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Biomedical subjects

A Imamura

Publications and source records attributed to A Imamura.

At least 19 recordsLinked to original sources
PubMed2000

Catalase-less peroxisomes. Implication in the milder forms of peroxisome biogenesis disorder.

We established a Chinese hamster ovary cell line having a temperature-sensitive phenotype in peroxisome biogenesis. This mutant (65TS) was produced by transforming a PEX2-defective mutant, Z65, with a mutant PEX2 gene, PEX2(E55K), derived from a patient with infantile Refsum disease, a milder form of peroxisome biogenesis disorder. In 65TS, catalase was found in the cytosol at a nonpermissive temperature (39 degrees C), but upon the shift to a permissive temperature (33 degrees C), catalase gradually localized to the structures containing a 70-kDa peroxisomal membrane protein, PMP70. In contrast to catalase, other matrix proteins containing typical peroxisome targeting signals, acyl-CoA oxidase and peroxisomal 3-ketoacyl-CoA thiolase, were co-localized with PMP70 in most cells, even at 39 degrees C. We found that these structures are partially functional peroxisomes and named them "catalase-less peroxisomes." Catalase-less peroxisomes were also observed in human fibroblasts from patients with milder forms of peroxisome biogenesis disorder, including the one from which the mutant PEX2 gene was derived. We suggest that these structures are the causes of the milder phenotypes of the patients. Temperature-dependent restoration of the peroxisomes in 65TS occurred even in the presence of cycloheximide, a protein synthesis inhibitor. Thus, we conclude that in 65TS, catalase-less peroxisomes are the direct precursors of peroxisomes.

ATP-Binding Cassette Transporters↗
PubMed2000

The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins.

The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera. Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins. We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro. These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B. subtilis.

Bacillus subtilis↗
PubMed2000

Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures.

Peroxisome biogenesis disorders, of which 13 complementation groups have been identified, are subdivided with regard to two major dysfunctions: peroxisomal matrix protein import and peroxisomal membrane synthesis. Detectable remnant membrane structures are evident only in the former. Molecular defects have been defined in 10 PEX genes, including eight related to protein import and two to membrane synthesis. We now have evidence that the human complete cDNA encoding Pex3p, a peroxisomal membrane protein (PMP) factor for the proper localization of PMPs, rescues the import of both PMP and the matrix protein in fibroblasts from a Zellweger syndrome patient of complementation group G. This patient was homozygous for a 1 base insertion in the codon for V182, which resulted in a change of codon (182-183) and introduced a termination codon (184), which inactivated PMP and matrix protein import by Pex3p. A PEX3-defective CHO mutant clone, ZPG208, was of the same complementation group as group G.

ATP-Binding Cassette Transporters↗
PubMed2000

Platelet-derived microparticles in patients with arteriosclerosis obliterans: enhancement of high shear-induced microparticle generation by cytokines.

We evaluated the plasma concentrations of cytokines and platelet-derived microparticles in patients with arteriosclerosis obliterans and studied the effect of cytokines on platelet-derived microparticle generation under high shear stress. Interleukin-6 levels peaked at 48 hours after vascular surgery, while thrombopoietin started to increase at 24 to 48 hours postoperatively and peaked on the seventh day. Platelet activation markers were increased in the arteriosclerosis obliterans patients preoperatively. Levels of P-selectin and CD63 both increased further, peaking at 6 to 24 hours postoperatively. Platelet-derived microparticle levels were also increased preoperatively. At 6 hours postoperatively, the plasma level of platelet-derived microparticles was significantly increased. Plasma platelet-derived microparticle level was lower at 12 hours but only returned to the preoperative value at 7 days after grafting. There was a difference in the platelet-derived microparticle level at 7 days between patients with or without antiplatelet therapy (cilostazol). The effect of cytokines on platelet activation under high shear stress was also studied. Interleukin-6 and thrombopoietin enhanced both P-selectin expression and platelet-derived microparticle generation under high shear stress. These results suggest that platelet-derived microparticles are released by platelet activation after vascular grafting when certain cytokines increase under high shear stress and that antiplatelet therapy may reduce platelet-derived microparticle levels postoperatively.

Aged↗
PubMed2000

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins.

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.

Animals↗
PubMed2000

Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient.

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

Amino Acid Sequence↗
PubMed2000

Restoration of biochemical function of the peroxisome in the temperature-sensitive mild forms of peroxisome biogenesis disorder in humans.

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.

Animals↗
PubMed2000

Genes encoding pseudo-response regulators: insight into His-to-Asp phosphorelay and circadian rhythm in Arabidopsis thaliana.

In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-to-Asp phosphorelay mechanisms are involved presumably in propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we identified and characterized a set of novel Arabidopsis genes whose products considerably resemble the authentic response regulators (ARR-series) of Arabidopsis in the sense that they have a phospho-accepting receiver-like domain. However, they should be discriminated from the classical ones in the strict sense that they lack the invariant phospho-accepting aspartate site. They were thus named APRRs (Arabidopsis pseudo-response regulators). Two such representatives, APRR1 and APRR2, were characterized extensively through cloning of the corresponding cDNAs, in terms of their structural designs, biochemical properties, subcellular localization in plant cells, and expression profiles at the transcriptional level. The result of in vitro phosphorylation experiment with the Arabidopsis AHP phosphotransmitter suggested that the pseudo-receivers have no ability to undergo phosphorylation. The result of transient expression assay with onion epidermal cells showed that the GFP-APRR1 fusion protein has an ability to enter into the nuclei. The C-terminal domain of APRR1, termed CONSTANS-motif, appears to be responsible for the nuclear-localization. The most intriguing result was that the accumulation of APRR1 transcript is subjected to a circadian rhythm. The APRR1 protein is identical to the one that was recently suggested to interact with the ABI3 (ABISCISIC ACID INSENSITIVE3) protein. These are discussed with special reference to the His-to-Asp phosphorelay signal transduction and circadian rhythm in Arabidopsis thaliana.

Amino Acid Sequence↗
PubMed2000

Clinical and bacteriological profiles of patients with typhoid fever treated during 1975-1998 in the Tokyo Metropolitan Komagome Hospital.

Patients with typhoid fever presenting to the Tokyo Metropolitan Komagome Hospital during the period 1975-1998 were retrospectively investigated. All cases were diagnosed by a positive culture for Salmonella typhi in either of their clinical specimens. Of the total number of 130 patients, 57% contracted the disease abroad; this population increased in later years as the total numbers of cases decreased. The period from disease onset to diagnosis averaged 14 days with 20% of the cases requiring over three weeks to establish a diagnosis. As for symptomatology relative bradycardia was seen in less than half of the cases, and rose spots or splenomegaly in less than one third. A positive blood culture was the most frequent test establishing the diagnosis followed by a positive stool culture. Intestinal bleeding was recognized in as many as 35 cases (27%) and even intestinal perforation occurred in two cases (1.5%). Chloramphenicol was most commonly employed during the early study period, however, during the late period it was replaced by fluoroquinolones. The clinical cure rate was 98% with regimens that include fluoroquinolones/quinolone; however it was 87% with the other antimicrobial regimens. Bacteriological relapse occurred in 25% of the non-fluoroquinolone group while only in 2.0% in the fluoroquinolone/quinolone group. Four strains of Salmonella typhi that were multi-resistant to chloramphenicol, ampicillin and cotrimoxazole were isolated in travelers from Asia. Early diagnosis by appropriate bacteriological examination regardless of classical symptomatology should be stressed and the use of fluoroquinolones is warranted in the treatment of typhoid fever.

Adolescent↗
PubMed2000

The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition.

The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.

Alanine↗
PubMed2000

Microsatellite instability in patients with multiple primary cancers of the gastrointestinal tract.

BACKGROUND: Little is known about the genetic alterations in multiple primary cancers of the gastrointestinal tract. Microsatellite instability (MSI) is frequently observed in hereditary non-polyposis colorectal cancer (HNPCC), and multiple primary cancers is a feature of this syndrome. AIMS: To identify MSI incidence, target gene mutation, and mismatch repair (MMR) protein status in patients with multiple primary cancers of the gastrointestinal tract. SUBJECTS: Fifty seven cancers from 22 Japanese patients with multiple primary cancers of the stomach, duodenum, colon, and rectum. METHODS: MSI was examined at 5-7 microsatellite loci. Mutation analysis for TGFbetaRII, IGFIIR, and BAX was performed in cancers with MSI. MMR protein status was examined by immunohistochemical analysis using a monoclonal antibody against hMSH2 and hMLH1. RESULTS: MSI was observed in 16 of 22 patients (73%) and in 29 of 57 lesions (51%). High frequency MSI (MSI-H) was found often in patients with multiple cancers in the same organ (p = 0.042), especially in multiple gastric cancer patients (p = 0.038). In contrast, patients with multiple cancers in different organs had a tendency to show low frequency MSI (MSI-L) or microsatellite stable (MSS) phenotype. Both target gene mutation and decreased expression of MMR protein were found only in seven lesions of three patients with MSI at more than four microsatellite loci. CONCLUSIONS: These results suggest that genetic instability may play an important role in the development of multiple gastrointestinal cancers but there may be different genetic alterations between multiple gastrointestinal cancers of the same and different organs.

Aged↗
PubMed2000

Temperature-sensitive mutation of PEX6 in peroxisome biogenesis disorders in complementation group C (CG-C): comparative study of PEX6 and PEX1.

Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as Pex1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30 degrees C but not at 37 degrees C. This patient was homozygous for a missense mutation, T-->C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1-deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1, the counterpart to L57P in PEX6, showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6, the counterpart to the temperature-sensitive mutation G843D in PEX1, revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and Pex1p.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective patients.

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

External iliac artery occlusion in posterior spinal surgery.

STUDY DESIGN: Report of a case of external iliac artery occlusion occurring as a rare complication of lumbar posterior spinal surgery. OBJECTIVE: To clarify the cause of this rare complication and recommend methods for preventing its recurrence. SUMMARY OF BACKGROUND DATA: Several cases of central retinal artery occlusion after posterior spinal surgery have been reported, but there has been no reported case of external iliac artery occlusion after posterior spinal surgery. METHODS: A 65-year-old woman who had a 32-year history of systemic lupus erythematosus underwent posterior decompression and spinal fusion for degenerative scoliosis. She was supported on a Hall frame during the operation. Three hours after surgery, paralysis and sensory impairment of the left leg and cyanosis of the toes of the left foot were noted. The popliteal, dorsalis pedis, and posterior tibial pulses were not palpable. Angiography showed complete occlusion of the external iliac artery, and emergency removal of the thrombus was performed. RESULTS: One year later, neurologic symptoms were absent, and the leg vessels were normal. CONCLUSIONS: Prolonged direct pressure on the inguinal region during posterior spinal surgery on a Hall frame may cause external iliac artery occlusion. Early recognition and adequate treatment can prevent serious sequelae.

Aged↗
PubMed1999

The 5'-flanking region of the mouse adenylyl cyclase type VIII gene imparts tissue-specific expression in transgenic mice.

The calcium-stimulated adenylyl cyclases (ACs) play a central role in stimulus-dependent modification of synaptic function. The type VIII AC (AC8) is one of three mammalian calcium-stimulated isoforms, each of which is expressed in a region-specific manner in the CNS. To delineate the DNA sequences responsible for appropriate targeting of AC8 expression, we report here the complete structure of the AC8 gene and define the pattern of expression of the full-length cDNA and its splice variants. In addition to expression within the brain, robust expression of AC8 was also found in the lung. By in situ hybridization, we have found the highest expression of AC8 mRNA within the olfactory bulb, thalamus, habenula, cerebral cortex, and hypothalamic supraoptic and paraventricular nuclei. By generating transgenic mice whose expression of beta-galactosidase is controlled by the AC8 5'-flanking DNA sequences, we demonstrate that the DNA sequences within the 10 kb preceding exon 1 are critical for establishment of this region-specific pattern. This spectrum of sites of production is unique to AC8 among the calcium-stimulated adenylyl cyclases and suggests nonredundant functions with other adenylyl cyclases in neuroendocrine regulation and/or behavior.

Adenylyl Cyclases↗
PubMed1999

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor-2) gene in patients with peroxisome biogenesis disorders.

The PEX6 (peroxisome assembly factor-2, PAF-2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487-496, 1999.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Ex vivo liver perfusion with arterial blood from a pig with ischemic liver failure.

To simplify liver support using an ex vivo perfused liver, an isolated pig liver was perfused with arterial blood from the recipient pig while monitoring the metabolic capacity of the ex vivo perfused liver. It was possible to perfuse the isolated liver for more than 24 h using arterial blood from a pig with ischemic liver failure. The viability of the isolated liver during support from the liver failure pig was well maintained as evidenced by the high adenylate energy charge (0.815) and a constant ketone body ratio (KBR) of over 1.0 sampled from the hepatic vein. Oxygen consumption (mean, 29.0 microl/min/g of liver) and bile production (mean, 24.2 microl/h/g of liver) were significantly higher in the isolated liver connected to the liver failure pig than in the organ connected to the pig without liver failure (15.5 microl/min/g and 7.3 microl/h/g, respectively). These findings suggest that this liver support system has sufficient metabolic capacity to support a failed liver. Further studies may provide the experimental basis necessary for the clinical application of this device in treatment of patients with acute liver failure.

Animals↗