There have been protocols reported concerning DNA extraction from various biological sources. A traditional DNA extraction procedure involves DNA separation from the other biological materials by ...organic extractions, followed by ethanol-precipitation of DNA from the resultant solution. We have developed a new simple extraction procedure for DNA purification in human serum in a single tube. The procedure involves simultaneous solubilization of proteins with a high concentration of NaI (chaotropic reagent) and N-lauroyl-sarcosinate, and precipitation of nucleic acids with isopropanol.
Although tumor necrosis factor α (TNFα) decreases the expression of peroxisome proliferator-activated receptor γ (PPARγ), its mechanism is not understood. We evaluated the effect of ceramide, the ...second messenger of TNFα, on the expression of PPARγ in primary cultured adipocytes. PPARγ mRNA and aP2 mRNA levels were measured with real-time PCR. The PPARγ protein level was measured with immunoblot. C6- and C2-ceramide, but not dihydroC6-ceramide, reduced the expression of PPARγ in a time and concentration dependent manner. The application of 1μM C6-ceramide for 36h reduced PPARγ mRNA level, aP2 mRNA level, and PPARγ protein level to 56.3%, 80.4% and 62.1%, respectively. Since ceramide is known to activate atypical PKC, we also studied the role of atypical PKC on the PPARγ reducing effect. Overexpression of wild type PKCζ magnified and accelerated the effect of TNFα and C6-ceramide on PPARγ mRNA levels, whereas overexpression of dominant negative PKCζ abolished the effect. We also found that the overexpression of constitutive active PKCζ reduced PPARγ mRNA level, aP2 mRNA level, and PPARγ protein level to 61.4%, 70.3% and 81.6%, respectively. Furthermore, TNFα activated nuclear factor-κB (NF-κB), known as a downstream effector of PKCζ to 256.6%, which was enhanced with overexpression of wild-type PKCζ. On the other hand, treatment with phorbol 12-myristate 13-acetate, another activator of NF-κB, also reduced the expression of PPARγ to 57.8%. These results indicate that the reducing effect of TNFα is mediated through ceramide, atypical PKC and NF-κB pathway.
The effects of histamine H
3-receptor antagonists, thioperamide, and clobenpropit on amygdaloid kindled seizures were investigated in rats. Both intracerebroventricular (i.c.v.) and intraperitoneal ...(i.p.) injections of H
3-antagonists resulted in a dose-related inhibition of amygdaloid kindled seizures. An inhibition induced by thioperamide was antagonized by an H
3-agonist (R)-α-methylhistamine and H
1-antagonists (diphenhydramine and chlorpheniramine). On the other hand, an H
2-antagonist (cimetidine and ranitidine) caused no antagonistic effect. Metoprine, an inhibitor of
N-methyltransferase was also effective in inhibiting amygdaloid kindled seizure, and this effect was augmented by thioperamide treatment.
The aim of this study was to investigate the expression of c-erbB2 in non-small cell lung cancers (NSCLCs) with attention both to membranous and cytoplasmic reaction, and to try to elucidate the ...meaning of cytoplasmic expression of c-erbB2 in NSCLCs. Immunohistochemical c-erbB2 expression and related clinico-pathological features were examined in 312 surgically resected patient tissues of NSCLCs, including 175 cases of adenocarcinoma and 137 cases of squamous cell carcinoma. Immunostaining of inner- and ecto-domain of c-erbB2, mRNA expression and the quantitation of soluble c-erbB2 in cultured media were performed in five NSCLC cell lines. Cytoplasmic expression of c-erbB2 was observed more frequently than membranous, both in patient tissues and cell lines. Neither membranous nor cytoplasmic expression of c-erbB2 was significantly correlated with short outcome in NSCLCs. Membranous c-erbB2 was expressed by both inner and ecto-domain, while cytoplasmic c-erbB2 was expressed by either or both inner and ecto-domain. c-erbB2 mRNA was produced in most cell lines; however, the soluble form was only detectable in a cell line that only presented a membranous c-erbB2. In conclusion, cytoplasmic c-erbB2 of NSCLCs was not a full-length protein only expressed in cellular membrane, but reflected degenerated c-erbB2 fragments with less functional ability.
The purpose of this study was to reveal the clinical validity of the Japanese version of the Functional Assessment of Cancer Therapy-Anemia scale (FACT-An) in relation to hemoglobin level. We also ...analyzed patients' scores for the related FACT-General scale (FACT-G), the FACT Anemia subscale, and the FACT Trial Outcome Index-Anemia scale (FACT TOI-An) to determine which was the most sensitive to anemia measurements.
Throughout Japan, we recruited 227 patients (mean+/-SD, 59+/-12.1 years old) diagnosed with a variety of cancers. We correlated the severity of anemia, as measured by hemoglobin levels, to scores on the FACT-An and on the other scales at baseline and at 3 months.
The questionnaire completion rate was more than 98% at both time points. The FACT-An had high internal consistency (Cronbach's alpha coefficient >0.8). FACT-An scores were significantly and positively correlated with hemoglobin levels both at baseline (r=0.24; 95% CI=0.12 to 0.36; n=225) and at 3 months (r=0.24; 95% CI=0.10 to 0.36; n=204). FACT-G, FACT Anemia subscale, and FACT TOI-An scores also successfully discriminated between patients with lower (Hb <11.0 g/dl) and higher (Hb > or =11.0 g/dl) hemoglobin levels. Moreover, the changes of these FACT scores over 3-months could discriminate changes in hemoglobin level.
The Japanese version of the FACT-An has higher clinical validity and can be used to appropriately assess health-related quality of life among Japanese cancer patients with anemia.
Previous phase II trials in Japan suggested that irinotecan was a promising agent for advanced or metastatic breast cancer pretreated with anthracycline. However, irinotecan has not yet been ...evaluated in the salvage setting for breast cancer pretreated with both anthracycline and taxane, which are two active agents for breast cancer.
The efficacy and safety of irinotecan were retrospectively evaluated in patients with breast cancer who had previously been treated with both doxorubicin and docetaxel. From 1996 to 1999, irinotecan was administered to 20 patients, all with a performance status of <2. Irinotecan treatment was repeated in approximately 6 week cycles consisting of the administration of irinotecan once weekly for 4 weeks followed by a 2 week rest. The median dose of irinotecan administered was 100 mg/m(2) weekly. The median number of irinotecan cycles given was 1 (range: 1-8 cycles). The median total dose was 388 mg/m(2) (range: 50-2400 mg/m(2)).
Performance status declined to >3 after treatment with irinotecan in four patients. Two patients had grade 3 leukopenia; three had grade 3 anemia and one had a creatinine elevation of grade 4. The objective response rate for all patients was 5.0% (95% CI: 0-15.5%). The median time to progression and overall survival were 35 days (range: 17-285 days) and 124 days (range: 17-667 days), respectively, since the start of the administration of irinotecan.
Salvage chemotherapy with irinotecan may be inactive against advanced and metastatic breast cancer pretreated with doxorubicin and docetaxel. We will evaluate irinotecan for advanced and metastatic breast cancer patients as first- or second-line chemotherapy combined with anthracycline or taxane.
We described here four patients diagnosed with Philadelphia chromosome positive (Ph+) leukemia, consisting of chronic myeloid leukemia (CML) (n=2) and Ph+ acute lymphoblastic leukemia (ALL) (n=2). ...All patients were treated with imatinib mesylate (300-400 mg/day) for the treatment of relapsed CML after allogeneic hematopoietic stem cell transplantation (SCT) (n=2), relapsed Ph+ ALL after SCT (n=1), and Ph+ ALL preceding SCT (n=1). Significant clinical and molecular responses were observed in all patients and three of them achieved sustained molecular remission. Imatinib was well tolerated and did not induce noticeable graft versus host disease although one patient presented severe skin rash (Grade III). Notably, serum cyclosporine A concentration increased after the initiation of imatinib treatment, probably through competitive inhibition of P450 3A4 isoenzyme. Our data suggest that imatinib in conjunction with SCT for the Ph+ leukemia may be a promising treatment strategy.
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