Biomedical subjects

S Kawamoto

Publications and source records attributed to S Kawamoto.

At least 19 recordsLinked to original sources

PubMed2003

Differential response to low temperature of two Delta6 fatty acid desaturases from Mucor circinelloides.

A recently identified Delta6 fatty acid desaturase in Mucor rouxii shows a low sequence homology (approximately 24% at the amino acid level) to that isolated from Mortierella alpina, but is phylogenetically closer to a plant enzyme, suggesting the occurrence of Delta6 desaturase isozymes in Mucorales molds. In the present study, two types of Delta6 desaturases, mcD6-1 ( Mo. alpina type) and mcD6-2 ( M. rouxii type), were cloned from Mucor circinelloides. When the cloned genes were expressed in the yeast Saccharomyces cerevisiae in the presence of a linoleic acid substrate (C18:2Delta9, 12), a newly generated gamma-linolenic acid (C18:3Delta6, 9, 12) was detected in the cells, which confirmed the suspected enzymatic function of the recombinant protein. This is the first report of Delta6 desaturase isozymes present in one organism. Northern analysis demonstrated that the amount of mcD6-2 mRNA was less than half of that of mcD6-1 mRNA in cells grown at 28 degrees C. However, upon cultivation of the cells at 15 degrees C for 0.5-1 h, mcD6-2 mRNA rapidly increased by up to 1.5-fold and then gradually decreased. By contrast, mcD6-1 transcripts levels did not fluctuate significantly for 1 h after the temperature shift, but declined by 75% over the next 2 h. The gamma-linolenic acid content in total fatty acid from M. circinelloides decreased at 28 degrees C, but was maintained at approximately 30% at 15 degrees C. These data suggest that Delta6 desaturase isozymes play physiologically distinct roles in the maintenance of cellular lipids and adaptation to low temperature.

Amino Acid Sequence↗

PubMed2003

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea.

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.

Amino Acid Sequence↗

PubMed2003

Incremental responses of compound muscle action potentials in normal rats produced by repeated electrical stimulation.

The purpose was to clarify the mechanism of normal incremental amplitude responses in compound muscle action potentials (CMAPs) and single fiber action potentials (SFAPs) produced by repeated high frequency electrical stimulation. Whereas the amplitude response of CMAPs and SFAPs increased during stimulation at 20 c.p.s. of the right flexor carpi ulnaris muscles of 50 rats, their durations gradually decreased. Incremental responses of the CMAPs and SFAPs evoked by repeated stimulation were suppressed in 25 rats by 30 mg/kg of magnesium sulfate and in 25 rats by 120 mg/kg of dantrolene sodium. We speculate that the incremental responses of CMAPs and SFAPs evoked by repeated stimulation were suppressed by the unchanged shape of the muscle after the injection of those chemicals.

Action Potentials↗

PubMed2003

[Spinal cord ischemia complicated with acute aortic dissection and intramural hematoma; report of two cases].

Spinal cord ischemic injury is one of te most serious complications in patients with aortic dissection. We experienced 2 cases with severe upper back pain and paraplegia of the lower extremities as initial manifestations of acute DeBakey type IIIb aortic dissection (case 1) and aortic intramural hematoma (IMH) from ascending aorta to abdominal aorta (case 2). Paraplegia was permanent and spinal cord atrophy was proved at Th 9-10 by the magnetic resonance imaging (MRI) in case 1. The aortic IMH regressed rapidly and paraplegia was transient in case 2. The MRI obtained 18 days after the onset showed scattered small lesions within the spinal cord at Th 4-7.

Adult↗

PubMed2002

Effects of oestrogen deficiency on osteoclastogenesis in the rat periodontium.

This study was designed to investigate quantitative changes in osteoclast generation in rat periodontium following ovariectomy. Wistar rats, aged 9 weeks, were subjected to either ovariectomy (OVX) or sham surgery. Osmotic pumps were implanted in 24 rats and either 17beta-oestradiol or vehicle solution were infused continuously. The rats were assigned to one of the following groups: (1) OVX+vehicle; (2) sham+vehicle; or (3) OVX+ 17beta-oestradiol. On the days 7 and 14 after surgery, four rats in each group were killed. Mandibles were demineralized and embedded in paraffin. Frontal sections of alveolar bone in the region of the first molar were cut for enzyme histochemistry and immunohistochemistry. On day 7, there was no significant difference in the number of tartrate-resistant acid phosphatase (TRAP)-positive cells located on bone surfaces in either group. However, the number of TRAP-positive mononuclear cells that were separated from the bone surface was significantly higher in group 1 than in groups 2 and 3. On the day 14, the number of TRAP-positive cells in group 1, which were attached to the bone surface, was significantly higher than had been apparent on day 7. There were also significant increases in the number of nuclei of TRAP-positive cells attached to the bone in group 1 compared with groups 2 and 3 on day 14. These findings demonstrate that oestrogen deficiency induces of osteoclastogenesis in the rat periodontium and that quantitative changes in osteoclastogenesis could be prevented by E2 infusion.

Acid Phosphatase↗

PubMed2002

Immunolocalization of osteoclast differentiation factor in rat periodontium.

The aim here was to observe the immunohistochemical localization of osteoclast differentiation factor (ODF)/receptor activator of NF kappa B ligand (RANKL) in the rat periodontium. Rat mandibles were demineralized and embedded in paraffin, and horizontal and frontal sections were prepared for immunohistochemical analysis. In horizontal sections, immunolocalization of RANKL was marked in the distal area of the periodontium of molars in which osteoclasts appeared, due to physiological tooth drift. In frontal sections, RANKL immunoreactivity was localized on spindle-shaped mesenchymal cells around blood vessels near the bone surface in the periodontium. In addition, immunoreaction for RANKL was detected on structures that appeared to be elongated cell processes near blood vessels in frontal sections. Immunohistochemical examination for the general antigen of nerve-specific protein suggested a similarity between these structures and nerve fibres.

Acid Phosphatase↗

PubMed2002

Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis.

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. Although combining direct DNA injection with in vivo electroporation increases the efficiency of gene transfer into muscle, applications of this method have remained limited because of the relatively low expression level. To overcome this problem, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated viral interleukin 10 (vIL-10) expression in vivo. The fusion cytokine vIL-10/mutFc was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine suppressed the phytohemagglutinin-induced IFN-gamma production by human peripheral blood mononuclear cells and decreased the mortality in a mouse viral myocarditis model as effectively as vIL-10 expression. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein genes.

Animals↗

PubMed2002

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.

Allergens↗

PubMed2002

Expression profile of active genes in the human pituitary gland.

To characterize transcripts abundantly expressed in the human pituitary gland in general as well as to isolate novel transcripts expressed specifically in this gland, we generated an expression profile of the active genes transcribed in it. A total of 1015 randomly collected 3prime prime or minute expressed sequence tags (ESTs) (gene signatures, GSs) were grouped into 527GS species. The results showed the relative expression levels of genes in the pituitary gland. The genes comprising more than 1% of total mRNA were prolactin, growth hormone and chromogranin B genes. When known genes were categorized, the genes for pituitary hormones were the most actively transcribed, followed by the genes for ribosomal proteins, nuclear proteins and secretory granule proteins. Through comparison of this gene expression profile with the BodyMap database containing profiles generated from 63 other human tissues, we obtained 11 genes which appeared to be specifically expressed in the pituitary gland. In addition to the eight known genes, we identified three novel pituitary-specific transcripts which encode putative proteins: pituitary gland specific factor 1a (PGSF1a), PGSF1b and PGSF2. This expression profile method is a novel approach to the isolation of pituitary-specific genes that may have important functions.

Amino Acid Sequence↗

PubMed2002

[Surgical treatment of aneurysm of the ductus arteriosus in the adult].

Aneurysm of the ductus arteriosus is rare, however, reports on this lesion have increased recently with the progress of thoracic aortic surgery in Japan. We report 3 male cases aged 58, 59, 73 years. Aneurysms and total aortic arch were replaced by artificial graft through median sternotomy using selective cerebral perfusion in 2 cases. Through left posterolateral thoracotomy using deep hypothermia with circulatory arrest, proximal descending thoracic aorta including the aneurysm was replaced in 1 case. Their postoperative courses were uneventful. Surgery for this lesion is safe and various surgical approaches and circulatory supporting methods can be selected depending on the size of aneurysm and associated lesions.

Aged↗

PubMed2002

[Pericardiectomy for post-coronary artery bypass grafting constrictive pericarditis; strategy for safe and complete pericardiectomy].

Surgery for constrictive pericarditis after coronary artery bypass grafting (CABG) needs complete pericardiectomy without injury to bypass grafts. We performed pericardiectomy for post-CABG constrictive pericarditis 15 months after the first surgery. Preoperative multislice helical 3-dimensional computed tomography (CT) clearly demonstrated the patent bypass grafts and anatomical relationship between grafts and surrounding organs. Among surgical approaches, we chose bilateral thoracotomy to avoid injury to the bypass grafts and to obtain a good surgical exposure, especially for pericardiectomy of the left side of the heart. Additionally, with the use of intraoperative doppler ultrasound blood flowmetry, we could safely achieve complete pericardiectomy. We conclude that the combined application of 3-dimensional CT, bilateral thoracotomy and doppler ultrasound blood flowmetry was a supreme strategy for the operation of constrictive pericarditis after CABG.

Coronary Artery Bypass↗

PubMed2001

Transplacental genetic immunization after intravenous delivery of plasmid DNA to pregnant mice.

A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.

AIDS Vaccines↗

PubMed2001

A new IFN-like cytokine, limitin, modulates the immune response without influencing thymocyte development.

A novel IFN-like molecule, limitin, was recently identified and revealed to suppress B lymphopoiesis through the IFN-alphabeta receptor, although it lacked growth suppression on myeloid and erythroid progenitors. Here we have studied diverse effects of limitin on T lymphocytes and compared limitin with previously known IFNs. Like IFN-alpha and -beta, limitin modified immunity in the following responses. It suppressed mitogen- and Ag-induced T cell proliferation through inhibiting the responsiveness to exogenous IL-2 rather than suppressing the production of IL-2. In contrast, limitin enhanced cytotoxic T lymphocyte activity associated with the perforin-granzyme pathway. To evaluate the effect of limitin in vivo, a lethal graft-versus-host disease assay was established. Limitin-treatment of host mice resulted in the enhancement of graft-versus-host disease. Limitin did not influence thymocyte development either in fetal thymus organ cultures or in newborn mice injected with limitin-Ig, suggesting that limitin is distinguishable from IFN-alpha and -beta. From these findings, it can be speculated that the human homolog of limitin may be applicable for clinical usage because of its IFN-like activities with low adverse effects on, for example, T lymphopoiesis, erythropoiesis, and myelopoiesis.

Adjuvants, Immunologic↗

PubMed2001

TFEC can function as a transcriptional activator of the nonmuscle myosin II heavy chain-A gene in transfected cells.

Transcription of the human nonmuscle myosin II heavy chain-A (NMHC-A) gene is regulated via multiple elements located in intron 1, including element F which contains an E-box. In this study we have identified and characterized the factors that are capable of binding to element F. Yeast one-hybrid screening using element F allowed isolation of cDNAs encoding transcriptional factors TFEC, TFE3, and USF2, each of which contains basic helix-loop-helix and leucine zipper motifs. Furthermore, cDNA cloning by polymerase chain reaction yielded cDNAs for two TFEC isoforms, designated TFEC-l and TFEC-s, which are generated by alternative pre-mRNA splicing. In addition to these four factors, USF1, which is known to share the same DNA binding elements with USF2, was isolated for comparison. Electrophoretic mobility shift assays and cotransfection studies of the expression constructs with reporter gene constructs revealed that the above five factors have different binding activities for element F with different transactivation potencies. USF1 and USF2 demonstrate the highest binding activity to element F, yet show the lowest element F-dependent transactivation. TFE3 has a high transactivation potency but the lowest binding activity. TFEC-l demonstrates a high binding activity with the highest transactivation potency, whereas TFEC-s has the same binding activity as TFEC-l with intermediate transactivation. We also demonstrate that an N-terminal activation domain exists only in TFEC-l, whereas a C-terminal activation domain is common to both the l and s isoforms. This study provides the first evidence of TFEC being an activator of transcription, with two separate activation domains.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2001

Cytokine gene therapy for myocarditis by in vivo electroporation.

Cytokines are important pathophysiologic and pathogenic factors in cardiovascular disorders, including viral myocarditis. We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral IL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle. Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak. Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL1ra or vIL-10 expression plasmid had significantly improved the survival rates of the animals. The expression of TNF-alpha was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ra treatment, and the expression of IFN-gamma in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls. These results show that gene therapy with IL-1ra or vIL-10 expression plasmid was effective in the treatment of viral myocarditis, and in vivo electroporation may be a useful method by which to deliver cytokine therapy in cardiovascular diseases.

Animals↗

PubMed2001

Dynamic micro-magnetic resonance imaging of liver micrometastasis in mice with a novel liver macromolecular magnetic resonance contrast agent DAB-Am64-(1B4M-Gd)(64).

DAB-Am64-(1B4M-Gd)(64) is a newly synthesized macromolecular liver magnetic resonance imaging (MRI) contrast agent with a polypropylenimine diaminobutane (DAB) dendrimer conjugated with a bifunctional diethylenetriaminepentaacetic acid (DTPA) derivative for complexing Gd(III) atoms. The characteristics of DAB-Am64-(1B4M-Gd)(64), which quickly accumulated in the liver, have been reported recently. In the present study, the dynamic micro-MRI with DAB-Am64-(1B4M-Gd)(64) was obtained in the mouse liver metastasis model using colon carcinoma cells to evaluate the ability to visualize the micrometastatic tumors compared with that using Gd-DTPA. The dynamic micro-MRI with DAB-Am64-(1B4M-Gd)(64) was able to homogeneously enhance the normal liver parenchyma and visualize micrometastatic tumors of 0.3-mm diameter in the liver of the mice with better contrast than that with Gd-DTPA. In conclusion, DAB-Am64-(1B4M-Gd)(64) is a new liver MRI contrast agent potentially useful for diagnosis of micrometastasis in the liver.

Animals↗

PubMed2001

Isolation of novel heart-specific genes using the BodyMap database.

Two novel heart-specific genes, C3orf3 (chromosome 3 open reading frame 3) and MMGL (myomegalin-like), were isolated using BodyMap, a gene expression database based on site-directed 3' expressed sequence tags (3'-ESTs) which were collected from nonbiased cDNA libraries of various tissues. The cDNA of C3orf3 was 1667 bp and was composed of 12 exons within a 10-kb-long genomic sequence. MMGL consisted of 8 exons within a genomic sequence of over 70 kb, leading to four alternatively spliced transcripts. Both genes were strongly expressed in heart and also in skeletal muscle. C3orf3 and MMGL were mapped to 3p22 and 1q1, respectively. Subcellular localizations of their putative proteins were determined as being in the cytoplasm for C3orf3 and in the cytoplasm and nucleus for MMGL. This study showed that BodyMap is a useful database for the isolation of tissue-specific genes.

Adaptor Proteins, Signal Transducing↗

PubMed2001

BodyMap incorporated PCR-based expression profiling data and a gene ranking system.

BodyMap is a human and mouse gene expression database that is based on site-directed 3'-expressed sequence tags generated at Osaka University. To date, it contains more than 300 000 tag sequences from 64 human and 39 mouse tissues. For the recent release, the precise anatomical expression patterns for more than half of the human gene entries were generated by introduced amplified fragment length polymorphism (iAFLP), which is a PCR-based high-throughput expression profiling method. The iAFLP data incorporated into BodyMap describe the relative contents of more than 12 000 transcripts across 30 tissue RNAs. In addition, a newly developed gene ranking system helps users obtain lists of genes that have desired expression patterns according to their significance. BodyMap supports complete transfer of unique data sets and provides analysis that is accessible through the WWW at http://bodymap.ims.u-tokyo.ac. jp.

Animals↗