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Biomedical subjects

K Hanada

Publications and source records attributed to K Hanada.

At least 181 records · Page 10Linked to original sources
PubMed1996

Characterization of single chain urokinase-type plasminogen activator with a novel amino-acid substitution in the kringle structure.

ECV304 is a cell line established by a spontaneous transformation of endothelial cells of a human umbilical vein. It was shown that ECV304 secretes single chain urokinase-type plasminogen activator (scu-PA). A subclone, ECV304 clone 15, was obtained by acclimatization of parental clone to serum-free medium followed by limiting dilution. The clone was found to produce approximately five times as much scu-PA (approximately 20 IU/10(6) cells per day) as the parental clone after a 40 days' culture. Though the biochemical characteristics of the purified scu-PA were indistinguishable from those of the native scu-PA, it had a lower affinity for fibrin clots under the employed conditions. Molecular cloning of a cDNA encoding the scu-PA has identified a novel substitution from C to T in the nucleotide sequence encoding the kringle structure. The substitution resulted in an alteration from Pro (CCG) to Leu (CTG) at amino-acid position 121, which may be directly or indirectly involved in the decrease in the apparent affinity.

Amino Acid Sequence↗
PubMed1996

K-ras and p53 mutations in stage I gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct.

BACKGROUND: An anomalous junction of the pancreaticobiliary duct (AJPBD) was thought to be an important risk factor for gallbladder carcinoma in Japan. In this report, we compared K-ras and p53 mutations in Stage I gallbladder carcinomas (GC) of patients with AJPBD with those in patients without AJPBD: METHODS: We examined 6 GC of patients with AJPBD and 20 GC of patients without AJPBD: Immunohistochemistry was performed for p53 protein. K-ras and p53 mutations were examined using genomic DNA extracted from the cancer regions. The methods of polymerase chain reaction (PCR) single strand conformation polymorphism analysis were performed for mutations in exons 5-8 of p53. The methods of PCR restriction fragment length polymorphism were performed for mutation in codon 12 of K-ras. RESULTS: p53 positivity was 67% in GC of patients with AJPBD and 65% in GC of patients without AJPBD: p53 mutations were found in exons 7 and 8 in GC of patients with AJPBD and in exons 5, 6, and 7 in GC of patients without AJPBD: The incidence of K-ras mutation in GC of patients with AJPBD (50%) was greater than that in patients without AJPBD (6%) (P < 0.05). CONCLUSIONS: These results suggest that K-ras mutation may be important in the early stage of carcinogenesis of the gallbladder mucosa with AJPBD, and that p53 mutations may also contribute to the early stage of carcinogenesis of the gallbladder mucosa, regardless of AJPBD:

Adenocarcinoma↗
PubMed1996

Expression of cripto in human gall bladder lesions.

The expression of cripto, a member of the epidermal growth factor (EGF) family, was examined by immunohistochemistry in benign lesions and carcinomas of the gall bladder. Cripto expression was detected in 6 (67 percent) of 9 hyperplasias, 4 (58 percent) of 7 adenomas, and 89 (68 percent) of 132 adenocarcinomas of the gall bladder. The degree of cripto expression was not correlated with depth of tumour invasion, tumour stage or patient prognosis. The incidence of cases with cripto expression was significantly higher in papillary and well-differentiated adenocarcinomas (positive 73 percent; strongly positive 38 percent) than in moderately and poorly differentiated adenocarcinomas (positive 54 percent; strongly positive 17 percent) (P < 0.05). These results suggest that cripto expression may not relate to progression in gall bladder carcinomas, but may be associated with tumour differentiation.

Adenocarcinoma↗
PubMed1996

GM-CSF-induced in vivo expansion of splenic dendritic cells and their strong costimulation activity.

Dendritic cells (DC), with their potent antigen-presenting and accessory activities, are involved in the stimulation of naive T cells. To examine the biological functions of DC, we developed a method for generating large numbers of murine splenic DC. First, DC were propagated in vivo by using a granulocyte-macrophage colony-stimulating factor-secreting tumor as a continuous in vivo source of the cytokine. The DC enriched in the spleen, especially in the white pulps, were purified after an overnight culture. We could reproducibly obtain 6 to 10 X 10(6) splenic DC per mouse. These DC were morphologically similar to interdigitating cells, expressed high levels of MHC class II and costimulatory molecules, and were highly allo-stimulatory in mixed lymphocyte reactions. Further analysis on T cell stimulation activity revealed that the DC had strong costimulatory activity on human T cells. Activated B cells, which express both B7-1 and B7-2, had little T cell costimulatory activity under the same assay conditions. A human histiocytic leukemia cell line, U937, that showed only weak costimulatory activity by itself, worked synergistically with DC and further intensified the T cell stimulation by DC. These findings suggest the presence of a T cell costimulation mechanism in DC, which is activated synergistically by monocytes or macrophages, and deserves further study.

Animals↗
PubMed1996

Changes in markers, receptors and adhesion molecules expressed on murine hemopoietic stem cells after a single injection of 5-fluorouracil.

Cytokines play a crucial role in the differentiation and proliferation of hemopoietic cells, and it has recently been found that adhesion molecules play crucial roles not only in differentiation and proliferation, but also in the homing and other functions of hemopoietic cells. We have very recently established a new method for purifying pluripotent hemopoietic stem cells (P-HSC) in mice by injecting 5-fluorouracil (5-FU). The P-HSC were found to be low-density, lineage marker-negative (Lin-), CD71- and major histocompatibility complex class I(high). In the present study, we analyze changes in the expression of various HSC markers (Sca-1 and CD34), receptors (c-kit and interleukin-6 receptor [IL-6R]) and adhesion molecules (very late activation antigen-4 [VLA-4], lymphocyte function-associated antigen-1 [LFA-1], and CD44) after 5-FU injection. The percentage of Sca-1+ cells increases after 5-FU treatment, reaching a maximum on day 3, whereas the percentage of IL-6R+ cells decreases, reaching a minimum on day 3. The percentage of CD34+ cells does not change after 5-FU treatment. The percentages of both c-kit(low) and c-kit(high) cells decrease, reaching a minimum on day 3 after 5-FU treatment, whereas the percentage of c-kit- cells reciprocally increases, reaching a maximum on day 3. However, there is no change in the expression of adhesion molecules (VLA-4, LFA-1 and CD44) on the P-HSC.

Animals↗
PubMed1996

Local application of basic fibroblast growth factor into the bone increases bone mass at the applied site in rabbits.

The effect of basic fibroblast growth factor (bFGF) applied locally into the bone under physiological conditions was investigated. An aqueous solution containing 0 microgram (vehicle), 100 micrograms or 400 micrograms recombinant human bFGF was percutaneously applied through a needle into the right ilium in rabbit, and the ilia were harvested 4 weeks after the application. Compared with vehicle-treated animals, bone mineral density measured by dualenergy X-ray increased in the 400 micrograms bFGF group. The width of trabeculae in the bFGF-treated groups was greater than in the vehicle group. These results showed that bFGF applied locally into the bone under physiological conditions affected bone formation, and suggested that such an application might have potential for increasing bone.

Absorptiometry, Photon↗
PubMed1996

The study of ultraviolet B-induced apoptosis in cultured mouse keratinocytes and in mouse skin.

The responsibility of ultraviolet B (UVB) radiation for the induction of apoptosis in epidermal cells in vitro and in vivo was examined. Using cultured mouse keratinocytes, PAM212 cells, the morphological development of apoptotic cells (AC) after UVB irradiation was observed, and their DNA status was also examined. In addition, histochemical analysis was performed to establish whether the UVB-mediated sunburn cells (SBC) were AC or not. The cultured cells exposed to UVB showed the morphological characteristics of AC, and the electrophoresis of DNA isolated from these cells showed characteristic fragmentation, i.e. 'DNA ladder'. DNA fragmentation was detectable with UVB doses of more than 50 mJ/cm2, and it appeared 12 h after irradiation, indicating endonuclease-mediated DNA damage. In vivo experimentation using the TdT-mediated dUTP-biotin nick end labeling method (TUNEL) for detection of AC showed scattered positive cells in the basal layer of the UVB-irradiated mouse ear skin. The distributed pattern of the TUNEL-positive cells was similar to that of SBCs. These findings suggest that UVB is a causative factor of apoptosis in the epidermal cells, and that SBC is formed as a result of the apoptosis.

Animals↗
PubMed1996

Successful treatment of mucosal melanosis of the lip with normal pulsed ruby laser.

Melanosis of the lip is not rare in Orientals. Although it causes cosmetic problems, treatment of this pigmentary disorder is often neglected because a satisfactory tool for complete removal of mucosal melanosis has not yet been developed. The pulsed ruby laser, which has a wavelength of 694 nm and a short pulse duration of 1-2 msec, selectively damages pigmented cells in the skin. In this study, six Japanese patients with labial melanosis, manifested as labial lentigo and Peutz-Jeghers syndrome, were successfully treated with the pulsed ruby laser. The therapy achieved rapid results without producing changes in mucosal texture or recurrence after operation.

Adolescent↗
PubMed1996

The effect of cold stress on UVB injury in mouse skin and cultured keratinocytes.

The effect of cold stress on skin damage caused by UVB irradiation was investigated both in vivo and in vitro. Ear skin of mice that had been exposed to cold stress at 0 degree C for 20 min and at 5 degrees C for 24 h was exposed to UVB radiation. Sunburn cell production was less in mice exposed to the lower temperature. In addition, the effect of cold stress on the survival rate of UVB-irradiated rat keratinocytes was examined in a cytotoxicity test, with the results showing that keratinocytes exposed to cold stress of 0 degree C had a higher survival rate than control cells. To pursue a promising clue for explaining the result, we examined metallothionein (MT) production in rat keratinocytes that had been exposed to cold stress at 0 degree C. Microfluorometric quantification showed a positive correlation between the time course and the intensity of immunofluorescence for MT, indicating that the molecule is inducible by exposure to cold stress in our experimental system. These results suggest that epidermal cells that have been exposed to cold stress maintain a higher resistance to UV radiation than nonexposed controls in vivo and in vitro, and that MT with radical-scavenging activity might contribute, at least in part, to photoprotection against UVB-induced oxidative damage in mammalian skin.

Animals↗
PubMed1996

A new isopatulin derivative pintulin produced by Penicillium vulpinum F-4148 taxonomy, isolation, physico-chemical properties, structure and biological properties.

During our screening program of natural products from fungal metabolites for drugs effective against tumor cell lines, we discovered a new isopatulin derivative, pintulin, from the fermentation broth of Penicillium vulpinum F-4148. Pintulin shows weak activity against tumor cell lines, compared to that of adriamycin.

Antibiotics, Antineoplastic↗
PubMed1996

Pathology and cellular kinetics of gallbladder with an anomalous junction of the pancreaticobiliary duct.

OBJECTIVES: Anomalous junction of the pancreaticobiliary duct (AJPBD) is thought to be an important risk factor for gallbladder carcinoma in Japan. In this report, we examine histopathology and cellular kinetics of gallbladder mucosae of patients with AJPBD and the possible risk of gallbladder carcinoma. METHODS: We examined 62 gallbladders from patients with AJPBD (group A), 16 gallbladder carcinomas from patients with AJPBD (group B), 60 gallbladder carcinomas from patients without AJPBD (group C), and six normal gallbladders from patients without AJPBD (group D). Histopathology, mucosal heights, and proliferative cell nuclear antigen-labeling index were obtained from routinely processed tissue specimens. RESULTS: The incidence of hyperplastic changes in group A and in the noncancerous regions (NCRs) of group B was greater than in the NCRs of group C (p < 0.05). The incidence of dysplastic changes in the NCRs of group B was greater than in the NCRs of group C (p < 0.05). The mucosal heights in group A and in the NCRs of group B were higher than in the NCRs of group C (p < 0.05). A high proliferative cell nuclear antigen-labeling index was observed in group A and in the NCRs of group B, where hyperplastic changes were frequently observed. CONCLUSIONS: These results suggest that a sequence of hyperplastic changes with a corresponding increase in cellular kinetics with progression through dysplasia to carcinoma may be important in carcinogenesis in gallbladders of patients with AJPBD. AJPBD itself may be a possible risk for gallbladder carcinoma.

Adolescent↗
PubMed1996

Effect of age on physiologic and mechanically stressed rat alveolar bone: a cytologic and histochemical study.

The effect of age on physiologic and mechanically stressed rat alveolar bone was assessed with special focus on bone formation and resorption in maxillary molar sections of 1-, 4-, 9-, 16-, and 24-month-old male Fischer rats. Tetracycline- and calcein-labeled undecalcified sections were used for observation by confocal laser scanning microscopy and contact microradiography. The localization of osteoclasts and the morphological change of osteoblasts were observed on decalcified sections. Mechanical stress was applied by an orthodontic elastic inserted between the first and second maxillary molars for 7 days. Results showed that bone-formative activity of osteoblasts and bone-resorptive activity of osteoclasts declined with age around the maxillary molar section of physiologic rat alveolar bone. However, results of mechanically stressed rat alveolar bone revealed no evidence of numerical difference in number, size, and activity of osteoclasts and osteoblasts among the ages, suggesting that mechanical stress provokes a higher activity of osteoblasts and osteoclasts similarly in both adult and younger rat alveolar bone.

Age Factors↗
PubMed1995

Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line.

Phospholipase A2 (PLA2) from human pancreas, designated hPLA2-I, functions as a digestive enzyme. Interestingly, the present study demonstrated that the mature form of hPLA2-I stimulated the growth of a human pancreatic cancer cell line MIAPaCa-2, whereas the pro-form was ineffective. PLA2s from Laticauda semifasciata fraction I, Crotalus adamanteus venom, Streptomyces violaceoruber and bee venom, showed no proliferative effect to the growth of MIAPaCa-2. The Scatchard plot analysis revealed that the MIAPaCa-2 cell had a specific binding site for the mature hPLA2-I. The equilibrium binding constant (Kd) and the maximum binding capacity (Bmax) were 2.6 nM and 0.4 fmol/10(6) cells, respectively. These results suggest that the mature hPLA2-I, but not the pro-form, may function as a growth factor of pancreas carcinoma via the specific binding site.

Bee Venoms↗
PubMed1995

Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes.

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

3T3 Cells↗
PubMed1995

Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes.

SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.

Acyltransferases↗
PubMed1995

Quantitative determination of unchanged cisplatin in rat kidney and liver by high-performance liquid chromatography.

A quantitative analytical method for measuring unchanged cisplatin (CDDP) and high- and low-molecular-mass metabolites (fixed and mobile metabolites) in rat kidney and liver was developed. Unchanged CDDP, separated from fixed and mobile metabolites in tissue homogenates by consecutive procedures of fractionation and ultrafiltration, was determined by high-performance liquid chromatography (HPLC) with post-column derivatization. Although unchanged CDDP was found to be partly metabolized to fixed metabolites during the preparation of cytosolic ultrafiltrates, the recovery of unchanged CDDP gave a constant value (about 70%), which was independent of tissue type and CDDP concentration (from 1 to 10 micrograms/ml). The detection limit for unchanged CDDP in the cytosolic ultrafiltrate was 20 ng/ml, corresponding to a concentration detection limit of 65 ng Pt per g of tissue in the kidney and liver. The concentrations of fixed and mobile metabolites were determined as platinum concentrations in the tissue homogenate and in the cytosolic ultrafiltrate using atomic absorption spectrometry after correcting for transformation of unchanged CDDP to fixed metabolites. The distribution of unchanged CDDP, mobile metabolites and fixed metabolites in rat kidney and liver, after bolus injection of CDDP (5 mg/kg), was determined using this method.

Animals↗