echocardiography and the plasma level of atrial natriuretic peptide (ANP) were measured in 32 dogs with left heart insufficiency. There was a significant correlation between plasma ANP concentration and LA/AO (r=0.66, p<0.001). The authors obtained the result that the degree of expansion of the left atrial diameter seemed to have a close relationship with plasma ANP concentration. Plasma ANP concentration decreased when the clinical signs of the patients improved. However, the LA/AO ratio after treatment did not decrease. From these findings, we concluded that plasma ANP concentration has the possibility to become a significant index in the diagnosis and prognosis of heart disease in dogs.

Animals↗

PubMed2001

Vegetarian diet ameliorates symptoms of atopic dermatitis through reduction of the number of peripheral eosinophils and of PGE2 synthesis by monocytes.

Many patients with atopic dermatitis are dissatisfied with conventional treatments based on topical steroids and have experienced some traditional remedies and alternative therapies. However, most of such therapies have not been evaluated scientifically and clinically by specialists. This study was designed to assess whether a certain vegetarian diet might be effective for atopic dermatitis and if so, to identify the mechanisms of this remedy through analyses of immunological parameters. An open-trial study was carried out in twenty patients with atopic dermatitis. An improvement of dermatitis was evaluated by SCORAD index and serological and immunological parameters were monitored. After a two-month treatment, the severity of dermatitis was strikingly inhibited, as assessed by SCORAD index and serological parameters including LDH5 activity and a number of peripheral eosinophils. A sharp reduction in eosinophils and neutrophils was observed prior to improvement in the skin inflammation. In addition, PGE2 production by peripheral blood mononuclear cells was reduced by this treatment. In contrast, serum IgE levels did not change during the same period. Although this study is an open-trial one, it suggests that this treatment may be useful for the treatment of adult patients with severe atopic dermatitis.

Adolescent↗

PubMed2001

Grafting of encapsulated genetically modified cells secreting GDNF into the striatum of parkinsonian model rats.

In order to deliver glial cell line-derived neurotrophic factor (GDNF) into the brain, we have established a cell line that produces GDNF in a continuous fashion by genetic engineering. These cells were encapsulated and grafted into parkinsonian model rats that had received unilateral intrastriatal injection of 6-hydroxydopamine 2 weeks earlier. Neurochemical analysis showed that GDNF has been produced from the capsule for 6 months after grafting and histological analysis revealed good survival of GDNF-producing cells in the capsule 6 months after grafting. The density of nigrostriatal dopaminergic fibers in the striatum as well as the number of dopaminergic cell bodies in the substantia nigra recovered significantly after GDNF-producing cell grafting. These results suggest the possible application of GDNF-producing cell grafting for the treatment of Parkinson's disease.

Animals↗

PubMed2001

Neurotrophic factor-secreting cell grafting for cerebral ischemia: preliminary report.

In this experiment, we examined a possible protective effect of encapsulated neurotrophic factor-secreting cell grafting for ischemic injury. We established a basic fibroblast growth factor (bFGF)-secreting cell line by genetic manipulation. We enveloped these cells into polymer capsules, which consist of a semipermeable membrane, and implanted them into the right striatum of rats. At 6 days after implantation, these rats received right middle cerebral artery occlusion (MCAO) using interluminal suture technique. At 24 h after MCAO, rats were sacrificed and their cerebral infarction volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and image analysis. We found approximately 30% reduction in infarct volume in the encapsulated bFGF-secreting cell grafting groups vs. the encapsulated naive BHK cell grafting group or the without implantation group. We measured bFGF secretion from encapsulated bFGF-secreting cells using enzyme-linked immunosorbent assay (ELISA). The retrieved capsules continued to secrete bFGF. There was no significant difference of bFGF secretion between the capsules before and after transplantation. A large number of viable BHK-bFGF cells was observed within the full length of the retrieved capsule. These results indicate that encapsulated bFGF-secreting cell grafting exerts a protective effect on ischemic injury.

Animals↗

PubMed2001

Multispecific substrate recognition of kidney-specific organic anion transporters OAT-K1 and OAT-K2.

We characterized the interactions of various compounds with OAT-K1 and OAT-K2, kidney-specific organic anion transporters. By using Madin-Darby canine kidney cells stably transfected with OAT-K1 or OAT-K2 cDNA, the antitumor drug methotrexate, the mycotoxin ochratoxin A, endogenous organic anions (thyroid hormones, taurocholic acid, and conjugated steroids), and the antiretroviral drug zidovudine were shown to be substrates for these transporters. Although the apparent Michaelis constant (Km) values of methotrexate for OAT-K1 and OAT-K2 were 2.1 and 1.8 microM, respectively, 2.5 mM methotrexate inhibited only 20% of the 125I-thyroid hormones uptake via these transporters. In addition, 100 microM methotrexate did not have any effect on [3H]zidovudine uptake via OAT-K1 or OAT-K2. Similarly, several substrates caused little or no mutual inhibition at concentrations much higher than their Km values for these transporters. Moreover, intracellular methotrexate trans-stimulated the OAT-K1- and OAT-K2-mediated uptake of [3H]folic acid, but not that of other compounds. Organic anion-transporting polypeptide 2 (oatp2), a liver-type homolog of OAT-K1 and OAT-K2, showed similar events. The inhibition constant values of triiodothyronine and taurocholic acid for [3H]digoxin uptake in oatp2-expressing oocytes resulted in 50.4 and 1.48 mM, respectively, which were about 9- and 40-fold higher than their Km values for oatp2, respectively. These findings suggested that several substrates interact with these transporters at different amino acid residue(s). Taken together, these observations suggested that OAT-K1 and OAT-K2 could serve as multispecific transporters, mediating transport of a wide variety of endogenous substances, xenobiotics, and their metabolites in the kidney, presumably via several interaction sites in their molecules.

Animals↗

PubMed2000

Primary structure of human hepatocellular carcinoma-associated aldehyde dehydrogenase.

Tumor-associated aldehyde dehydrogenase (T-ALDH) is strongly expressed in hepatocellular carcinoma (HCC) but undetectable in normal liver. In the present study, this enzyme from human HCC, HCC T-ALDH, was purified and the partial amino acid sequences (384 residues) determined by direct protein sequencing matched the amino acid sequence (453 residues) deduced from cloned HCC T-ALDH cDNAs with an open reading frame. The coding sequences of HCC T-ALDH cDNA, human stomach ALDH3A1 cDNA [Hsu et al., J. Biol. Chem. 267 (1992) 3030-3037] and human squamous cell carcinoma (SCC) T-ALDH cDNA (Schuuring et al., GenBank I.D. M74542) matched one another except for discrepancies at four positions, with consequent P12R, I27F and S134A substitutions. R and A were found in HCC and SCC T-ALDHs, whereas P and S were present in stomach ALDH3A1. To confirm that these discrepancies would have general occurrence, coding sequences of HCC T-ALDH cDNAs from six patients and stomach ALDH3A1 cDNAs from two individuals were examined and all were found to encode ALDH3A1 having R, I and A at protein positions 12, 27 and 134, respectively, indicating HCC T-ALDH to be variant ALDH3A1 which is common in human stomach tissues.

Aldehyde Dehydrogenase↗

PubMed2000

A Salmonella detection system using an engineered DNA binding protein that specifically captured a DNA sequence.

We have developed a novel method for the detection with high selectivity of a double-stranded DNA fragment using an engineered DNA-binding protein, DnaA IV, a fusion protein of the DNA-binding domain of DnaA and glutathione S-transferase. The DNA fragment detection system is based on DNA-protein interaction and consists of sequence-specific binding of DnaA IV with a DNA fragment containing the DnaA box. DnaA IV, while not capturing other DNA fragments, specifically captured that containing the DnaA box. Because the oriC fragment containing the DnaA box could be specifically amplified by PCR from the genus Salmonella, the DNA fragment detection system was adapted for the detection of Salmonella. The Salmonella detection system using PCR amplification and the engineered DNA-binding protein could distinguish 104 cfu/mL Salmonella from 106 cfu/ mL contaminating bacteria.

Culture Media↗

PubMed2000

PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters.

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

Animals↗

PubMed2000

Mapping of a particular element using an absorption edge with an X-ray fluorescence imaging microscope.

An X-ray fluorescence imaging microscope with a Wolter-type objective mirror (magnification: 13) has been constructed at beamline 39XU of SPring-8. Monochromatic X-rays (DeltaE/E approximately 10(-4)) in the energy range 6-10 keV were used for X-ray fluorescence excitation of the specimens. Using two monochromatic X-rays above and below the absorption edge of a particular element, a two-dimensional image of the element could be obtained. As a result, two-dimensional element mapping of the test specimens (Cu, Co, Ni, Fe and Ti wires) and constituent minerals (Fe, Mn and Ti) of a rock specimen (a piemontite-quartz schist) became possible.

Journal Article↗

PubMed2000

Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury.

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.

Animals↗

PubMed2000

The potential role for nephritis-associated plasmin receptor in acute poststreptococcal glomerulonephritis.

Immunoglobulin G from a patient convalescing from acute poststreptococcal glomerulonephritis (APSGN) bound specific antigenic sites in early APSGN glomeruli. A streptococcal cytoplasmic antigen (preabsorbing antigen, PA-Ag), could selectively preabsorb fluorescein isothiocyanate (FITC)-labeled IgG and prevented glomerular staining. The antigen was purified and identified as an M(r) approximately 43,000 protein with a pI of 4.7 that strongly activated complement C3 (N. Yoshizawa, S. Oshima, I. Sagel, J. Shimizu, and G. Treser, 1992, J. Immunol. 148, 3110-3116). In the present study, a nephritogenic antigen was purified by affinity chromatography using APSGN IgG-immobilized Sepharose followed by chromatography on an anion-exchange resin. Purification was monitored by ELISA and Western blotting using the binding characteristics of the specific antibodies present in APSGN serum. The molecular weight of the purified antigen, named nephritis-associated plasmin receptor (NAPlr), was an M(r) approximately 43,000 protein and the internal amino acid sequence was found to be homologous to those of the plasmin receptor (Plr) of group A streptococci strain 64/14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The purified NAPlr exhibited GAPDH activity and plasmin(ogen) binding activity. Using FITC-labeled rabbit anti-NAPlr, the antigen was found to be present in the glomeruli of 22 of 22 patients in the early stage of APSGN. Bacterial Plr was also demonstrated in human APSGN glomeruli for the first time using monoclonal antibody to the recombinant Plr protein. Antibody to NAPlr was found in the sera of 46 of 50 (92%) patients within 3 months of onset. These results led us to speculate that NAPlr bound to the glomeruli may contribute to the pathogenesis of APSGN via plasmin and complement activation.

Adult↗

PubMed2000

Early and delayed Tc-99m ECD brain SPECT in SLE patients with CNS involvement.

We compared early and delayed Tc-99m ECD SPECT scans in 32 SLE patients (Group 1, definite neuropsychiatric disorders; Group 2, minor neurologic symptoms or normal) with those of normal controls by visual inspection and semi-quantitative evaluation. With visual interpretation, 13 out of 14 patients in Group 1 (93%) and 7 out of 18 patients in Group 2 (39%) had diffuse uneven decrease in early scans. Seven patients in Group 2 (39%) who had normal early scans demonstrated focal decrease in the medial frontal lobe in delayed scans. With cerebral region to cerebellar ratios, in early scans, the medial frontal lobe in Group 1 and Group 2 was significantly lower than in normal controls, and lateral frontal lobe and occipital lobes in Group 1 were significantly lower than in normal controls. Nevertheless, in delayed scans, every cortical region except for the parietal lobe in Groups 1 and 2 was significantly lower than in normal controls. The retention rates in all regions in SLE patients were significantly lower than in normal controls. No case showed SPECT improvement on follow-up studies in either group in spite of clinical improvement. Delayed Tc-99m ECD brain SPECT of high sensitivity might be useful in detecting CNS involvement. Although the SPECT findings did not correlate with the neuropsychiatric symptoms, early and delayed Tc-99m ECD SPECT seems to provide useful objective diagnostic information in SLE patients.

Adult↗

PubMed2000

Inhibition of membrane-bound carbonic anhydrase enhances subretinal fluid absorption and retinal adhesiveness.

BACKGROUND: The clinical use of currently available carbonic anhydrase (CA) inhibitors is limited by systemic side-effects, thought to result from the inhibition of intracellular CA isoenzymes. This study investigates how benzolamide, a carbonic anhydrase inhibitor which does not readily penetrate cell membranes, modulates retinal pigment epithelium functions relative to acetazolamide, which diffuses into the cytosol. METHODS: Small retinal detachments were made in Dutch rabbits by injecting saline into the subretinal space. Detachment height was measured using a dual He-Ne beam YAG laser focusing system, and the fluid absorption rate was calculated before and after intravenous injections of saline, acetazolamide or benzolamide. Retinal adhesiveness was determined by peeling the retina from the RPE and measuring the amount of adherent pigment. RESULTS: The baseline fluid absorption rate of 0.04 microl/mm(2)/h was unchanged after injection of 0.9% NaCl or low-dose benzolamide (5 mg/kg). The absorption increased to about 0.14 microl/mm(2)/h after higher benzolamide doses (20-40 mg/kg) and to 0.13 microl/mm(2)/h after acetazolamide (20 mg/kg). Both acetazolamide and benzolamide significantly slowed the post-enucleation failure of retinal adhesiveness. CONCLUSION: Since benzolamide had effects similar to acetazolamide, inhibition of membrane-bound CA appears to be sufficient to enhance subretinal fluid absorption and retinal adhesiveness. Membrane-specific CA inhibitors may therefore be of clinical value if they minimize side-effects from intracellular CA inhibition.

Absorption↗