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Biomedical subjects

M Kusakabe

Publications and source records attributed to M Kusakabe.

At least 19 recordsLinked to original sources
PubMed2006

Volumetric coronary angiography using the 256-detector row computed tomography scanner: comparison in vivo and in vitro with porcine models.

PURPOSE: To investigate the feasibility of volumetric cine imaging in human cardiac studies by comparing in vivo and in vitro coronary angiography using a 256-detector row computed tomography (CT) without ECG gating. MATERIAL AND METHODS: The left and right coronary arteries of two domestic pigs were scanned in vivo and in vitro in cine mode using the 256-detector row CT. The device scanned approximately 100 mm in the cranio-caudal direction with one rotation, with a slice thickness of 0.5 mm. RESULTS: The coronary arteries could be observed to the third-degree branches in vitro, but could be visualized clearly only to the proximal portion (first-degree or second-degree branches) in vivo. CONCLUSION: Application of cardiac volumetric cine imaging with 256-detector row CT may be a promising means of obtaining diagnostic information and has potential for adoption to human studies.

Animals↗
PubMed2006

Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes.

In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase, cytochrome P450 17alpha-hydroxylase/17,20-lyase, cytochrome P450 11beta-hydroxylase, 11beta-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17alpha-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17alpha-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Xenopus FRS2 is involved in early embryogenesis in cooperation with the Src family kinase Laloo.

FRS2 has been identified in mammalian cells as a protein that is tyrosine phosphorylated and binds to Grb2 and Shp2 in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. But neither its existence in other vertebrate classes or invertebrates nor its function during embryonic development has been defined. Here we have identified and characterized a Xenopus homolog of FRS2 (xFRS2). xFRS2 is tyrosine phosphorylated in early embryos, and overexpression of an unphosphorylatable form of xFRS2 interferes with FGF-dependent mesoderm formation. The Src family kinase Laloo, which was shown to function in FGF signaling during early Xenopus development, binds to xFRS2 and promotes tyrosine phosphorylation of xFRS2. Moreover, xFRS2 and Laloo are shown to bind to Xenopus FGF receptor 1. These results suggest that xFRS2 plays an important role in FGF signaling in cooperation with Laloo during embryonic development.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Animals↗
PubMed2001

Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene.

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.

Animals↗
PubMed2001

Immunohistochemical characterization of hepatoblastomas in B6C3F1 mice treated with diethylnitrosamine and sodium phenobarbital.

Hepatoblastomas (HBs) were induced in B6C3F1 male mice by diethylnitrosamine (DEN) and sodium phenobarbital (PB). Six-week-old mice received a single intraperitoneal dose of DEN followed by a continuous treatment with PB in diet at a concentration of 0 (group 1) or 500 (group 2) ppm for 50 weeks. HBs were observed in 13 of 21 (62%) group 2 mice, with typical histologic features as reported previously, while no such tumors were observed in group 1. Seven of 13 (54%) HBs were found in and/or adjacent to hepatocellular adenomas (HCAs) or hepatocellular carcinomas (HCCs). Immunohistochemically, all HBs were positive for S-100 protein but negative for keratin, alpha-fetoprotein (AFP), albumin (ALB) and vimentin, while HCC cells occasionally reacted positively for AFP with a mosaic pattern. HCC and HCA cells were occasionally positive for ALB. Non-neoplastic hepatocytes and normal bile ducts were positively stained for ALB and keratin/S-100 protein, respectively. S-100 protein is known to be expressed in many mesenchymal tissues and neoplasms including neuroectodermal elements but negative in cells of the hepatic lineage. Thus, the present immunohistochemical results suggested that mesenchymal differentiation occurs in mouse HB cells as observed in human HBs, one of the most frequent infant liver tumors in humans. Although the susceptibility of mouse HBs to PB-promotion suggests a hepatocytic histogenesis, the present immunohistochemical results support the hypothesis that the mouse HB is derived from pluripotent endodermal stem-like cells.

Albumins↗
PubMed2001

[A case of multiple liver metastases from sigmoid colon cancer effectively treated by hepatic intra-arterial administration of levoforinate and 5-fluorouracil].

A 62-year-old female patient, who was diagnosed with sigmoid colon cancer with multiple liver metastases, was admitted to our hospital. She underwent sigmoidectomy with D3 lymph node dissection on January 31, 2000. In addition to that, she received hepatic intra-arterial infusion of levoforinate (l-LV) 250 mg and 5-fluorouracil (5-FU) 500 mg for combined multiple hepatic metastases starting on postoperative day 14, and these medications were administered over 48 hours once weekly by infuser pump. The tumor diminished by 59% 2 months after the start of administration and further diminished at 4 months. PR was achieved. Cancer metastasis to the cerebellum and metastasis to the lung were detected at month 9 and month 11, respectively, but the liver metastatic tumor continued to diminish in size, ultimately becoming undetectable by CT scan at month 10. Surgery and radiotherapy were performed for the cerebellar metastasis, and intravenous administration of a combination of l-LV and 5-FU was performed systemically for the pulmonary metastatic tumor. At present, the patient receives regular outpatient treatment continuously. To our knowledge, there has been no report on the combination therapy with l-LV and 5-FU through the hepatic artery. Since good antitumor efficacy was demonstrated in the present patient, this case is described in this report together with four other cases of hepatic metastasis from colorectal cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Mutation induction by heavy-ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

Animals↗
PubMed2000

Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation.

Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.

3T3 Cells↗
PubMed2000

Tumor formation suppressed in gammadeltaT knock-out mice.

Tumor formation was examined in mice in which the beta-chain gene of alphabetaT was knocked out (alphabetaT-KO mice) or the delta-chain gene of gammadeltaT was knocked out (gammadeltaT-KO mice). Development of Hepa 1-6 cell hepatoma was observed in six of six alphabetaT-KO mice, in four of six wild-type mice, and in only one of six gammadeltaT-KO mice. These results imply that gammadeltaT cells play a role in suppression of killer cell activity in tumor-bearing mice.

Animals↗
PubMed2000

The cell cycle control gene ZAC/PLAGL1 is imprinted--a strong candidate gene for transient neonatal diabetes.

We describe a screen for new imprinted human genes, and the identification in this way of ZAC (zinc finger protein which regulates apoptosis and cell cycle arrest)/ PLAGL1 (pleomorphicadenoma of the salivary gland gene like 1) as a strong candidate gene for transient neonatal diabetes mellitus (TNDM). To screen for imprinted genes, we compared parthenogenetic DNA from the chimeric patient FD and androgenetic DNA from hydatidiform mole, using restriction landmark genome scanning for methylation. This resulted in identification of two novel imprinted loci, one of which (NV149) we mapped to the TNDM region of 6q24. From analysis of the corresponding genomic region, it was determined that NV149 lies approximately 60 kb upstream of the ZAC / PLAGL1 gene. RT-PCR analysis was used to confirm that this ZAC / PLAGL1 is expressed only from the paternal allele in a variety of tissues. TNDM is known to result from upregulation of a paternally expressed gene on chromosome 6q24. The paternal expression, map position and known biological properties of ZAC / PLAGL1 make it highly likely that it is the TNDM gene. In particular, ZAC / PLAGL1 is a transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide, which is the most potent known insulin secretagog and an important mediator of autocrine control of insulin secretion in the pancreatic islet.

Cell Cycle↗
PubMed2000

Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M).

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.

Animals↗
PubMed2000

Internalization and down-regulation of muscarinic acetylcholine receptors in cerebellar granule cells of tenascin-gene deficient mice.

The expression of tenascin-C on oligodendrocytes parallels the migration of granule cells in the developing cerebellum, indicating a role for tenascin-C as a guide for granule neurons to find their proper locations. In this study, cultured cerebellar granule neurons from tenascin-C-knockout mice were used to examine the role of tenascin-C in agonist-induced muscarinic acetylcholine receptor down-regulation. Exposure of granule cells from wild-type or tenascin-C-negative mice to the muscarinic acetylcholine receptor agonist carbachol (1 mM) resulted in normal sequestration of cell-surface muscarinic acetylcholine receptors as assessed by [3H]N-methylscopolamine binding; however, down-regulation of total muscarinic acetylcholine receptors, measured with [3H]quinuclidinyl benzilate, was inhibited in granule cells from tenascin-C-negative mice. Remarkably, incubation of the tenascin-C-negative cells with the microtubule stabilizer taxol (10 microM) restored down-regulation of total muscarinic acetylcholine receptors to normal levels. We speculate that agonist-induced down-regulation of muscarinic acetylcholine receptors is functionally associated with tenascin-C-regulated microtubule structures in the developing cerebellum.

Animals↗
PubMed2000

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells.

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.

3T3 Cells↗
PubMed2000

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Amino Acid Sequence↗
PubMed2000

Postoperative delirium in patients with chronic lower limb ischaemia: what are the specific markers?

OBJECTIVES: we determined the incidence and specific markers of postoperative delirium in elderly patients with chronic lower limb ischaemia. PATIENTS AND METHODS: since April, 1995, 110 patients aged 60 years or older (mean: 71.6+/-6.6 years) who underwent bypass surgery were assessed regarding aetiological factors of delirium: age, sex, dementia, body-mass index, hypertension, diabetes, cerebral disease, laboratory test results, severity of limb ischaemia, type of arterial occlusion, operative time, and blood transfusion. RESULTS: discriminant analysis showed statistical significance in the following five variables: age >/=70 years; critical limb ischaemia (and/or ankle pressure <40 mmHg); dementia; duration of operation >/=7 hours; low serum albumin. The overall percentage of cases correctly classified was 78.2% (Wilks>> Lambda=0.695, p<0.001); the standardized regression coefficients of the five variables were 0.648, 0.500, 0.329, 0.218, and 0.200, respectively. In logistic regression, the regression coefficients for old age and critical limb ischaemia were 2.646 (14.1 of odds ratio; 95% confidence interval, 2.7-72.0) and 1.337 (3.8; 1.3-10.9), respectively. CONCLUSIONS: the incidence of postoperative delirium in elderly patients with chronic lower-limb ischaemia was as high as 42.3%, and an age of over 70 years and critical limb ischaemia were identified as specific markers, with 14.1 times and 3.8 times the odds of suffering from delirium after bypass surgery.

Age Distribution↗
PubMed2000

Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver.

Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.

Animals↗