Search PubMed⌕ Search

Biomedical subjects

K Takehara

Publications and source records attributed to K Takehara.

At least 73 records · Page 4Linked to original sources
PubMed2000

Expression of a bioactive bovine interleukin-12 using baculovirus.

Recombinant baculoviruses that express recombinant bovine interleukin-12 (rboIL-12) subunits, p35 and p40 subunits were constructed. A recombinant virus containing the p40 subunit gene expressed the p40 subunit as a 40kDa monomer and an 80kDa disulfide-linked homodimer in the infected insect cells and in the culture supernatant. The p35 subunit was expressed in a 30kDa monomer in the infected cells but not in the supernatant. Superinfection of both recombinant viruses into the cells in a spinner flask resulted in the formation of a 70kDa disulfide-bonded heterodimer detected in the supernatant by immunoblotting using anti-p40 and anti-p35 subunits antibodies. The superinfected culture supernatant showed induction of IFNgamma mRNA synthesis and IFNgamma production in bovine peripheral blood mononuclear cells. Thus, the bioactive rboIL-12 was produced in large scale using a baculovirus expression system.

Animals↗
PubMed2000

Smad6 is a Smad1/5-induced smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter.

Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. We have isolated the mouse Smad6 promoter and identified the regions responsible for transcriptional activation by BMPs. The proximal BMP-responsive element (PBE) in the Smad6 promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for Drosophila Mad and Medea. We generated a luciferase reporter construct (3GC2-Lux) containing three repeats of the GC-rich sequence derived from the PBE. BMPs and BMP receptors induced transcriptional activation of 3GC2-Lux in various cell types, and this activation was enhanced by cotransfection of BMP-responsive Smads, i.e. Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE was observed. These results indicate that the expression of Smad6 is regulated by the effects of BMP-activated Smad1/5 on the Smad6 promoter.

Animals↗
PubMed2000

Clonal heterogeneity in sporadic melanomas as revealed by loss-of-heterozygosity analysis.

The major obstacle preventing effective treatment of melanoma is the biological heterogeneity of tumor cells. This study was performed to determine clonal genetic heterogeneity within primary melanoma and the evolution of these heterogeneous sub-clones during disease progression. DNA samples were obtained from 44 morphologically distinct areas identified within 10 primary tumors and from 15 metastases in the same patients. Loss of heterozygosity (LOH) analyses were performed using 17 microsatellite markers that mapped to chromosomes 6q, 9p, 10q and 18q, the most frequently deleted in melanoma. Of 10 primary tumors, 8 were revealed to have intratumoral genetic heterogeneity in terms of LOH of the 4 chromosome arms examined, 7 containing at least 2 different sub-clones harboring LOH of different chromosome areas, while the remaining one tumor showed prominent intratumoral genetic heterogeneity consisting of at least 6 genetically distinct sub-clones. LOH of 6q was detected only in a sub-set of multiple microdissected samples in most of the primary tumors, but was most frequently detected in metastases, suggesting that loss of this chromosome arm occurred late and played an important part in metastatic progression. Comparison of LOH between sub-clones within primary tumors and within metastases showed the divergence of metastatic clones from dominant populations within the primary tumor in 5 patients, whereas in the remaining three patients parent sub-clones were not identified, or constituted only a minor sub-population within the primary tumors. These results, showing considerable genetic heterogeneity in sporadic melanoma, have profound implications for the choice of future therapeutic strategies.

Adult↗
PubMed2000

Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression.

Inflammatory cells play a crucial role in wound healing, but the role of adhesion molecules including L-selectin and intercellular adhesion molecule-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisional wounds in mice lacking L-selectin, ICAM-1, or both. The loss of ICAM-1 inhibited wound healing, keratinocyte migration from the edges of the wound toward the center, and granulation tissue formation. By contrast, L-selectin deficiency alone did not affect any of these parameters. However, the loss of both L-selectin and ICAM-1 resulted in inhibition of keratinocyte migration and granulation tissue formation beyond those caused by loss of ICAM-1 alone. Treatment of platelet-derived growth factor to the wounds normalized delayed wound healing in ICAM-1(-/-) mice, but not in L-selectin/ICAM-1(-/-) mice. Therefore, although ICAM-1 contributes to wound repair to a greater extent than L-selectin, a role for L-selectin was revealed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiltration of neutrophils and macrophages in ICAM-1(-/-) and L-selectin/ICAM-1(-/-) mice. These results demonstrate a distinct role of ICAM-1 and L-selectin in wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site.

Animals↗
PubMed2000

Growth regulation of skin fibroblasts.

The growth of skin fibroblasts is regulated in a complex manner by various growth factors. Representative growth factors are platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-beta (TGF-beta), and connective tissue growth factor (CTGF). These growth factors have various biological activities besides growth regulation of skin fibroblasts, and are involved in wound healing and in the pathogenesis of various disorders. For example, PDGF and CTGF stimulate chemotaxis of skin fibroblasts, b-FGF stimulates angiogenesis, and TGF-beta stimulates production of matrix proteins. First, the properties of these growth factors are reviewed briefly. Our skin fibrosis model in newborn mice are also described here. In 1986, Roberts et al. reported that subcutaneous injection of TGF-beta in newborn mice caused granulation tissue formation followed by fibrosis (Roberts et al. Proc Natl Acad Sci USA 1986;83:4167-71). We conducted similar experiments, and found that TGF-beta1, beta2 or beta3 caused skin fibrosis after 3 consecutive days of injection; this change was transient and disappeared after 7 consecutive days of injection. In contrast, irreversible fibrosis was observed upon stimultaneous injection of TGF-beta and b-FGF or TGF-beta and CTGF, or TGF-beta injection for the first 3 days and b-FGF or CTGF injection for the next 4 days (Shinozaki et al. Biochem Biophys Res Commun 1997;237:292-7; Mori et al. J Cell Physiol 1999;181:153-9). These observations suggest that TGF-beta induces skin fibrosis and b-FGF or CTGF maintains it in various skin fibrotic disorders. In the 21st century, we speculate that cocktails of various growth factors may permit subtle growth regulation of skin fibroblasts; such technology would have applications in the treatment of many skin diseases.

Animals↗
PubMed2000

Genetic changes in sweat gland carcinomas.

The molecular pathogenesis of malignant appendageal tumors is poorly understood. Immunohistochemical staining, polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) and sequencing analyses were performed in a mixed group of 21 sweat gland carcinomas. LOH was mostly confined to the chromosome arm 17p. None of the remaining 17 tumors showed LOH at any loci. Nuclear accumulation of p53 protein was observed in 3 tumors, all of which also showed LOH of 17p. One eccrine gland adenocarcinoma showed allelic loss of 17p and a Cys 176 Arg mutation in the p53 gene. The other three tumors that showed LOH of 17p, however, had wild-type p53 genes. A clear transition from benign eccrine poroma to porocarcinoma that was associated with p53 protein stabilization and allelic loss was observed in one tumor. One eccrine porocarcinoma/undifferentiated adnexal carcinoma showed prominent microsatellite instability, probably reflecting an underlying defect in DNA mismatch repair. Overexpression of erbB-2 was observed in three tumors. The low frequencies of LOH and p53 alterations in sweat gland carcinomas contrasted with the multiple genetic defects normally observed in cutaneous squamous cell carcinomas, and may be partly explained by the relative protection of cutaneous appendages from ultraviolet light and other environmental mutagens.

Adenocarcinoma↗
PubMed2000

Expression of structurally unaltered androgen receptor in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is an uncommon neoplasm of the skin that shows differentiation to an apocrine sweat gland. Although we previously showed that erbB-2 overexpression may play a part in the progression of EMPD, molecular genetic defects underlying the development of EMPD are poorly understood. In the study described here, we examined androgen receptor expression and gene alterations in 30 cases of EMPD without internal malignancy. Immunohistochemistry revealed that 24 of 30 (80%) cases of EMPD variably expressed nuclear androgen receptor. Semi-quantitation of receptor content by scoring immunostained sections showed no difference between in situ (n = 17) and invasive (n = 13) EMPD tumors. Androgen receptor expression was also observed in four of six lymph node metastases. In these lymph nodes, expression of androgen receptor mRNA was confirmed by reverse transcriptase-polymerase chain reaction. Direct sequencing of exon 2 through exon 8, which encodes DNA- and hormone-binding domains of the androgen receptor gene, revealed no mutation in any of the 10 advanced stage tumors. Neither amplification nor deletion of the androgen receptor gene locus was detected by dual color fluorescence in situ hybridization analysis in 14 tumors. The present findings showing frequent expression of structurally unaltered androgen receptor in an advanced stage of EMPD may provide a rational basis for hormone therapy, which is widely used in the treatment of metastatic prostate cancer and androgen receptor-positive breast cancer recurrence.

Aged↗
PubMed2000

Hereditary non-polyposis colorectal cancer associated with disseminated superficial porokeratosis. Microsatellite instability in skin tumours.

A 73-year-old man presented with typical lesions of disseminated superficial porokeratosis (DSP) and multiple seborrhoeic keratoses on his face, trunk and extremities, and later developed a keratoacanthoma on his lip. He belonged to a cancer-prone pedigree susceptible to colonic, uterine and other internal cancers, and had a personal history of early gastric cancer and advanced adenocarcinoma of the descending colon without adenomatous polyps at age 59 years. Polymerase chain reaction amplification of skin samples for seven separate microsatellite polymorphisms revealed microsatellite instability (MSI) at multiple loci in five of six seborrhoeic keratoses and the keratoacanthoma, strongly suggesting underlying defects in DNA mismatch repair. Although no germline mutations in two mismatch repair genes hMSH2 and hMLH1 were found, our patient was recognized as having hereditary non-polyposis colorectal cancer (HNPCC) based on the family history and the findings of the microsatellite analysis of skin tumours. This confirmed the usefulness of detection of MSI in prevalent and readily accessible skin lesions, including non-sebaceous non-dysplastic tumours such as seborrhoeic keratosis in the screening of HNPCC families. Although DSP may also be inherited as an autosomal dominant condition, this particular skin disease appeared to be sporadic in our patient and, to our knowledge, no association of DSP or other forms of porokeratosis with HNPCC has previously been reported. In contrast to the seborrhoeic keratoses and keratoacanthoma, no MSI was observed in two samples from DSP lesional epidermis examined.

Adult↗
PubMed2000

Characterization of autoantibodies to endothelial cells in systemic sclerosis (SSc): association with pulmonary fibrosis.

To determine the prevalence and the characterization of antibodies to endothelial cells in patients with SSc, serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy control subjects were examined by ELISA using cultured human umbilical vein endothelial cells (HUVEC), indirect immunofluorescence analysis (IIF), and immunoblotting using cytoplasmic extract of HUVEC. IgG and/or IgM isotype anti-endothelial cell antibodies (AECA) were demonstrated by ELISA in 43 of 80 patients with SSc (54%), in 15 of 20 patients with SLE (75%), and in none of 20 healthy control subjects. Immunofluorescence analysis on HUVEC substrate showed homogeneous cytoplasmic staining. Immunoblotting demonstrated that these patients had antibodies directed to one or several antigens of approximately 60, 90, 110 and 140 kD, and the most common responses were to the 90-kD antigen. By the immunofluorescence method using HUVEC, affinity-purified anti-90-kD antibodies showed identical cytoplasmic staining to that produced by sera positive for AECA. Furthermore, AECA were closely correlated with pulmonary fibrosis in patients with SSc. These findings suggest that patients with SSc have abnormal antibodies to endothelial cell antigens, and support the hypothesis that endothelial dysfunction is involved in the development of this disease.

Adult↗
PubMed2000

Decreased expression levels of L-selectin on subsets of leucocytes and increased serum L-selectin in severe psoriasis.

L-selectin is a leucocyte adhesion molecule involved in leucocyte interactions with vascular endothelial cells. Following leucocyte activation L-selectin is endoproteolytically released from the cell surface. To assess whether psoriasis vulgaris results in systemic leucocyte activation, we examined expression levels of L-selectin on subsets of peripheral blood leucocytes from patients with psoriasis (n = 25) and normal control subjects. Serum levels of soluble L-selectin were quantified by ELISA in patients with psoriasis (n = 75), pustulosis palmaris et plantaris, and contact dermatitis, as well as normal control subjects. Psoriasis severity was evaluated by psoriasis area and severity index (PASI). L-selectin expression levels on CD4+ T cells, B cells, monocytes, and neutrophils from patients with severe-type psoriasis (PASI > or = 15) was significantly decreased compared with leucocytes from normal control subjects. Furthermore, L-selectin expression on CD4+ T cells showed good inverse correlation with PASI scores. Monocyte L-selectin expression was restored when the skin lesions of psoriasis were remitted. The frequencies of L-selectin+ CD4+ T cells or L-selectin+ CD8+ T cells from patients with psoriasis were almost normal. Serum L-selectin levels in patients with severe-type psoriasis were significantly higher than those in normal control subjects. These results suggest that subsets of leucocytes may be activated in psoriasis, and that L-selectin expression levels on some leucocyte subsets, especially CD4+ T cells, tend to correlate with disease severity of psoriasis.

Adult↗
PubMed2000

Impaired collagen gel contraction with cultured skin fibroblasts from patients with systemic sclerosis.

We investigated the capacity of skin fibroblasts, derived from 9 patients with systemic sclerosis (SSc), to contract collagen lattices in a three-dimensional culture system. In comparison with control fibroblasts (N = 8), more than 30% of SSc fibroblasts exhibited markedly impaired ability to contract collagen lattices. The expression of alpha2beta1 integrins and integrin-mediated signals were not significantly different between normal and SSc fibroblasts. Although the underlying mechanisms remain to be determined, our present data provide evidence that certain aspects of interaction with collagen are impaired in SSc fibroblasts.

Adolescent↗
PubMed2000

Elevated serum KL-6 levels in patients with systemic sclerosis: association with the severity of pulmonary fibrosis.

BACKGROUND: Serum KL-6 has been suggested to be a useful marker for the evaluation of interstitial lung disease activity. OBJECTIVE: To determine the correlation between serum KL-6 levels and pulmonary fibrosis in patients with systemic sclerosis (SSc). METHODS: Serum samples from patients with limited cutaneous SSc (lSSc; n = 19), diffuse cutaneous SSc (dSSc; n = 26) and normal individuals (n = 15) were examined by ELISA. RESULTS: Serum KL-6 levels in SSc patients were significantly higher than those in normal controls. KL-6 levels in dSSc patients were significantly elevated compared with those in lSSc patients. Elevated KL-6 levels were associated with the presence of pulmonary fibrosis in SSc patients or dSSc patients. Furthermore, KL-6 levels inversely correlated with percentages of diffusion capacity of carbon monoxide and vital capacity in SSc patients or dSSc patients. CONCLUSION: KL-6 may be a simple, serologic indicator for the severity of pulmonary fibrosis in SSc.

Adult↗
PubMed2000

Subsite structure and catalytic mechanism of a new glycosyltrehalose-producing enzyme isolated from the hyperthermophilic archaeum, Sulfolobus solfataricus KM1.

A glycosyltrehalose-producing enzyme from Sulfolobus solfataricus KM1 catalyzes a conversion of maltooligosaccharides to glycosyltrehaloses and also hydrolyzes maltooligosaccharides to liberate glucose, as a side reaction. From the sum of the conversion and hydrolysis reaction rates, the rate parameters involved in the "splitting" of the alpha-1,4 glucosidic linkage were calculated. From the data obtained, the subsite structure for maltooligosaccharides was identified. From the analysis of the hydrolysate of maltotriose in [18O labeled H2O, the hypothesis of the C1-O bond splitting and the formation of a glycosyl (maltosyl)-enzyme intermediate was strongly supported. From the analysis of the reaction product in the presence of [3H] labeled glucose, the occurrence of intermolecular transglycosylation was confirmed. These data strongly support the suggestion that the catalytic mechanism of this enzyme is a transglycosylation.

Catalysis↗
PubMed2000

Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis.

OBJECTIVE: To determine the serum levels and clinical correlation of connective tissue growth factors (CTGF) in patients with systemic sclerosis (SSc). METHODS: Serum samples from patients with limited cutaneous SSc (lSSc, n = 32), diffuse cutaneous SSc (dSSc, n = 28), systemic lupus erythematosus (SLE, n = 30), polymyositis/dermatomyositis (PM/DM, n = 20), and healthy control subjects (n = 30) were examined by ELISA for detection of CTGF. RESULTS: Serum CTGF levels in patients with SSc were significantly higher than those in patients with SLE or PM/DM, and in controls. CTGF levels in patients with dSSc were significantly higher than those in patients with lSSc. As for clinical correlation of CTGF, SSc patients with elevated CTGF had pulmonary fibrosis, decreased DLCO, and decreased vital capacity more frequently than those with normal CTGF levels. Further, DLCO and vital capacity were inversely and directly correlated with serum CTGF levels in patients with SSc. The dSSc patients with disease duration of 1-3 years had significantly elevated levels of CTGF compared with dSSc patients with duration < 1 year or more than 3 years. CONCLUSION: Serum CTGF levels were increased in patients with SSc, and correlated with the extent of skin sclerosis and the severity of pulmonary fibrosis. In addition, it appears that production of CTGF is involved in the development or maintenance of fibrosis rather than in initiation of fibrosis in SSc. These data suggest that CTGF plays a critical role in the development of fibrosis in SSc.

Adolescent↗
PubMed2000

Quantitative blood velocity mapping in glomerular capillaries by in vivo observation with an intravital videomicroscope.

We have evaluated rat glomerular microcirculation under in-vivo condition with an intravital videomicroscope. To measure erythrocyte velocities, a line segment was set along the glomerular capillaries in time-sequential videotaped images and then a spatiotemporal image was constructed along that segment. The angle of striped pattern in the spatiotemporal image which reflects the erythrocyte velocity, was estimated to compute erythrocyte velocity vector mapping. We found that erythrocyte velocities in rat glomeruli were significantly faster in diabetic rats than in control rats (p < 0.05). In conclusion, we have succeeded in quantitatively evaluating the in-vivo renal microcirculation with our videomicroscope system and by spatiotemporal image analyzing method.

Animals↗
PubMed2000

Serum levels of soluble interleukin 6 receptor and soluble gp130 are elevated in patients with localized scleroderma.

OBJECTIVE: To investigate the clinical relevance of serum soluble interleukin 6 receptor (sIL-6R) and soluble gp130 (sgp130) in localized scleroderma. METHODS: Serum levels of sIL-6R and sgp130 were examined by ELISA in 45 patients with localized scleroderma. Twenty patients with systemic sclerosis (SSc) and 20 healthy individuals served as controls. RESULTS: Serum levels of both sIL-6R and sgp130 were significantly elevated in patients with localized scleroderma compared with healthy controls. Moreover, serum sgp130 levels in patients with localized scleroderma were significantly higher than in patients with SSc. In patients with localized scleroderma, elevated sIL-6R levels significantly correlated with levels of IgM antihistone antibodies, the presence of rheumatoid factor, the number of linear lesions, and the number of body areas involved. Elevated sgp130 levels were significantly associated with levels of IgG antihistone antibodies, the number of plaque lesions, the total number of lesions, and the number of body areas involved. CONCLUSION: These results suggest that elevated serum sIL-6R and sgp 30 may reflect activation of the IL-6 system that may be associated with the development of sclerotic lesions and autoantibody production in localized scleroderma.

Adolescent↗
PubMed2000

Levels of interleukin 12, a cytokine of type 1 helper T cells, are elevated in sera from patients with systemic sclerosis.

OBJECTIVE: To determine serum levels and spontaneous production by peripheral blood mononuclear cells (PBMC) of interleukin 12 (IL-12), a potent inducer of type 1 helper (Th1) T cells, in patients with systemic sclerosis (SSc). METHODS: Serum IL-12 levels and spontaneous production levels of IL-12 in culture supernatants from PBMC were examined by ELISA. Serum levels of IL-4, IL-6. IL-10, and IL-13 and production levels by PBMC of IL-6 and IL-10 were also examined by ELISA. Renal vascular damage was determined as a pulsatility index (PI) by color flow Doppler ultrasonography of kidneys. RESULTS: Serum IL-12 levels were significantly elevated in patients with SSc (n = 62) compared with healthy controls (n = 20). Similarly, spontaneous production levels of IL-12 by PBMC in patients with SSc (n = 47) were higher than those in controls (n = 20). Serum IL-12 levels did not correlate with serum levels of any Th2-type cytokines such as IL-4, IL-6, IL-10, and IL-13. However, spontaneous production levels of IL-10 by PBMC significantly correlated with serum IL-12 levels in patients. Patients with elevated serum IL-12 levels had the increased PI values more frequently than those with normal IL-12 levels. Further, serum levels of IL-12 and production levels of IL-12 by PBMC correlated significantly with the PI values in patients with SSc. CONCLUSION: These results suggest that the increased levels of IL-12 may relate to the activation of Th1 cells in SSc and that IL- 12 overproduction may be associated with renal vascular damage.

Adolescent↗