Isotopic investigations for the origin of relic from the Matsusaki site, Japan.

We measured the environmental radioactivity and isotope ratio, (87)Sr/(86)Sr ratio, (234)U/(238)U, delta(13)C, and (228)Ra/(226)Ra activity ratio, of adhesion that adhered to a wooden tubing vessel obtained from the Matsusaki site, one of the salt-production sites where Moshio is manufactured, in Aichi Prefecture, Japan. The environmental radioactivity and isotope ratio verified that the adhesion originated from the seaweed and/or sea grass.

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing.

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.

Structure-activity studies on nociceptin analogues: ORL1 receptor binding and biological activity of cyclic disulfide-containing analogues of nociceptin peptides.

Nociceptin/Orphanin FQ is an endogenous peptide ligand for the opioid receptor-like 1 (ORL1) receptor. To investigate the structural and conformational requirements of the nociceptin (NC)-receptor interaction, six cyclic analogues containing Cys disulfide linkages were designed and synthesized. Analogues cyclized at the N-terminal part, cyclo[Cys(0), Cys(7)]NC(1-13)-NH(2) (2) and cyclo[Cys(0), Cys(11)]NC(1-13)-NH(2) (4), and their corresponding linear peptides had very low activities in both the receptor binding and the GTP gamma S functional assays using human ORL1 transfected cell membranes. On the contrary, analogues cyclized at the C-terminal parts by the disulfide linkages at positions 6-10, 7-11, 7-14, and 10-14 sustained relatively high potencies in both assays. Notably, cyclo[Cys(10), Cys(14)]NC(1-14)-NH(2) (12) was found to be a potent NC agonist nearly as active as the parent peptide or NC. The maximum efficacy (Emax) of the C-terminally cyclized analogues and their linear counterparts in the GTP gamma S functional assay showed more than 94% (vs NC as 100%), suggesting that these analogues are full agonists. Analogue 12 is the first conformationally constrained NC analogue with almost full activity, and thus may serve to analyze the bioactive conformations of NC at the receptor site as well as serving as a template for more potent NC agonists.

Transforming growth factor beta triggers two independent-senescence programs in cancer cells.

Transforming growth factor-beta (TGF-beta)TG has been shown to play a multifunctional role in tumorigenesis. Here we demonstrate that TGF-beta induces a morphological change and expression of senescence-associated beta-galactosidase activity in the human lung adenocarcinoma cell line A549 cells within a week after the addition. These TGF-beta induced phenotypic changes are thought to characterize the rapid onset of senescence. When A549 cells were treated with TGF-beta, cell growth was not completely arrested, but the activity of telomerase was down regulated via transcriptional repression of telomerase reverse transcriptase, which led to a shortening of the telomere during long-term culture and finally resulted in replicative senescence. These results indicate that TGF-beta is able to induce a rapid senescence in A549 cells without significantly inhibiting cell growth and can further direct A549 cells to a replicative senescence state via the suppression of telomerase which culminates in telomere shortening. All these experimental results suggest that TGF-beta transmits several separate and independent signals to shift A549 cells back to a normal senescent cell.

Light-induced pigment aggregation in xanthophores of the medaka, Oryzias latipes.

The response mechanism of medaka xanthophores to light was examined at the cellular level. Innervated and denervated xanthophores of adult medakas responded to light (9,000 lux) within 30 sec by pigment aggregation, and this aggregation was not mediated through alpha-adrenoceptors on the cell membrane. Maximum sensitivity to light was at wavelengths of 410-420 nm, and the direct effect of light was reversible. Xanthophore responsiveness to light in summer was higher than that in winter. Ca2+ and calmodulin were not involved in the response, but rather, an important role for cAMP and phosphodiesterase (PDE) was suggested. It seems likely that photoreception by visual pigment which is sensitive to light at wavelengths of 410-420 nm increases PDE activity, probably via a G-protein, such as occurs with visual cells in the retina, which causes a decrease in levels of cytosolic cAMP, in turn leading to pigment aggregation within medaka xanthophores.

Production of a monoclonal antibody reacted broadly with feline calicivirus field isolates.

A monoclonal antibody (MAb) reactive with 36 field isolates and 2 laboratory strains of feline calicivirus (FCV) was produced by immunizing mice with the mixture of FCVs. The MAb (4D7) reacted with FCVs in an enzyme-linked immunosorbent assay (ELISA), but had no neutralizing activity against the F4 strain of FCV. MAb 8G1, previously produced against the FCV F4 strain, also reacted in ELISA with all FCVs used in the present study. However, the epitopes recognized by 4D7 and 8G1 were different. Using these two MAbs and a polyclonal rabbit antibody, we attempted to develop a sandwich ELISA for detection of FCV antigen. The combination of 4D7 and the polyclonal rabbit IgG was most sensitive. Using this system, all the field isolates of FCV cultured in vitro were detected. However, among the 36 swab samples, from which FCV was isolated, 4 were negative.

Bidirectional regulation of telomerase activity in a subline derived from human lung adenocarcinoma.

Telomerase is active in germline cells and tumor cells, but is either not expressed or is repressed in a majority of somatic cells. The regulation mechanism of telomerase activity has lately drawn considerable attention for the possible use of telomerase inhibitors for anti-cancer therapy. We analyzed the regulation mechanism of telomerase activity in the human lung adenocarcinoma cell line A549 and its subline A5DC7 which shows impaired tumor phenotypes. Although A549 and A5DC7 cells have similar growth potentials, when cultured in medium supplemented with 5% fetal bovine serum A5DC7 cells demonstrated a remarkable negative regulation of telomerase activity and telomere shortening. After the long-term culture, A5DC7 cells showed a large senescent cell-like morphology, arrested growth at the 106 population doubling level and entered senescence which was demonstrated by expression of the beta-galactosidase senescence marker. When the serum concentration was raised from 5% to 10% for the senescent A5DC7 cells, telomerase reactivation and telomere lengthening occurred as well as resumption of proliferation. These results demonstrate that the growth arrest seen in senescent A5DC7 cells is reversible and that telomerase activity is also bi-directionally regulated in A5DC7 cells, an event which could not be observed in normal senescent cells. This indicates that stringent telomerase repression and complete growth arrest concomitant with cellular senescence is collapsed in senescent A5DC7 cells in response to exogenous signals. Our study suggests that cellular senescence progresses and/or is regulated at multiple levels.

[Combination therapy of high dose 5'-DFUR+MMC for advanced or recurrent gastric cancer. 5'-DFUR Joint Research Group in the Osaka District for Gastric Cancer].

We conducted multi-center clinical trials of 'Treatment with high dose 5'-DFUR+MMC' in order to evaluate the effects, the possibility for outpatient treatment and the survival effects in patients with advanced or recurrent gastric cancer. The treatment schedule was as follow; 5'-DFUR 1,600 mg/body/day was given orally for five consecutive days every week and MMC 6 mg/m2 was injected intravenously once every four weeks. Forty-eight patients were enrolled and 34 cases were evaluated for anti-tumor responses. The overall response rate was 29.4% (10/34 cases). Side effects were mainly diarrhea, but outpatient treatment was possible in most cases. The median survival period was 249 days, which is longer than with other treatments. This result suggests that this combination chemotherapy is useful in patients with advanced or recurrent gastric cancers.

Proximal subtotal gastrectomy for the treatment of carcinoma of the upper third of the stomach: its indications based on lymph node metastasis and perigastric lymphatic flow.

To clarify the indications for a proximal subtotal gastrectomy in the treatment of carcinoma in the upper third of the stomach based on lymph node metastases, 1055 patients in whom either a D2 or greater lymph node removal was performed were reviewed. In the patients in which the lesion was confined to the upper stomach and did not invade beyond the muscularis propria of the stomach wall, no metastases to either the lymph nodes above and below the pylorus or the lymph nodes along the greater curvature were observed. A lymphatic flow study revealed a minimal flow to these nodes from the upper stomach in patients without lymph node metastasis, but in cases with lymph node metastases the lymphatic flow changed. The indications for a proximal subtotal gastrectomy for a carcinoma of upper third of the stomach therefore must fulfill the following two conditions: (1) The deepest layer of cancerous invasion does not extend beyond the muscularis propria of the stomach wall, and (2) No macroscopic evidence of lymph node metastasis can be detected during surgery.

Magnetic resonance imaging in a dog with choroid plexus carcinoma.

Choroid plexus carcinoma was diagnosed in a 10-year-old maltese dog with chief complaint of progressive ataxia and head tilt. No abnormalities was observed on hemogram, radiographs of the skull, and electroencepharograph (EEG). Neurological examination suggested central vestibular lesions. On the magnetic resonance imaging (MRI) examination, images after contrast enhancement with Gadolinium DTPA-dimeglumine showed a rough circular lesion with an increased signal intensity in caudal fossa. This lesion was histopathologically confirmed to be choroid plexus carcinoma.

Prognostic value of the number of metastatic lymph nodes in gastric cancer with radical surgery.

To evaluate the effectiveness of extended lymph node dissection in gastric cancer, the relationship between the number of lymph nodes with metastasis and the long-term outcome was studied retrospectively in 761 patients who underwent curative resection with extensive lymph node dissection. The cumulative 5-year survival rate was 85.8% in patients without lymph node metastasis, 60.2% in those with 1-4 lymph nodes, 35.6% with 5-10 nodes, and 12.3% with 11 or more nodes involved. Concerning N2 or N3 patients in whom metastatic lymph nodes would have remained without extensive dissection, the 5-year survival rate was 70.5% in those with 1-4 lymph nodes involved and 44.0% with 5 or more lymph nodes involved when no serosal invasion was observed. It was 38.5% with 1-4 lymph nodes and 5% with 5 or more lymph nodes involved when serosal invasion was observed. These results suggest that extensive lymph node dissection is effective in gastric cancer patients without serosal invasion and, when only a few lymph nodes are involved, also in those with serosal invasion. However, it is not considered to be effective in patients with serosal invasion and metastasis to many lymph nodes. Combination therapies are thought to be required in such patients.

Endoscopic evaluation of the depth of invasion of the depressed type early gastric cancer and apparently early advanced cancer in cases of a peptic ulcer within the cancer lesion.

Depressed-type early and apparently early advanced cancers were divided into two groups according to whether or not an associated peptic ulcer was present within the cancer lesion, and the accuracy of preoperative endoscopic diagnosis was reviewed. As a result it was found that evaluation of depth of invasion was more difficult in the ulcer [UL(+)] group than in the group without an ulcer [UL(-)]. In the UL(+) group it was difficult to identify endoscopic findings which indicate depth of invasion at the tip of the folds, the volume of cancer invasion of the deeper layers was small, and there many cases of invasion of deeper layers in a portion of the margins of the peptic ulcers.

[The indication for lymph node dissection around abdominal aorta in gastric cancer].

In 77 cases with gastric cancer, for which lymph node dissection around the left renal vein was performed in the past 5 years, the sites and routes of metastasis were investigated to determine the indication for dissection around the left renal vein. The rate of metastasis to the lymph nodes around the left renal vein was 23.4%. The typical routes of metastasis were 1) the left lower phrenic route, 2) via the splenic artery, 3) via the celiac artery, 4) via the superior mesenteric artery and 5) the postpancreatic route. Dissection was considered to be indicated for the patient with N2 or more distant lymph node involvement and for cases with carcinoma of the upper part of the stomach or whole stomach with N2 positive lymph node. It was also indicated for N3(+) cases or No. 9(+) cases of carcinoma of the middle and lower parts of the stomach. Needless to say the absence of other non curative factors is required.

[Hepatic arterial infusion chemotherapy of hepatocellular carcinoma].

In 24 cases of unresectable hepatocellular carcinoma, we performed hepatic arterial catheterization and intra-arterial infusion chemotherapy. Adriamycin (ADM), Mitomycin C (MMC), 5-FU and Lipiodol (LPD) were administered an average of 13.5 times over a mean period of 106 days. Except for 5 unevaluable cases, there were 0 CR, 5 PR, 4 MR, 7 NC and 2 PD cases, for an efficiency rate of 27.8%. Complications thought to be due to the catheter included catheter blockade in 1 case (4.3%) and dermal infection of insertion site in 3 cases (13.0%). As for the results of follow-up study, one-year survival rate with this therapy was 47.8%, which compares favorably with a one-year survival rate of 30.0% in 30 cases treated only with TAE. From the above results, hepatic arterial infusion chemotherapy can be repeatedly performed on an outpatient basis, and it is considered to be a useful therapeutic method for treating unresectable hepatocellular carcinoma.