bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective Evaluation of Skin Surface Electropotentials in Japanese Patients with Suspicious Breast Lesions
Authors: Fukuda Mamoru , Shimizu Kaname , Okamoto Norihiko , Arimura Toshihiro , Ohta Tomohiko , Yamaguchi Susumu , Faupel Mark L.
Published: August 2005

The biofield breast examination (BBE) is a new, noninvasive and cost‐effective method for diagnosing breast lesions currently undergoing multicenter evaluation in the USA and Europe. The test analyzes subtle differences in electrical potential caused by dysregulated epithelial proliferation. This report summarizes a prospective evaluation of BBE in a population of 101 patients with suspicious breast lesions scheduled either for open surgical biopsy or fine needle aspiration biopsy. Of the 101 patients included in the study, 49 were found to have a breast malignancy and 52 were found to have a benign breast lesion. BBE correctly identified 44 of 49 biopsy‐proven cancers (sensitivity = 90%) and correctly indicated no cancer in 31 of 52 biopsy‐proven benign cases (specificity = 60%). Sensitivity increased to 95% for cancers less than 2.5 cm in size. These results indicate that BBE may be an effective adjunctive test to help to resolve abnormalities discovered by physical examination or other screening methods.
Expression of p21waf‐1/cip‐1 Is Significantly Induced in the Livers of LEC Rats with Chronic Liver Injury
Authors: Sawada Norimasa , Kojima Takashi , Obata Hiroyuki , Isomura Hiroshi , Atsumi Shin‐ichi , Sawaki Masakuni , Tsuzuki Naoto , Tobioka Hirotoshi , Kokai Yasuo , Satoh Masaaki , Mori Michio
Published: August 2005

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21waf‐1/cip‐1 and p27, cyclin‐dependent kinase inhibitors, by northern blot analysis. The expression of p21waf‐1/cip‐1 in the LEC rat liver was 3‐fold higher than that of age‐matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p2lwaf‐1/cip‐1 in the nuclear matrix fraction of the LEC rat liver. Immunohisto‐chemically, p21waf‐1/cip‐1 was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.
Deletion of Src Homology 3 Domain Results in Constitutive Activation of Tec Protein‐Tyrosine Kinase
Authors: Yamashita Yoshihiro , Miyazato Akira , Ohya Ken‐ichi , Ikeda Uichi , Shimada Kazuyuki , Miura Yasusada , Ozawa Keiya , Mano Hiroyuki
Published: August 2005

Tec protein‐tyrosine kinase (PTK) is the prototype of a new subfamily of non‐receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine‐phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (TecΔSH3). The activity of TecΔSH3 was confirmed in 293 cells, as well as in cytokine‐dependent hematopoietic cells (BA/F3). TecΔSH3 should be a useful tool to study the in vivo substrates of Tec PTK.
Involvement of Vascular Endothelial Growth Factor in Kaposi's Sarcoma Associated with Acquired Immunodeficiency Syndrome
Authors: Sakurada Shinsaku , Kato Tetsuji , Mashiba Kohichi , Mori Shigeo , Okamoto Takashi
Published: August 2005

To examine the role of vascular endothelial growth factor (VEGF) in the development of edema associated with Kaposi's sarcoma (KS) in acquired immunodeficiency syndrome (AIDS), we exploited animal model systems to detect the activity that induces vascular hyper‐permeability (VHP) using cultured AIDS‐KS spindle cells. Cultured AIDS‐KS spindle cells and conditioned medium (AIDS‐KS‐CM) that had been semi‐purified through a heparin affinity column were tested for the ability to induce VHP in animals. The AIDS‐KS spindle cells and AIDS‐KS‐CM induced VHP that was histamine‐independent. The VHP‐inducing activity was detected in the 0.5 M NaCl fraction from the heparin affinity column and was blocked by anti‐VEGF neutralizing antibody. In addition, the production of VEGF was demonstrated in fresh AIDS‐KS tissue as well as in cultured AIDS‐KS cells, while control cells were negative for VEGF production. From these observations, we concluded that AIDS‐KS cells produce a factor(s) that promotes VHP, and this factor could be VEGF.
Inactivation of the E‐Cadherin Gene in Primary Gastric Carcinomas and Gastric Carcinoma Cell Lines
Authors: Tamura Gen , Sakata Ken , Nishizuka Satoshi , Maesawa Chihaya , Suzuki Yasushi , Iwaya Takeshi , Terashima Masanori , Saito Kazuyoshi , Satodate Ryoichi
Published: August 2005

We investigated the E (epithelial)‐cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet‐ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN‐45, GCIY, HGC‐27 and GT3TKB). We utilized PCR‐SSCP and RT‐PCR followed hy direct sequencing to detect gene mutations and skipped exons, and RT‐PCR‐SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons, were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet‐ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E‐cadherin gene included an 18 bp deletion (codon 418‐423) and a 3 bp deletion (codon 400, calcium‐binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN‐45 cell line exhibited an 18 bp deletion at the exon 6‐intron 6 boundary with loss of the wild‐type allele, and 2 of the remaining 3 cell lines (HGC‐27 and GT3TKB) had lost expression without detectable structural alteration of the E‐cadherin gene. These data provide support for classic two‐hit inactivation of the E‐cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.
Establishment and Characterization of Three Androgen‐independent, Metastatic Carcinoma Cell Lines from 3,2′‐Dimethyl‐4‐aminobiphenyl‐induced Prostatic Tumors in F344 Rats
Authors: Nakanishi Hayao , Takeuchi Shinichi , Kato Koji , Shimizu Satoru , Kobayashi Kiyoshi , Tatematsu Masae , Shirai Tomoyuki
Published: August 2005

Three stable carcinoma cell lines, designated PLS10, PLS20 and PLS30, have been established from 3,2′‐dimethyl‐4‐aminobiphenyl plus testosterone‐induced carcinomas in the dorsolateral prostate of male F344 rats. The cells are keratin‐positive and grow as typical epithelial monolayers in culture. When injected into intact male nude mice, PLS10 and PLS30 cells form well‐differentiated adenocarcinomas with abundant connective tissue stroma, while PLS20 cells give rise to poorly differentiated adenocarcinomas. Growth of all PLS cell lines in nude mice is not affected by castration and the cells are immunohistochemically negative for androgen receptors. Tumor growth rates in nude mice were found to be PLS20 > PLS10 > PLS30, with significant in vitro stimulation by insulin/transferrin, but not epidermal growth factor, dexamethasone or basic fibroblast growth factor. Spontaneous lung metastases were observed in all cases. However, skeletal invasion including bone is essentially observed only with the PLS20 tumors. Gelatin zymography showed predominant secretion of the active form of gelatinase B (Mr 92,000 type IV collagenase) by all the cell lines. Karyotype analysis revealed PLS10, PLS30 and PLS20 to be diploid, hyperdiploid and hypertetraploid, respectively. The results demonstrate that the three PLS cell lines are androgen‐independent and metastatic in common, but have different histology, growth potential and invasiveness. They may therefore be useful models for understanding progression and metastasis of human prostatic carcinomas.
A Comparative Study between the Gastric Mucosa of Chileans and Other Dwellers of the Pacific Basin
Authors: Rubio Carlos A. , Pisano Raúl , Llorens Pedro , Duarte Ignacio
Published: August 2005

A total of 3,289 sections of 120 gastrectomy specimens from Chile were reviewed. Intramucosal cysts were found in 61.7% of the specimens, ciliated‐metaplastic cells in 33.3%, large vacuolated cells in 20.8% and extensive intestinal metaplasia in 51.7%. The frequency of these non‐neoplastic changes was significantly higher in specimens with early adenocarcinoma of intestinal type than in those with early adenocarcinoma of diffuse type or having a peptic ulcer. Similar results have been recorded in other inhabitants of the Pacific basin, but not in inhabitants of the Atlantic basin. Interestingly, the gastric cancer incidence in the various populations studied in the Pacific basin is much higher than in those studied in the Atlantic basin. Environmental factors acting in Chile appear to have induced those non‐neoplastic changes in the gastric mucosa. The question arises as to whether environmental factors (promoters?) acting in Chile (as well as in Japan, in Hawaii and in New Zealand) have favored the necessary non‐neoplastic mucosal conditions required for the subsequent development of gastric adenocarcinoma (in particular of intestinal type).
Usefulness of Lymphokine‐activated Killer Cells Generated from Bone Marrow Mononuclear Cells for the Purging of Residual Tumor Cells in Peripheral Blood Stem Cell Graft
Authors: Komatsu Fumio , Kajiwara Michiko
Published: August 2005

Lymphokine‐activated killer (LAK) cells were generated from bone marrow mononuclear cells (BM), and the usefulness of the BM‐LAK for purging of residual tumor cells in autologous peripheral blood stem cell (PBSC) graft was determined. The BM and peripheral blood lymphocytes (PBL) were obtained from the same bone marrow donors. The BM‐LAK and PBL‐LAK were generated by incubation with interleukin‐2 for 7 days. The BM‐LAK demonstrated higher killer activity against a lymphoma cell line Raji than the PBL‐LAK. The BM‐LAK also had a higher percentage of CD4‐CD8‐CD16+ cells than the PBL‐LAK, which suggests that their high killer activity is related to these cells. The BM‐LAK did not show any killer activity against the PBSC graft. However, they killed tumor cells which contaminated the PBSC graft, and in particular, killed chimeric ber/abI messenger RNA‐positive residual leukemic cells. These results suggest that the BM‐LAK may be applicable for purging. As the BM‐LAK possess higher killer activity than the PBL‐LAK, they may be more useful than the PBL‐LAK.
Relationship between Cell‐killing Efficiency and Number of Platinum Atoms Binding to DNA, RNA, and Protein Molecules in HeLa Cells Treated with cis‐Diamine(glycolato)platinum(II)
Authors: Akaboshi Mitsuhiko , Kawai Kenichi , Kinashi Yuko , Masunaga Shinichiro , Ono Koji
Published: August 2005

HeLa S‐3 cells were treated with 195mPt‐radiolabeled cis‐diamine(glycolato)platinum(II) (254‐S) under various conditions, and the relationship between the lethal effect and the numbers of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentrations for the cells treated with 254‐S at 37°C for 0.5, 1, 2, and 3 h were 67.1, 47.0, 26.8 and 8.1 μM1, respectively. Using identically treated cells, we determined the numbers of Pt atoms combined with DNA, RNA, and protein molecules after fractionation of the cells. In this way, the D0 values (D0, the dose that causes an average of one lethal event per member of the population), expressed as the drug concentration, could he related to the number of Pt atoms combined with each fraction. The efficiency of the Pt atom in killing the cells, expressed as the reciprocal of the D0 values, was then calculated for each fraction. The results suggested that DNA was the primary target for cell killing by 254‐S. The target volumes for DNA were 3.96, 4.97, and 11.77 × 104 nucleotides for 1‐, 2‐, and 3‐h treated cells, respectively. In terms of the target volume, the cell‐killing effects of 254‐S were comparable to those of cis‐diamine‐dichloroplatinum(II) (CDDP), for which the target volumes under identical conditions were determined to be 5.17, 5.71, and 10.3 × 104 nucleotides, respectively, while in terms of the mean lethal dose (D0), the cell‐killing effects of 254‐S were lower than those of CDDP by a factor of 5.1 (47.0/9.3), 4.0 (26.8/6.7), or 2.5 (8.1/3.2) for 1‐, 2‐, or 3‐h treatment, respectively.
Modulation of Multidrug Resistance by SDZ PSC 833 in Leukemic and Solid‐tumor‐bearing Mouse Models
Authors: Watanabe Toru , Naito Mikihiko , Oh‐hara Tomoko , Itoh Yohjiro , Cohen Dalia , Tsuruo Takashi
Published: August 2005

P‐Glycoprotein inhibitors, including the nonimmunosuppressive cyclosporin D analog SDZ PSC 833 (PSC 833), have been developed to circumvent multidrug resistance. In the present study, the potential of PSC 833 in reversing multidrug resistance was evaluated in various systemic treatment models with leukemic and solid‐tumor‐bearing mice. Having a relatively wide therapeutic window of daily p.o. doses from 12.5 to 75 mg/kg, PSC 833 significantly improved the antileukemic activity of the anticancer drugs adriamycin (ADM), vincristine (VCR) and etoposide (VP‐16) given i.p. or i.v. against i.p.‐inoculated vincristine‐resistant P388 tumor (P388/VCR). PSC 833 in combination with i.p.‐injected anticancer drugs in optimal schedule and dosage induced apparent cures in some leukemic mice, whereas no cures were obtained with the cyclosporin A/anticancer drug combinations. PSC 833 combined with i.v.‐injected anticancer drugs was highly active, but not curative, against P388/VCR and parental P388 tumors (maximum T/C>175%). PSC 833 in combination with intravenous treatment with ADM showed prominent anti‐solid‐tumor activity against s.c.‐inoculated colon adenocarcinoma 26 and human colorectal adenocarcinoma HCT‐15. Against colon adenocarcinoma 26, the PSC 833/ADM combinations induced cure in two or three of six mice. PSC 833/ADM combinations significantly inhibited the growth of the tumor with maximum percent inhibitions of 83 and 73% in the early and advanced stages of the HCT‐15 tumor models, respectively. The present study demonstrated that PSC 833 is highly active in potentiating the antitumor activity of systemically administered ADM, VCR and VP‐16 against four murine and human tumors with a relatively wide therapeutic window of daily p.o. dose range of 12.5–100 mg/kg.
A Prospective Randomized Trial of the Preventive Effect of Pre‐operative Transcatheter Arterial Embolization against Recurrence of Hepatocellular Carcinoma
Authors: Yamasaki Susumu , Hasegawa Hiroshi , Kinoshita Hiroaki , Furukawa Masato , Imaoka Shingi , Takasaki Ken , Kakumoto Youichiro , Saitsu Hideki , Yamada Ryusaku , Oosaki Yukio , Arii Shigeki , Okamoto Eizo , Monden Morito , Ryu Munemasa , Kusano Shouichi , Kanematsu Takashi , Ikeda Kenji , Yamamoto Masayuki , Saoshiro Takeo , Tsuzuki Toshiharu
Published: August 2005

To clarify whether pre‐operalive transcatheter arterial embolization (TAE) improves survival after hepatectomy, a prospective randomized comparative study was done. Of a total of 115 registered patients having solitary hepatocellular carcinoma (HCC) 2 to 5 cm in diameter, 18 (15.7%) were excluded after randomization. As a result, 97 patients were chosen as subjects and divided into two groups: hepatectomy with (group A: n = 50) and without (group B: n=47) pre‐operative TAE. The period of observation of the patients who survived the surgery was between 4.0 and 6.6 years. The randomization appeared to have provided well‐balanced groups of patients and the clinico‐pathological characteristics of the two groups were quite similar. The necrotic part of the cancerous lesions, as confirmed by operative specimens, amounted to 74.8 ±33.4% (mean±SD) in group A and 6.8 ±7.2% in group B (P<0.01). However, the cancer‐free survival rates after hepatectomy in both groups showed little difference (39.1±7.0 (%±SE) and 31.1±0.1, respectively). We speculate that TAE is not effective against such HCC accessory lesions as minute intrahepatic metastasis and tumor thrombus and that pre‐operative TAE does not improve post‐operative survival.
Calcium and Magnesium in Drinking Water and Risk of Death from Colon Cancer
Authors: Yang Chun‐Yuh , Chiu Hui‐Fen , Chiu Jeng‐Fen , Tsai Shang‐Shyue , Cheng Ming‐Fen
Published: August 2005

The possible association between the risk of colon cancer and the levels of calcium and magnesium in drinking water from municipal supplies was investigated in a matched case‐control study in Taiwan. All eligible colon cancer deaths (1714 cases) of Taiwan residents from 1989 through 1993 were compared with deaths from other causes (1714 controls), and the levels of calcium and magnesium in drinking water of these residents were determined. Data on calcium and magnesium levels in drinking water throughout Taiwan were obtained from the Taiwan Water Supply Corporation. The control group consisted of people who died from other causes and the controls were pair‐matched to the cases by sex, year‐of‐birth, and year‐of‐death. The adjusted odd ratios (95% confidence interval) were 0.79 (0.64–0.98) for the group with water calcium levels between 24.4 and 42.3 mg/liter and 0.58 (0.47–0.73) for the group with calcium levels of 42.4 mg/liter or more. The adjusted odd ratios were not statistically significant for the relationship between magnesium levels in drinking water and colon cancer. The results of the present study show that there is a significant protective effect of calcium intake from drinking water against colon cancer
Inhibitory Effects of Dietary Protocatechuic Acid and Costunolide on 7,12‐Dimethylbenz[a]anthracene‐induced Hamster Cheek Pouch Carcinogenesis
Authors: Ohnishi Masami , Yoshimi Naoki , Kawamori Toshihiko , Ino Natsuko , Hirose Yoshinobu , Tanaka Takuji , Yamahara Johji , Miyata Hideo , Mori Hideki
Published: August 2005

The modifying effects of dietary exposure to two natural products, protocatechuic acid (PCA) and Costunolide during the development of neoplasms in oral carcinogenesis initiated with 7,12‐dimethyl‐benz[a]anthracene (DMBA) were investigated in male Syrian golden hamsters. All hamsters except those in the test chemical alone and control groups received DMBA (0.5%) in mineral oil to the right buccal pouch 3 times per week for 4 or 6 weeks. At 13 weeks of age, the groups exposed to DMBA were fed diet containing PCA or Costunolide at a dose of 0.2 g/kg diet (200 ppm) for 17 weeks. The other groups consisted of hamsters given mineral oil alone for 6 weeks, or given 200 ppm PCA or Costunolide alone, or untreated. All animals were necropsied at the termination of the experiment (week 24). PCA or costunolide significantly decreased the tumor burden (P<0.001‐P<0.05) and the extent of dysplastic areas (%) (P<0.001‐P<0.05). PCA significantly decreased the mean number of AgNORs/nucleus (P<0.05). The BrdUrd‐labeling index was reduced by dietary administration of test compounds, though not significantly. These results suggest that PCA and costunolide inhibited hamster buccal pouch carcinogenesis and such inhibition may be related to suppression of cell proliferation in the buccal mucosa. It was also found that telomerase activity expressed in neoplastic and preneoplastic lesions of hamster buccal pouch epithelium after DMBA treatment correlated with the histopathological degree of malignancy.
Cytotoxicity of Histocompatibility Leukocyte Antigen–DR8–restricted CD4+ Killer T Cells against Human Autologous Squamous Cell Carcinoma
Authors: Miyazaki Akihiro , Sato Noriyuki , Takahashi Shuji , Sasaki Aya , Kohama Gen–iku , Yamaguchi Akira , Yagihashi Atsuhito , Kikuchi Kokichi
Published: August 2005

Although CD8+ killer T cells reacting against human autologous tumor cells have recently been studied in detail, little is known about the cytotoxic mechanism of CD4+ T cells against such tumor cells. In order to investigate this, we have established CD4+ cytotoxic T lymphocyte TcOSC–20 lines. TcOSC–20 showed selective cytotoxic activity against autologous OSC–20 cells, derived from a cancer of the tongue, in an HLA–DR–restricted fashion. HLA–DR8 (DRB1* 08032) is the only DR molecule expressed on OSC–20 cells, and anti–DRS monoclonal antibody could inhibit the Cytotoxicity, suggesting that HLA–DRB1★ 08032 is the tumor rejection antigen–presenting moleculeto TcOSC–20. The Fas ligand was expressed on TcOSC–20 lines, and its expression was induced upon mixed lymphocyte–tumor cell culture of autologous peripheral blood lymphocytes. Furthermore, the Cytotoxicity of TcOSC–20 was inhibited by anti–Fas ligand antibody.These data imply that TcOSC–20 lines recognize the tumor antigenic peptide presented by HLA–DR8, and exert Cytotoxicity against autologous tumor cells via a Fas–mediated cytotoxic pathway.
Enhancement of Tumor Radio‐response by Irinotecan in Human Lung Tumor Xenografts
Authors: Tamura Kenji , Takada Minoru , Kawase Ichirou , Tada Takuhito , Kudoh Shinzoh , Okishio Kyouichi , Fukuoka Masahiro , Yamaoka Naoki , Fujiwara Yasuhiro , Yamakido Michio
Published: August 2005

We investigated the ability of 7‐ethyl‐10‐[4‐(l‐piperidino)‐l‐piperidmo]carbonyloxycamptothecin (CPT‐11) to increase tumor radio‐response in vivo using human lung tumor xenografts. The xenografts were treated with (1) CPT‐11 (10 mg/kg) intraperitoneally on days 1, 5 and 9, (2) single dose radiation (10 Gy/leg) on day 1, or (3) a combination regimen of both treatments in which radiation was given 1 h after the first dose of CPT‐11. DNA flow cytometry studies were performed to define the cell cycle changes following treatment for 1 to 12 h with 0, 0.5, 2.0 or 8.0 ng/ml SN‐38, the major active metabolite of CPT‐11. In both small cell lung cancer (MS‐1) and small cell/large cell carcinoma (LX‐1) xenografts, combination treatment resulted in significant tumor regression compared with the use of CPT‐11 (P=0.0005, 0.0053) or radiation treatment (P=0.00221, 0.0035) alone. Neither severe body weight loss nor enhanced skin reaction was observed following the combined treatment. In flow cytometry studies, the proportion of cells in G2/M‐phase, the most radio‐sensitive phase, increased after 1 h exposure to the lowest dose of SN‐38 (0.5 ng/ml). These findings suggest that CPT‐11 is a potent radiosensitizing agent, and that its activity is related to the cell cycle. This is the first report to indicate that CPT‐11 serves as a radiosensitizer in vivo.
Infrequent Overexpression of p53 Protein in Epstein‐Barr Virus‐associated Gastric Carcinomas
Authors: Ojima Hitoshi , Fukuda Toshio , Nakajima Takashi , Nagamachi Yukio
Published: August 2005

Epstein‐Barr virus (EBV) infection was studied in a total of 412 patients with poorly differentiated gastric adenocarcinoma by in situ hybridization for EBV‐encoded small RNA. EBV‐speciflc RNA was detected in tumor cell nuclei of 83 (20.1%) of 412 gastric carcinomas, of which 60 were histologically subclassified as gastric carcinoma with lymphoid stroma (GCLS). All EBV‐positive gastric carcinomas as well as 90 randomly selected EBV‐negative gastric carcinomas were further studied for p53 protein expression by immunohistocbemistry. The Overexpression of p53 protein was demonstrated in only 7 (8.4%) of 83 EBV‐positive gastric carcinomas. This was in marked contrast to the frequency of 34.4% in EBV‐negative gastric carcinomas. In addition, a few p53‐positive nuclei were characteristically scattered in the tumors of many EBV‐positive GCLS, but this was not regarded as p53 Overexpression arising from mutation of the gene. Our findings suggested that EBV‐associated gastric carcinomas may arise through a different mechanism from other types of gastric carcinomas without EBV infection.
Antitumor Activity of TZT‐1027, a Novel Doiastatin 10 Derivative
Authors: Kobayashi Motohiro , Natsume Tsugitaka , Tamaoki Satoru , Watanabe Jun‐ichi , Asano Hajime , Mikami Takashi , Miyasaka Katsuhiko , Miyazaki Koichi , Gondo Masaaki , Sakakibara Kyoichi , Tsukagoshi Shigeru
Published: August 2005

Dolastatin 10, a pentapeptide isolated from the marine mollusk Dolabella auricularia, has antitumor activity. TZT‐1027, a dolastatin 10 derivative, is a newly synthesized antitumor compound. We evaluated its antitumor activity against a variety of transplantable tumors in mice. Intermittent injections of TZT‐1027 were more effective than single or repeated injections in rake with P388 leukemia and B16 melanoma. Consequently, TZT‐1027 shows schedule dependency. TZT‐1027 was effective against P388 leukemia not only when administered i.p., but also when given i.v. However, although TZT‐1027 given i.v. was active against murine solid tumors, TZT‐1027 administered i.p. was ineffective against all the tumors tested with the exception of colon 26 adenocarcinoma. The i.v. injection of TZT‐1027 at a dose of 2.0 mg/Ag remarkably inhibited the growth of three murine solid tumors; colon 26 adenocarcinoma, B16 melanoma and M5076 sarcoma, with T/C values of less than 6%. The antitumor activities of TZT‐1027 against these tumors were superior or comparable to those of the reference agents; dolastatin 10, cisplatin, vincristine, 5‐fluorouracil (5‐FU) and E7010. In experiments with drug‐resistant P388 leukemia, TZT‐1027 showed good activity against cisplatin‐resistant P388 and moderate activity against vincristine‐ and 5‐fluorouracil‐resistant P388, but no activity against adriamycin‐resistant P388. TZT‐1027 was also effective against human xenografts, that is, tumor regression was observed in mice bearing MX‐1 breast and LX‐1 lμng carcinomas. TZT‐1027 at 10 μM almost completely inhibited the assembly of porcine brain microtubules. Therefore, its mechanism of antitumor action seems to he, at least in part, ascrihable to the inhibition of microtubule assembly. Because of its good preclinical activity, TZT‐1027 has been entered into phase I clinical trials.
MAD‐related Genes on 18q21.1, Smad2 and Smad4, Are Altered Infrequently in Esophageal Squamous Cell Carcinoma
Authors: Maesawa Chihaya , Tamura Gen , Nishizuka Satoshi , Iwaya Takeshi , Ogasawara Satoshi , Ishida Kaoru , Sakata Ken , Sato Nobuhiro , Ikeda Kenichiro , Kimura Yusuke , Saito Kazuyoshi , Satodate Ryoichi
Published: August 2005

The MAD (mothers against decapentaplegic)‐related genes, Smad2 (former name MA DR2 or JV18‐1) and Smad4 (former name DPC4), have been identified on chromosome 18q21.1, We analyzed 30 primary esophageal squamous cell carcinomas (ESCC) and 7 cell lines derived from ESCC for intragenic mutations and loss of heterozygosity (LOH) of the Smad2 and Smad4 genes. LOH was detected in 5 of 14 (35%) informative cases. However, no mutations in either gene were detected in either the primary carcinomas or the cell lines, and only a G‐to‐A base transition within the 3′‐untranslated region of the Smad4 gene was observed in a carcinoma. There were no homozygous deletions in either of the genes in the cell lines. MAD‐related genes on chromosome 18q21.1 are altered infrequently in ESCC.
A Randomized Controlled Trial for Chemoprevention of Gastric Cancer in High‐risk Japanese Population; Study Design, Feasibility and Protocol Modification
Authors: Tsubono Yoshitaka , Okubo Shunji , Hayashi Masato , Kakizoe Tadao , Tsugane Shoichiro
Published: August 2005

We have initiated a population‐based, double‐blind, randomized controlled trial to examine the effects of supplementation of beta‐carotene and vitamin C on the incidence of gastric cancer. The subjects were participants in an annual health screening program conducted by four municipalities in Akita prefecture, one of the regions with the highest mortality from gastric cancer in Japan. We measured their serum levels of pepsinogens (PGs) I and II, and asked persons diagnosed with chronic atrophic gastritis (defined as PGI < 70 ng/ml and PGI/PGII ratio <3.0) to take diet supplements containing 0 or 15 mg/day beta‐carotene and 50 or 500 mg/day vitamin C for 5 years. During the first year of recruitment conducted in one village from June through September, 1995, 52% (635/1214) of screening participants had chronic atrophic gastritis and 73% (439/602) of eligible persons responded. However, in response to a National Cancer Institute press report released on January 18, 1996, indicating that two beta‐carotene trials had shown no benefit and potential harm from the supplement, we discontinued the beta‐carotene and continued with the trial using only vitamin C. Of 397 participants remaining at this point, 77% (305) consented to stay in the study. The results indicate that a randomized controlled trial for cancer prevention is feasible in the Japanese asymptomatic population.
Rare Occurrence of ras and p53 Gene Mutations in Mouse Stomach Tumors Induced by N–Methyl–N–nitrosourea
Authors: Furihata Chie , Tatematsu Masae , Saito Masae , Ishida Shizuka , Nakanishi Hayao , Inada Ken‐ichi , Tei Hajime , Hattori Masahira , Ito Takashi , Sakaki Yoshiyuki
Published: August 2005

The incidence of point mutations of H–, K– and N–ras and p53 oncogenes in male BALB/c mouse stomach tumors induced with Ar–methyl–Ar–nitrosourea (MNU) was examined by direct sequencing and PCR single–strand conformation polymorphism (PCR–SSCP). A mutation of GGT to AGT at K–ras codon 12 was found by SSCP in one adenocarcinoma from a total of 19 specimens including 5 adenocarcinomas, 9 adenomatous hypcrplastic regions, 1 squamous cell carcinoma and 4 normal–like stomach regions from 4 mice. No mutations were detected by direct sequencing of H–, K– and N–ras oncogenes at exons 1 (codons 12 and 13) and 2 (codon 61) in a total of 26 specimens comprising 10 adenocarcinomas, 10 adenomatons hyperplastic regions, 2 squamous cell carcinomas and 4 normal–like stomach regions from 6 mice. No mutations were detected by direct sequencing ofp53 oncogene at exons 5, 6, 7 and 8 in a total of 30 specimens including 13 adenocarcinomas, 8 adenomatous hyperplastic regions, 2 squamous cell carcinomas, 1 papilloma and 6 normal–like stomach regions from 7 mice. These results suggest that ras and p53 oncogenes do not play a role in mouse stomach carcinogenesis induced by MNU.
Expression of Interstitial Collagenase (Matrix Metalloproteinase‐1) in Gastric Cancers
Authors: Sakurai Yoshihiko , Otani Yoshihide , Kameyama Kaori , Hosoda Yasuhiro , Okazaki Isao , Kubota Tetsuro , Kumai Koichiro , Kitajima Masaki
Published: August 2005

The expression of matrix metalloproteinase‐1 (MMP‐1) gene and the presence of MMP‐1 protein in gastric cancer were examined by in situ hybridization and immunohistochemistry. Expression of the interstitial Collagenase (MMP‐1) gene was detected within the stroma of the neoplastic glands, and infiltration of eosinopilic was observed to be associated with regions of MMP‐1 gene expression. The degree of eosinophils infiltration correlated with the level of MMP‐1 mRNA expression. Immunostaining showed localization of MMP‐1 protein in the stromal cells, and additionally in the neoplastic glands. These findings indicate that the stromal cells may play an important role in the expression of MMP‐1, and suggest a pathophysiological role for MMP‐1 in the invasion and metastasis of gastric cancer.
Detection of MAGE‐4 Protein in Sera of Lung Cancer Patients
Authors: Shichijo Shigeki , Hoshino Tomoaki , Koufuji Kikuo , Hayashi Akihiko , Kawamoto Mayumi , Kikuchi Megumi , Higuchi Takafumi , Ichiki Masao , Oizumi Kotaro , Itoh Kyogo
Published: August 2005

We investigated the level of MAGE‐4 protein in sera of patients with primary lung cancer to understand better the biological roles of the MAGE proteins. MAGE‐4 protein was detected as a non‐degraded form in botb the supernatant of a MAGE‐4+ tumor cell line and in a patient's serum. Serum level of the MAGE‐4 protein in lung cancer patients (n= 100, mean = 1.17 ng/ml) was significantly higher than that in either patients with benign pulmonary diseases (n= 80, 0.33 ng/ml) or healthy donors (n=68, 0.32 ng/ml). It was higher than the cutoff level (1.15 ng/ml) in 34 of 100 cancer patients, but not in anyone in the other groups.
Reduced Expression of Cyclin–dependent Kinase Inhibitor p27Kipl Is Associated with Advanced Stage and Invasiveness of Gastric Carcinomas
Authors: Yasui Wataru , Kudo Yasusei , Semba Shuho , Yokozaki Hiroshi , Tahara Eüchi
Published: August 2005

Reduced expression of a cyclin–dependent kinase inhibitor p27Kipl has recently been shown to predict poor survival of patients with breast and colorectal cancers. We studied the expression of p27Kipl in gastric carcinomas by northern blotting, western blotting and immunohistochemistry to determine whether lack of p27 has implications for aggressiveness of gastric cancer. Reduced expression of p27 was detected in 40% of the gastric carcinomas at the mRNA level, while it was detected in 57% at the protein level. No gross alterations of the p27 gene were observed in any of the cases examined by Southern blot analysis. Inimunohistochemical studies revealed that the expression of p27 was well preserved in most of the gastric adenomas, whereas it was so in only 26% of the gastric carcinomas. Fifty–six percent of the carcinomas showed almost no p27–positive cells. Decrease of p27–positive cells significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. The expression of p27 showed an inverse correlation with the expression of cyclin E. These findings suggest that reduction of p27Klpl protein may reflect the progression of gastric carcinomas and may be an indicator of high–grade malignancy.
Characterization of the Extracellular Domain in Vascular Endothelial Growth Factor Receptor‐1 (Flt‐1 Tyrosine Kinase)
Authors: Tanaka Keiji , Yamaguchi Sachiko , Sawano Asako , Shibuya Masabumi
Published: August 2005

Flt‐1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor‐1, binds VEGF and a new VEGF‐related ligand, placenta growth factor, but KDR/Flk‐1 (VEGF receptor‐2) binds only VEGF. To characterize the functional regions in the Flt‐1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt‐1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N‐terminal 1st to 3rd immunoglobulin (Ig)‐like domains of Flt‐1 binds both ligands with high affinity. However, for dimer formation of soluble Flt‐1, a region further downstream in the Flt‐1 extracellular domain was required. Mutant Flt‐1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt‐1 tyrosine kinase. Soluble Flt‐1 carrying the N‐terminal 1st to 3rd Ig region suppressed VEGF‐dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt‐1, suggesting that the binding of one soluble Flt‐1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.
Intelligence in relation to later beverage preference and alcohol intake
Authors: Mortensen Laust H. , Sørensen Thorkild I. A. , Grønbæk Morten
Published: August 2005

Aims  The health effects of drinking may be related to psychological characteristics influencing both health and drinking habits. This study aims to examine the relationship between intelligence, later beverage preference and alcohol intake.

Design  Prospective cohort study.

Setting  Zealand, Denmark.

Participants  A total of 900 obese men and a random population sample of 899 young men.

Measurements  Intelligence testing at the draft board examinations over a 22‐year period between 1956 and 1977. Percentage of wine of total alcohol intake (wine pct), preference for wine (wine pct >50), heavy drinking (>21 drinks per week) and non‐drinking (<1 drink per week), and vocational education from follow‐ups of the initial study sample in 1981–83 and 1992–94.

Findings  A strong dose–response‐like association was found between intelligence quotient (IQ) in young adulthood and beverage preferences later in life in both the obese and the random population sample. At the first follow‐up a 30‐point advantage in IQ [2 standard deviations (SD)] was found to be associated with an odds ratio (OR) for preferring wine over beer and spirits of 1.7 (1.3–2.4). At the second follow‐up the corresponding OR was 2.8 (2.0–3.9). A 30‐point advantage in IQ was found to be associated with an OR for being a non‐drinker of 0.5 (0.3–0.8) at the first follow‐up and second follow‐up. We examined whether, at the second follow‐up, the association between IQ, beverage preferences and non‐drinking could be explained by socio‐economic position (SEP). The association between IQ and non‐drinking disappeared when controlling for SEP. The association between IQ and beverage preferences was attenuated, but remained statistically significant. IQ was not associated with heavy drinking.

Conclusion  Irrespective of socio‐economic position, a high IQ was associated with preference for wine to other beverages, but IQ was not related similarly to alcohol consumption.
A new antihistamine levocetirizine inhibits eosinophil adhesion to vascular cell adhesion molecule‐1 under flow conditions
Authors: Wu P. , Mitchell S. , Walsh G. M.
Published: August 2005

BackgroundWe previously demonstrated that low concentrations of a new antihistamine levocetirizine inhibited eosinophil transmigration through human microvascular endothelial cells.

ObjectiveHere, the inhibitory effect of levocetirizine on eosinophil adhesion to recombinant human vascular cell adhesion molecule‐1 (rhVCAM)‐1 was examined under conditions of shear stress using anin vitromodel of the post‐capillary venules.

MethodsEosinophils isolated from normal subjects were pre‐incubated with a concentration range of levocetirizine (10−6–10−10 m) or negative dilution control. Resting or granulocyte macrophage‐colony stimulating factor (GM‐CSF)‐stimulated cells were pumped through rhVCAM‐1 (10 μg/mL) coated capillary tubes using a microfluidic syringe pump at a precise and constant flow rate (1 dyn/cm2). Images of rolling and firmly adherent eosinophils were captured using real‐time video microscopy.

ResultsLevocetirizine significantly inhibited resting eosinophil adhesion to rhVCAM‐1 with maximal effect at 10−8 M with an EC50of 10−9 m. Levocetirizine almost abolished resting eosinophil adhesion by the 15 min time‐point. GM‐CSF significantly enhanced eosinophil adhesion and their ability to flatten on rhVCAM‐1. Both phenomena were inhibited by levocetirizine in a dose‐dependent manner, at both 5 and 15 min (optimal concentration of 10−8 mwith an EC50of 10−9 m). Real‐time imaging revealed that the effect of levocetirizine on post‐adhesion behaviour (detachment, flatness) contributed to its inhibitory action on eosinophil adhesion to rhVCAM‐1. In contrast, very late antigen (VLA)‐4 mAb inhibited eosinophil adhesion to rhVCAM‐1 from the earliest time‐points.

ConclusionPhysiologically relevant concentrations of levocetirizine inhibit resting and GM‐CSF‐stimulated firm eosinophil adhesion to rhVCAM‐1 under flow conditions.
Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins
Authors: Finlay W. J. J. , DeVore N. C. , Dobrovolskaia E. N. , Gam A. , Goodyear C. S. , Slater J. E.
Published: August 2005

Background
Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour‐intensive. Therefore, we have examined recombinant antibody development by phage‐display against single allergens and protein mixtures.

Objective
We used the avian immunoglobulin system (generated from single VH and VL genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals.

Methods
A single‐chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage‐display and their properties investigated by ELISA and Western blot.

Results
Mono‐specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody‐concentration‐dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti‐Amb a 1 scFvs also mapped Amb a 1‐isoform location in Western blot of ragweed extracts separated by 2D SDS‐PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti‐Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t‐test analysis) to those obtained using conventional assays (radial immunodiffusion).

Conclusion
Phage‐display technology may generate multiple allergen‐specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.
Modulation of Anopheles gambiae gene expression in response to o’nyong‐nyong virus infection
Authors: Sim C. , Hong Y. S. , Vanlandingham D. L. , Harker B. W. , Christophides G. K. , Kafatos F. C. , Higgs S. , Collins F. H.
Published: August 2005

To determine if gene expression of An. gambiae is modulated in response to o’nyong‐nyong virus (ONNV) infection, we utilized cDNA microarrays including about 20 000 cDNAs. Gene expression levels of ONNV‐infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days postinfection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two‐fold up‐ or down‐regulated. Quantitative real‐time PCR analysis (qRT‐PCR) further substantiated the differential expression of six of these genes in response to ONNV infection. These genes have similarity to a putative heat shock protein 70, DAN4, agglutinin attachment subunit, elongation factor 1 alpha and ribosomal protein L35. One gene, with sequence similarity to mitochondrial ribosomal protein L7, was down‐regulated in infected mosquitoes. The expression levels and annotation of the differentially expressed genes are discussed in the context of host/virus interaction including host translation/replication factors, and intracellular transport pathways.
Evidence for alternative electron sinks to photosynthetic carbon assimilation in the high mountain plant species Ranunculus glacialis
Authors: STREB PETER , JOSSE EVE‐MARIE , GALLOUËT EMILY , BAPTIST FLORENCE , KUNTZ MARCEL , CORNIC GABRIEL
Published: August 2005

The high mountain plant species Ranunculus glacialis has a low antioxidative scavenging capacity and a low activity of thermal dissipation of excess light energy despite its growth under conditions of frequent light and cold stress. In order to examine whether this species is protected from over‐reduction by matching photosystem II (PSII) electron transport (ETR) and carbon assimilation, both were analysed simultaneously at various temperatures and light intensities using infrared gas absorption coupled with chlorophyll fluorescence. ETR exceeded electron consumption by carbon assimilation at higher light intensities and at all temperatures tested, necessitating alternative electron sinks. As photorespiration might consume the majority of excess electrons, photorespiration was inhibited by either high internal leaf CO2 molar ratio (Ci), low oxygen partial pressure (0.5% oxygen), or both. At 0.5% oxygen ETR was significantly lower than at 21% oxygen. At 21% oxygen, however, ETR still exceeded carbon assimilation at high Ci, suggesting that excess electrons are transferred to another oxygen consuming reaction when photorespiration is blocked. Nevertheless, photorespiration does contribute to electron consumption. While the activity of the water –water cycle to electron consumption is not known in leaves of R. glacialis, indirect evidence such as the high sensitivity to oxidative stress and the low initial NADP‐malate dehydrogenase (NADP‐MDH) activity suggests only a minor contribution as an alternative electron sink. Alternatively, the plastid terminal oxidase (PTOX) may transfer excess electrons to oxygen. This enzyme is highly abundant in R. glacialis leaves and exceeds the PTOX content of every other plant species so far examined, including those of transgenic tomato leaves overexpressing the PTOX protein. Finally, PTOX contents strongly declined during deacclimation of R. glacialis plants, suggesting their important role in photoprotection. Ranunculus glacialis is the first reported plant species with such a high PTOX protein content.
Ethene (ethylene) production in the marine macroalga Ulva (Enteromorpha) intestinalis L. (Chlorophyta, Ulvophyceae): effect of light‐stress and co‐production with dimethyl sulphide
Authors: PLETTNER INA , STEINKE MICHAEL , MALIN GILL
Published: August 2005

Ethene (ethylene; H2C = CH2) is one of a range of non‐methane hydrocarbons (NMHC) that affect atmospheric chemistry and global climate. Ethene acts as a hormone in higher plants and its role in plant biochemistry, physiology and ecology has been the subject of extensive research. Ethene is also found in seawater, but despite evidence that marine microalgae and seaweeds can produce ethene directly, its production is generally attributed to photochemical breakdown of dissolved organic matter. Here we confirmed ethene production in cultured samples of the macroalga Ulva (Enteromorpha) intestinalis. Ethene levels increased substantially when samples acclimatized to low light conditions were transferred to high light, and ethene addition reduced chlorophyll levels by 30%. A range of potential inhibitors and inducers of ethene biosynthesis were tested. Evidence was found for ethene synthesis via the 1‐aminocylopropane‐1‐acrylic acid (ACC) pathway and ACC oxidase activity was confirmed for cell‐free extracts. Addition of acrylate, a potential ethene precursor in algae that contain the compatible solute dimethylsulphoniopropionate, doubled the ethene produced but no acrylate decarboxylase activity was found. Nonetheless the data support active production of ethene and we suggest ethene may play a multifaceted role in algae as it does in higher plants.