bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and characterization of soluble amino‐terminal domain of NR2B subunit of N‐methyl‐d‐aspartate receptor
Authors: Wong Esther , Ng Fui‐Mee , Yu Chye‐Yun , Lim Peiqi , Lim Leng‐Hiong , Traynelis Stephen F. , Low Chian‐Ming
Published: August 2005

N‐methyl‐d‐aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino‐terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis‐ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis‐ATD2B adopted well‐defined secondary structures and monodispersity. More significantly, the soluble 6xHis‐ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis‐ATD2B with a dissociation constant (KD) of 127.5±45 nM, which was within the range of the IC50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized KD.
Effects of Phe‐to‐Trp mutation and fluorotryptophan incorporation on the solution structure of cardiac troponin C, and analysis of its suitability as a potential probe for in situ NMR studies
Authors: Wang Xu , Mercier Pascal , Letourneau Paul‐Jean , Sykes Brian D.
Published: August 2005

19F NMR spectroscopy is potentially a powerful tool for probing protein properties in situ. However, results obtained using this technique are relevant only if the 19F probe offers minimal perturbation to the surrounding environment. In this paper, we examine the effect of 5‐fluorotryptophan (5fW) incorporation on the three‐dimensional structure of cardiac troponin‐C (cTnC), with the intention of developing a 19F‐labeled TnC for use in in situ 19FNMR. We find that, in general, 5fW does not perturb the structure of the protein significantly. Replacement of residue Phe 153 with 5fW produces no noticeable change in protein conformation. However, replacement of residue Phe 104 with 5fW produces a folding behavior that is dependent on the Escherichia coli strain used to express the mutant. The orientations of the indole rings in these mutants are such that the Trp residue adopts a χ2 of ∼90° in the F104W mutant and ∼−100° in the F153W mutant. Using results from 19F‐1H heteronuclear NOE experiment, we show the replacement of L‐Trp with 5fW at these positions does not change the orientation of the indole ring and the spread of the 5fW side‐chain dihedral angles increases moderately for the F104(5fW) mutant and not at all for the F153(5fW) mutant. Based on these structures, we conclude that the substitution of Phe by 5fW at these two positions has minimal effects on the structure of cTnC and that the 5fW indole rings in both mutants have well defined orientation, making the two mutants viable candidates for use in in situ 19F NMR spectroscopy.
The European standard series in 9 European countries, 2002/2003 – First results of the European Surveillance System on Contact Allergies
Authors: Uter W. , Hegewald J. , Aberer W. , Ayala F. , Bircher A. J. , Brasch J. , Coenraads P.‐J. , Schuttelaar M.‐L. A. , Elsner P. , Fartasch M. , Mahler V. , Belloni Fortina A. , Frosch P. J. , Fuchs T. , Johansen J. D. , Menné T. , Jolanki R. , Krêcisz B. , Kiec‐Swierczynska M. , Larese F. , Orton D. , Peserico A. , Rantanen T. , Schnuch A.
Published: August 2005

Since January 2001, the European Surveillance System on Contact Allergies (ESSCA), supported by European Union funding (contract QLK4‐CT‐2001‐00343), has started to collect patch‐test data. This comprises a standardized clinical history and the patch‐test results using the European standard series, from 17 centres in 9 European countries listed above. In 2002 and 2003, 10 511 patients' test results have been pooled and analysed. The anamnestic data partly reflect the subspecialties of some centres. The most common allergen was nickel sulfate (17.3%); however, large international variations were observed. The prevalence of contact allergy to Myroxylon pereirae resin (balsam of Peru) (5.8%) is coming close to the frequency found with the fragrance mix (6.4%). Regarding contact allergy to chromium compounds, different frequencies were noted in the 2 centres focused on occupational dermatitis (2.3% in the FIOH versus 7.4% in the Nofer Institute). These most likely reflect the beneficial effect of addition of ferrous sulfate in one, but not the other country. As differences may partly be due to different patch‐test reading, standardization may need to be refined further. By providing post‐marketing surveillance in the field of contact allergy, ESSCA will meet its objective of increased consumer safety across Europe.
Contact dermatitis due to clarifying cream
Authors: Serra‐Baldrich E. , Dalmau J. , Pla C. , Muntañola A. A.
Published: August 2005

A 45‐year‐old woman developed a severe eczematous reaction localized on the application site after using a clarifying cream. Patch testing showed positively to vitamin K1.
No Evidence of HTLV‐1 Infection in Japanese Multiple Sclerosis Patients in Polymerase Chain Reaction
Authors: Watanabe Toshiki , Iwasaki Yuzo , Tashiro Kunio , Yoshida Mitsuaki
Published: August 2005

To test possible association of human T cell leukemia virus type 1 (HTLV‐1) with multiple sclerosis (MS), polymerase chain reaction was applied to detect HTLV‐1 DNA integrated into peripheral blood mononuclear cell DNA. Nine Japanese MS patients all gave a negative result, indicating no HTLV‐1 infection in these MS patients.
Mutagenicity Arising from Boiled Rice on Treatment with Nitrous Acid
Authors: Hayatsu Hikoya , Hayatsu Toshiko
Published: August 2005

When an aqueous homogenate of boiled rice was treated with nitrous acid at pH 3, direct‐acting mutagens were formed. The presence of the mutagens was demonstrated by isolating the mutagenic fractions through blue‐rayon adsorption, a method used to extract polycyclic compounds, and subsequent high‐performance liquid chromatography (HPLC). The mutagens were active in Salmonella typhimurium TA100 and TA98 without metabolic activation. Several different mutagens were formed, as judged from the HPLC profile.
Mass Screening for Breast Cancer: Comparison of the Clinical Stages and Prognosis of Breast Cancer Detected by Mass Screening and in Out‐patient Clinics
Authors: Ota Jun , Horino Toshio , Taguchi Tetsuo , Ishida Tsunehiro , Izuo Masaru , Ogita Masami , Abe Rikiya , Watanabe Hiromu , Morimoto Tadaoki , Itoh Sueyoshi , Tashiro Hideya , Yoshida Koichi , Honda Kazuyoshi , Sasakawa Michizou , Enomoto Kohji , Kashiki Yoshitomo , Kido Choichiro , Kuroishi Tetsuo , Tominaga Suketami
Published: August 2005

To establish the criteria for assessing the life‐prolonging effect of mass screening for breast cancer, clinical stage and prognosis of breast cancer detected by mass screening in 11 regions of Japan were compared with those for matched patients in out‐patient clinics. A total of 728 patients detected by mass screening and 1,450 found in the out‐patient clinics were reviewed. The stage of the disease was Tis or I in 40.9% of the patients detected by mass screening, and 28.7% of those found in the out‐patient clinics. In contrast, stage III was found in 9.3% and 14.6%, respectively, indicating that early stages were significantly more common in the patients detected by mass screening. The overall survival curve for the patients detected by mass screening was compared with that for those found in the out‐patient clinics. The 5‐year survival rate was significantly higher in the patients detected by mass screening (91.7% vs. 85.6%; P<0.01), while the 10‐year survival rate was slightly higher in the same group of patients, but the difference from the other group was not significant (80.5% vs. 78.1%). Women who had conducted breast self‐examination (BSE) showed a higher survival rate than those who had not conducted BSE.
Nucleolar Organizer Regions in Hepatocarcinogenesis Induced by N‐2‐Fluorenylacetamide in Rats: Comparison with Bromodeoxyuridine Immunohistochemistry
Authors: Tanaka Takuji , Takeuchi Toshimi , Nishikawa Akiyoshi , Takami Tsuyoshi , Mori Hideki
Published: August 2005

The number of silver‐stained nucleolar proteins (AgNOR) was counted in preneoplastic and neoplastic rat liver lesions induced by N‐2‐fluorenylacetamide (FAA) and was compared with that of bromodeoxyuridine (BrdU)‐incorporating cells detected immunohistochemically using monoclonal antibody against BrdU. Male ACI/N rats were given diet containing 200 ppm FAA for 12, 16 or 20 weeks to induce hepatocellular foci and tumors. The mean numbers of AgNOR stained by a one‐step silver colloid method and BrdU‐labeling indices in various liver cell lesions were as follows: nontreated liver (n = 20), 1.20 and 0.08; nonlesional areas (n=20), 1.33 and 0.13; altered liver cell foci (n = 80), 2.04 and 4.05 [eosinophilic cell type (n = 20), 1.78 and 1.82; clear cell type (n=20), 1.45 and 1.77; basophilic cell type (n=20), 1.99 and 4.58; hyperbasophilic cell type (n=20), 2.94 and 8.02]; neoplastic nodules (n = 10), 3.11 and 2.99; hepatocellular carcinomas (n = 10), 7.22 and 8.29. Thus, the mean number of AgNOR and the value of BrdU‐labeling index were well correlated and both values showed a stepwise increase from normal liver cells to liver cell carcinoma, although some scatter was present. These data suggest that mean number of AgNOR may reflect the cellular kinetics in rat hepatocarcinogenesis, and the one‐step silver colloid method for demonstration of AgNOR may therefore be a simple and useful staining to examine the proliferative nature of cells.
Granulocyte‐colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro
Authors: Nakamaki Tsuyoshi , Sakashita Akiko , Sano Motoharu , Hino Ken‐ichiro , Suzuki Kazuo , Tomoyasu Shigeru , Tsuruoka Nobuyoshi , Honma Yoshio , Hozumi Motoo
Published: August 2005

The interaction of granulocyte‐colony stimulating factor (G‐CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G‐CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10−8M RA induced granulocytic differentiation of APL cells. Although G‐CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G‐CSF significantly enhanced the RA‐induced granulocytic differentiation of APL cells in vitro. Enhancement by G‐CSF was not due to the prolongation of survival of RA‐induced differentiated cells, but the differentiation‐inducing effects of G‐CSF might be evident only in the presence of RA. Since G‐CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G‐CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.
Expression of DNA Polymerase α and Leu3a Molecules in Growing and Saturated Cultures of Human Leukemic Cells: Phenotype Analysis of Proliferative Cells by Flow Cytometry
Authors: Shibata Masao , Hirono Yukari , Takahashi Masaaki , Kaneda Tsuguhiro
Published: August 2005

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase α (pol α), was selected as a marker for proliferative cells, and Leu3a molecule as a cell‐surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein‐conjugated anti‐pol α antibody, developed by us, and commercially available phycoerythrin‐conjugated anti‐Leu3a antibody. The optimal fixative for both proteins was phosphate‐buffered 2% paraformaldehyde. The pol α‐positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol α‐linked fluorescence was observed. On the other hand, the pol α‐positive population in the saturated culture was ca. 70%, with weaker pol α‐linked fluorescence. Thus, the population of pol α‐positive cells and the amount of pol α in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a‐positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate‐conjugated anti‐pol α antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non‐proliferative cells became easier by simultaneous labeling with antibodies against pol α and phenotype‐speciflc proteins.
Synergy of Nocardia rubra Cell Wall Skeleton and Interleukin 2 in the in vivo Induction of Murine Lymphokine‐activated Killer Cell Activity
Authors: Kawase Ichiro , Komuta Kiyoshi , Shirasaka Takuma , Hara Hideki , Tanio Yoshiro , Watanabe Masatoshi , Saito Shin'ichi , Ikeda Toshiyuki , Masuno Tomiya , Kishimoto Susumu , Yamamura Yuichi
Published: August 2005

Combination of an ip injection of Nocardia rubra cell wall skeleton (N‐CWS) and 3 daily sc injections of human recombinant interleukin 2 (rIL 2) into C3H/HeN mice resulted not only in a significant increase in the number of peritoneal cells (PC) but also in a potent induction of their lymphokine‐activated killer (LAK) activity, compared with results obtained with N‐CWS or rIL 2 alone. The augmented LAK activity of PC was mediated by nonadherent, nonphagocytic, Thy‐1.2+∼− and asialo GM1+ cells. Nonadherent PC induced by an ip injection of N‐CWS bound more 125I‐labeled rIL 2 than did normal, nonadherent PC, and generated high LAK activity when cultured overnight with rIL 2. In contrast, normal, nonadherent PC responded only weakly to the overnight stimulation with rIL 2. The phenotype of N‐CWS‐induced PC with an elevated IL 2 responsiveness was Thy‐1.2+∼−, Lyt‐1.1−, Lyt‐2.1− and asialo GM1+, suggesting that the N‐CWS‐stimnlated LAK precursors were derived mainly from the NK cell lineage. However, mature T cells may also be involved in this mechanism, because N‐CWS failed to augment the IL 2 responsiveness of nonadherent PC in BALB/c nu/nu mice. Treatment of CS7BL/6N mice bearing solid Lewis lung carcinoma (3LL) tumors with an intratumoral injection of N‐CWS followed by 6 daily sc injections of rIL 2 resulted in the apparent suppression of tumor growth, while N‐CWS or rIL 2 alone produced no such suppression. These results suggest that N‐CWS augments the antitumor effect of rIL 2 by accumulating LAK precursors and elevating their responsiveness to rIL 2 at the injection site.
Detection of Multidrug Resistance (MDR1) Gene RNA Expression in Human Tumors by a Sensitive Ribonuclease Protection Assay
Authors: Ueda Kazumitsu , Yamano Yoshiaki , Kioka Noriyuki , Kakehi Yoshiyuki , Yoshida Osamu , Gottesman Michael M. , Pastan Ira , Komano Tohru
Published: August 2005

The human MDR1 gene encoding P‐glycoprotein, an energy‐dependent drug‐efflux pump, was initially isolated from a multidrug‐resistant KB carcinoma cell. When a 3 kb genomlc sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5′ KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug‐resistant cell line, KB‐8–5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.
A Novel Multidrug Resistance in Cultured Leukemia and Lymphoma Cells Detected by a Monoclonal Antibody to 85‐kDa Protein, MRK20
Authors: Ohtsu Tomoko , Ishida Yasushi , Tobinai Kensei , Minato Keisuke , Hamada Hirofumi , Ohkochi Etsuko , Tsuruo Takashi , Shimoyama Masanori
Published: August 2005

A monoclonal antibody, MRK20, in F(ab′)2 form [MRK20‐F(ab′)2], which reacts with 85‐kDa membrane protein in a doxorubicin (ADM)‐resistant subline (K562/ADM) of human myelogenous leukemia cell line, K562, was examined for reactivity with 41 cultured human leukemia and lymphoma cell lines. None of these cell lines had ever been exposed to any anticancer agent in vitro except K562/ ADM. The relative resistance index to various drugs was calculated by dividing the 50% growthinhibitory concentration (IC50) of the test cell line by IC50 of K562 (the negative control in the antibody experiment). MRK20‐F(ab′)2 reacted with seven cell lines, KYO‐1 derived from chronic myelogenous leukemia in blastic crisis (CMLbc), CMK from acute megakaryoblastic leukemia, HEL from erythroleukemia, P31/FUJ from acute monocytic leukemia, KOPM‐28 from CMLbc, PL‐21 from acute promyelocytic leukemia and K562/ADM, MRK20‐F(ab′)2 did not react with 34 other cell lines. All seven MRK20‐F(ab′)2‐positive cell lines had relative resistance index values of 2 or more to anthracyclines (ADM, pyrarubicin, daunorubicin), mitoxantrone, etoposide, bleomycin, and pepleomycin. There was no distinct correlation between the reactivity to MRK20‐F(ab′)2 and a higher relative resistance index than 2 to vinca alkaloids, actinomycin‐D, cisplatin, 4‐hydroperoxycyclophos‐phamide, nimustine hydrochloride, methotrexate or cytarabine. These results indicate that MRK20‐F(ab′)2 detects a novel multidrug resistance to anthracyclines, mitoxantrone, etoposide, bleomycin and pepleomycin in cultured human leukemia and lymphoma cells.
Geographical Variance in the Risk of Gastric Stump Cancer: No Increased Risk in Japan?
Authors: Tersmette Anne C. , Giardiello Francis M. , Offerhaus G. Johan A. , Tersmette Kasper W. F. , Ohara Kumiko , Vandenbroucke Jan P. , Tytgat Guido N. J.
Published: August 2005

Geographical differences may exist in the risk of gastric stump cancer. Therefore, we performed meta‐analysis of literature reports in Japan (n=3), the USA (n=4), and Europe (n = 20) on the risk of postgastrectomy cancer. The weighted mean relative risk of stump cancer in Japan was 0.28, 95% confidence limits 0.21–0.38 as compared to 1.53, 95% confidence limits 0.98–2.41 in the USA and 1.66, 95% confidence limits 1.55–1.79 in Europe. Thus, the risk of gastric cancer in the postgastrectomy patient seems decreased in Japan and is significantly less than in the USA or Europe, where an increased risk exists. Since there is a high risk of gastric cancer of the intact stomach in Japan, the discovery of a low cancer risk in the gastric stump may provide evidence that these gastric cancers are two different entities.
Point Mutation of c‐Ki‐ras Oncogene in Gastric Adenoma and Adenocarcinoma with Tubular Differentiation
Authors: Kihana Toshimasa , Tsuda Hitoshi , Hirota Teruyuki , Shimosato Yukio , Sakamoto Hiromi , Terada Masaaki , Hirohashi Setsuo
Published: August 2005

The presence of point mutation at codons 12,13 and 61 of the c‐Ki‐ras oncogene was investigated in 7 cases of gastric adenoma and 35 cases of gastric adenocarcinoma using DNA samples from formalin‐fixed and paraffin‐embedded tissues. Oligonucleotides encompassing the three codons were amplified by using the polymerase chain reaction (PCR), and then examined for point mutation by the selective oligonucleotide hybridization technique. Point mutation was detected in three of the 7 adenomas (43%) and three of the 35 carcinomas (9%). All the gastric adenomas showed the histology of tubular adenoma, being very similar to that of colonic adenoma. The 35 cases of gastric adenocarcinoma were classified into 17 cases of differentiated type and 17 cases of undifferentiated type including signet‐ring cell carcinoma. The point mutation of c‐Ki‐ras oncogene was detected only in the differentiated type (3/17, 18%), and there was no case with point mutation in the undifferentiated type. These results suggest that the genetic mechanism of carcinogenesis differs between the differentiated type and the undifferentiated type of gastric adenocarcinoma, and also that c‐Ki‐ros activation is possibly involved in a relatively early step of the “adenoma‐carcinoma sequence,” which leads to the development of a portion of differentiated adenocarcinomas in the stomach.
Inhibitory Effect of 8‐Chloro‐cyclic Adenosine 3′,5′‐Monophosphate on Cell Growth of Gastric Carcinoma Cell Lines
Authors: Takanashi Atsushi , Yasui Wataru , Yoshida Kazuhiro , Yokozaki Hiroshi , Saito Daizo , Abe Kaoru , Urakami Kenichi , Mild Keizaburo , Tahara Eiichi
Published: August 2005

A cAMP analogue, 8‐chloro‐cAMP (8‐Cl‐cAMP), selectively binds to site 1 receptor of type II regulatory subunit (RII) of cAMP‐dependent protein kinase. The effects of 8‐Cl‐cAMP on human gastric carcinoma cell lines were studied. Twenty μM 8‐CI‐cAMP clearly inhibited cell growth in six cell lines (TMK‐1, KATO‐III, MKN‐7, ‐28, ‐45, and ‐74) but not in MKN‐1. Cell population in the G1 phase was increased in KATO III cells, which were most responsive to 8‐Cl‐cAMP, while cell cycle progression in TMK‐1 and MKN‐1 cells was apparently not influenced by 8‐Cl‐cAMP. The various changes induced by 8‐Cl‐cAMP were further analyzed in TMK‐1 cells. Decrease of type I regulatory subunit (RI) of cAMP‐dependent protein kinase and translocation of RII from cytosol to nucleus were induced by 8‐Cl‐cAMP treatment. 8‐CI‐cAMP increased the level of cAMP‐response element (CRE) binding protein in addition to inducing FOS mRNA, whose promoter contains CRE. 8‐Cl‐cAMP decreased the expression of mRNA for transforming growth factor‐α (TGF‐α), while the expression of epidermal growth factor receptor was not changed. Expression of HRAS and MYC mRNAs was slightly increased, whereas the amounts of HRAS and MYC proteins remained unchanged. Our results overall suggest that 8‐Cl‐cAMP might be a useful tool for antitnmor therapy of gastric cancers and that cell growth inhibition by 8‐Cl‐cAMP might account for the decrease of TGF‐α expression by tumor cells.
Enhancement by Verapamil of Neocarzinostatin Action on Multidrug‐resistant Chinese Hamster Ovary Cells: Possible Release of Nonprotein Chromophore in Cells
Authors: Miyamoto Yoichi , Maeda Hiroshi
Published: August 2005

Multidrug‐resistant CHRC5 cells were about 10‐fold more resistant to the proteinaceous anticancer drug neocarzinostatin (NCS) and its nonprotein chromophore (NPC) than the parental AUXB1 cells. There was little difference in cell growth, glutathione content, or activities of several antioxidant enzymes between the two cell lines. The degree of intracellular incorporation and extracellular excretion of fiuorescein isothiocyanate‐labeled NCS by CHRC5 cells was similar to that of AUXB1 cells. On the other hand, 20 μM verapamil or 27 μM cepharanthine restored the susceptibility of CHRC5 cells to NCS and NPC to the level of AUXB1 cells. In addition, NPC was found to suppress the photolabeling of [3H]azidopine (a known P‐glycoprotein‐binding ligand) to plasma membranes of CHHC5 cells. All these findings favor the possibility that NPC was excreted via P‐glycoprotein, which may contribute to the resistance of CHRC5 cells to NCS.
Cancer Mortality Survey in a Spa Area (Misasa, Japan) with a High Radon Background
Authors: Mifune Masaaki , Sobue Tomotaka , Arimoto Hiroko , Komoto Yoshiaki , Kondo Sohei , Tanooka Hiroshi
Published: August 2005

The 1952–88 cancer mortality records for inhabitants of the Misasa spa area, Japan, which has a high radon background, and a neighboring control area without any radon spa were analyzed (average outdoor Rn concentration: 26 mBq‐liter−1 in Misasa vs. 11 mBq‐liter−1 in the control area). Standardized mortality ratios (SMRs) for cancers of all sites were significantly lower among the inhabitants of both Misasa (male 0.538; female 0.463) and the control area (male 0.850; female 0.770), than in the whole Japanese population. Poisson regression analysis showed that the relative risks among the inhabitants of Misasa were significantly lower than in the control area for deaths from cancers of all sites (0.67) and stomach cancer (0.59). The relative risk of lung cancer death was also lower (0.55 times) in Misasa than in the control area, although the difference was not statistically significant. These results suggest that the linear no‐threshold hypothesis for radiation risk may not be valid for exposure to low doses of radon.
Insertion/Deletion Polymorphism and Other Restriction Fragment Length Polymorphisms in the MCC Gene
Authors: Miyoshi Yasuo , Nishisho Isamu , Miki Yoshio , Mori Takesada , Kinzler Kenneth W. , Vogelstein Bert , Nakamura Yusuke
Published: August 2005

The MCC gene is a candidate as a tumor suppressor gene for colorectal neoplasms. Further, MCC is tightly linked to the familial adenomatous polyposis (FAP) locus by linkage and physical analysis. Hence, restriction fragment length polymorphisms (RFLPs) of this gene might be very useful for presymptomatic diagnosis of individuals in families segregating mutant alleles of the APC gene. Here we report the identification of five polymorphic systems in MCC gene (both cDNA and genomic), one of which is an insertion/deletion polymorphism that is detectable by a polymerase chain reaction method. These five RFLP systems should be useful for linkage studies in FAP and for examining loss of heterozygosity at this locus in colonic polyps and tumors.
Detection of the Ligand Activity of the c‐erbB‐2 Protein in Calf Serum
Authors: Shan RuJiao , Matsuda Satoru , Ichino Motohide , Yamamoto Tadashi
Published: August 2005

We established NIH3T3 derivatives in which wild‐type c‐erbB‐2 or activated c‐erbB‐2 having a point mutation in the sequence coding for the transmembrane domain was expressed. These cell lines were termed RC and A4, respectively. A4 cells but not RC or NIH3T3 cells grew even in the presence of a low concentration (0.05%) of calf serum (CS), although the rate of their proliferation was low. In media containing 0.1% CS, both A4 cells and RC cells but not NIH3T3 cells could proliferate. Furthermore, RC cells induced foci formation when cultured in media containing 0.5% and 5% CS, while growth of the parental NIH3T3 cells was contact‐inhibited under these conditions. These data suggest the presence of a factor(s) which activates protein‐tyrosine kinase activity of the c‐erbB‐2 protein. In fact, the c‐erbB‐2 protein prepared from RC cells showed CS‐dependent protein‐tyrosine kinase activity when assayed in membrane fractions.
Carcinogenicity of 1,2‐DimethyIhydrazine in Colorectal Tissue Heterotopically Transplanted into the Glandular Stomach of Rats
Authors: Nakagawa Yoshiki , Watanabe Hiromitsu , Takahashi Tadateru , Ito Akihiro , Dohi Kiyohiko
Published: August 2005

The present study was designed to examine the effect of the intestinal carcinogen 1,2‐dimethyl‐hydrazine (DMH) on grafted colorectal mucosa implanted into the glandular stomach of rats. Four groups were studied: Group 1 received the operation and DMH, Group 2 received the operation alone, Group 3 received DMH alone and Group 4 (controls) received only a sham operation. For Groups 1 and 2, about 8‐mm diameter segments of colorectal tissue obtained from various sites in the large intestine of 8‐week‐old male F344 rats were isologously implanted into the fundic region of the stomachs of age‐matched rats. DMH was injected at a dose of 20 mg/kg body weight i.m. per week for 20 weeks beginning 4 weeks after the operation. The animals were then observed for 8 months after the initial DMH treatment. In Group 1, adenocarcinomas developed in 41 of 60 successful implants (68%). Furthermore, poorly differentiated type tumors were observed in the grafts obtained from the rectum. This finding was contrary to that for intrinsic rectal tumors, all of which were well differentiated. The histochemical staining of mucin in the tissues from different sites of the large intestine revealed that sulfomucin, which normally exists essentially only in the intrinsic descending colon or rectum, was present in the grafts from the proximal ascending or ascending colon. No gastric tumors were observed in the control rats, which received either DMH or sham operations alone. Tumors in the intrinsic large intestine were observed only in rats that received DMH. These results indicate that colorectal mucosa implanted into the glandular stomach, like the intrinsic large intestine, is still sensitive to tumorigenesis caused by DMH.
Expression and Characterization of Kinase‐active v‐erbB Protein Using a Baculovirus Vector System
Authors: Morishita Kaoru , Iwamoto Masahiro , Murakami Kenji , Kubota Minoru , Maeda Susumu , Toyoshima Kumao , Yamamoto Tadashi
Published: August 2005

The v‐erbB gene is an oncogene of the avian erythroblastosis virus encoding a protein that is a truncated version of the epidermal growth factor receptor. The v‐erbB protein was expressed alone or as polyhedrin‐erbB fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The expression level of the fusion protein whose polyhedrin portion consisted of only 8 amino‐terminal amino acids was more than ten times higher than that of the non‐fusion protein. Studies with tunkamycin showed that the recombinant v‐erbB proteins were glycosylated. The recombinant protein autophosphorylated tyrosine residues, and phosphorylated a synthetic tyrosine‐containing peptide and lipocortin I. These observations indicate that functional v‐erbB protein can he expressed in silkworm‐derived cells, and furthermore, that this system can be used for large‐scale production.
c‐Myc Interferes with the Commitment to Differentiation of Murine Erythroleukemia Cells at a Reversible Point
Authors: Takada Shinji , Yamamoto Tohru , Ohmori Yasufumi , Matsui Yasuhisa , Obinata Masuo
Published: August 2005

When murine erythroleukemia (MEL) cells, containing the transferred rat c‐myc gene under the control of human metallothionein II gene promoter, are induced to differentiate with dimethyl sulfoxide, the level of differentiation is dependent on the c‐Myc level, which is modulated by the addition of Zn ions. In this work, we examined the point of inhibition of differentiation by elevated levels of c‐Myc in one (clone 38‐2) of the typical transformants. Commitment assay indicated that elevated levels of c‐Myc interfere with entry of the transformant into the commitment event, but when c‐myc expression was reduced by removing Zn ions from the medium, the cells could reenter the commitment program. However, once the cells were committed, such cells could not return to the uncommitted state. In addition, time‐dependent expression of two erythroid specific genes was inhibited by elevated levels of c‐Myc in time‐dependent manner. These results suggest that c‐Myc modulates MEL cell differentiation at a reversible point of commitment.
Induced Synthesis of O6‐Methylguanine‐DNA Methyltransferase in Rat Hepatoma Cells Exposed to DNA‐damaging Agents
Authors: Fukuhara Masao , Hayakawa Hiroshi , Sakumi Kunihiko , Sekiguchi Mutsuo
Published: August 2005

When the rat hepatoma cell line H4IIE was treated with DNA‐damaging agents such as N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), ultraviolet light and γ‐rays, the O6‐methylguanine‐DNA methyltransferase activity increased 2 to 3 times over the level seen in non‐treated cells. SDS/polyacrylamide gel electrophoresis followed by fluorography revealed that a single species of methyltransferase protein with a molecular weight of 25,500 was present in both non‐treated and treated cells. Northern blot analysis using a cloned rat cDNA as a probe revealed that the enzyme activity increased because transcription of the gene was enhanced. The level of enzyme activity increased within 48 h after UV irradiation and remained at a higher level for 150 h. Following UV irradiation, the cells become more resistant than the normal cells to MNNG.
Reduced Levels of Transforming Growth Factor‐beta Type I Receptor in Human Gastric Carcinomas
Authors: Ito Masanori , Yasui Wataru , Nakayama Hirofumi , Yokozaki Hiroshi , Ito Hisao , Tahara Eiichi
Published: August 2005

The expressions of transforming growth factor beta (TGF‐β) and its receptor and TGF‐β inhibitory element (TIE)‐binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF‐β mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF‐β expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF‐β when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE‐binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK‐1, whose growth was inhibited by TGF‐β, had only type I receptor for TGF‐β and showed a high level of TIE‐binding protein. Conversely, MKN‐1, a TGF‐β ‐resistant cell line, exhibited an extremely low level of TGF‐β receptor and had no TIE‐binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF‐β, they showed a reduction in TGF‐β type I receptor and a low level of TIE‐binding protein, resulting in escape from growth inhibition by TGF‐β.
Adenocarcinoma of the Lung with Selective Metastasis to the Lung: Clinical, Histologic and DNA‐Cytofluorometric Analyses
Authors: Nomori Hiroaki , Matsuno Yoshihiro , Noguchi Masayuki , Tsugane Shoichiro , Shimosato Yukio , Hirohashi Setsuo
Published: August 2005

In order to examine selective lung metastasis in lung adenocarcinoma, 7 patients who had died due to metastasis exclusively to the lung were analyzed. Because all of the tumors had been well differentiated adenocarcinoma, 21 cases of well differentiated adenocarcinoma which had metastasized to both the lung and other distant organs were selected as controls. Histologically, 6 of the 7 study cases were bronchiolo‐alveolar carcinoma (BAC) of variable cytological type or papillary adenocarcinoma with a thin fibrovascular stroma without apparent destruction of the alveolar wall. There was no difference in clinical findings or nuclear DNA content (NDC) between the BAC and papillary adenocarcinoma with thin fibrovascular stroma. The fibrovascular stroma in the study cases was significantly thinner than that in the control cases (P<0.01). Cytofluorometrically, the study cases had a significantly lower and less abnormal NDC than the control cases in terms of the mean NDC and DNA histogram patterns (P<0.01 and P<0.05, respectively). On the basis of these findings, we conclude that 1) BAC can be classified within the same spectrum as well differentiated papillary adenocarcinoma, and 2) selective lung metastasis occurs in well differentiated adenocarcinomas showing mild NDC abnormalities and having a thin fibrovascular stroma without apparent destruction of the alveolar wall.
Effect of Physical Exercise on the Content of 8‐Hydroxydeoxyguanosine in Nuclear DNA Prepared from Human Lymphocytes
Authors: Inoue Tamotsu , Mu Zhouseng , Sumikawa Kazuhide , Adachi Kozaburo , Okochi Toshikazu
Published: August 2005

Effects of exercise on the formation of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a biomarker of oxidative DNA damage, and other purine metabolites such as hypoxanthine, xanthine and uric acid were examined. Venous blood and urine were collected from swimmers and distance runners before and after the usual training. The amount of 8‐OH‐dG obtained from nuclear DNA of lymphocytes decreased remarkably after intermittent swimming. The amount of nuclear 8‐OH‐dG also declined after distance running, but this difference is statistically not significant. After each exercise, plasma concentrations of hypoxanthine, xanthine and uric acid rose significantly. Urinary excretion of hypoxanthine increased, and xanthine and uric acid decreased after exercise. The 8‐OH‐dG‐to‐creatinine ratio in urine increased slightly after swimming or running. It is supposed that the repair of oxidative DNA damage is augmented by exercise. As far as we know, this is the first report concerning the effect of exercise on oxidative damage in nuclear DNA.
Lack of Correlation between the Gap Junctional Communication Capacity of Human Colon Cancer Cell Lines and Expression of the DCC Gene, a Homologue of a Cell Adhesion Molecule (N‐CAM)
Authors: Mesnil Marc , Piccoli Colette , Klein Jean‐Louis , Morand Isabelle , Yamasaki Hiroshi
Published: August 2005

In many human colorectal cancers, the DCC gene encoding for a homologue of the neural cell adhesion molecule (N‐CAM) is found to be deleted. Previous work suggested that gap Junctional intercellular communication (GJIC) might play an important role in carcinogenesis and could be regulated by the expression of cell adhesion molecules such as E‐cadherin in some epithelial cell systems. In order to examine whether the deletion of the putative cell adhesion molecule DCC is related to the level of GJIC, which might, in turn, be important in human colorectal cancers, we compared levels of expression of the DCC gene with the GJIC capacity of a panel of human colorectal adenocarcinoma cell lines isolated from different stages of tumor progression. While the level of GJIC varied between the cell lines studied, we found no correlation between their communication capacity and DCC expression revealed by a reverse‐transcriptase/polymerase chain reaction method. This lack of correlation suggests that DCC is not a crucial regulator of GJIC.
Levels of Complement Regulatory Molecules in Lung Cancer: Disappearance of the D17 Epitope of CD55 in Small‐cell Carcinoma
Authors: Sakuma Takahiko , Kodama Ken , Kara Tomoko , Eshita Yoshimi , Shibata Nobuhiko , Matsumoto Misako , Seya Tsukasa , Mori Yoichi
Published: August 2005

The levels of complement‐regulatory molecules (complement receptor type one [CR1], decay‐accelerating factor [DAF], membrane cofactor protein [MCP], and an inhibitor of membrane attack complex [CD59]) in lung cancer cells were analyzed to investigate the relation between their expression and histological subtypes, and the possibility of homologous complement deposition on cancer cells. In 25 cell lines (10 adenocarcinoma, 3 large‐cell carcinoma, 7 small‐cell lung cancer [SCLC], and 5 squamous cell carcinoma), flow cytometric analysis revealed that MCP was expressed in all cell lines, whereas none of the cell lines was CR1‐positive. CD59 was detected in all cells. The DAF epitope defined by IA10 was expressed in all cells except one large cell carcinoma cell line. However, another epitope for anti‐DAF monoclonal antibody, D17, was not detected in 5 (71.4%) SCLC and in 4 (22.2%) non‐small‐cell lung cancer. This disparity was seen in most cell lines, irrespective of histological subtypes. The loss of D17 reactivity seemed to be pertinent to malignant phenotype, because most of the normal pulmonary cells possessed the D17 epitope. Furthermore, a cell line lacking DAF (IA10−/D17−) allowed alternative pathway‐mediated homologous complement (C3) deposition after pretreatment with anti‐MCP antibody. This raises a new possibility for immuno‐targeting of cancer. These cell lines should be useful in studying the biology of lung cancer.
Expression Behavior of 85‐kDa Membrane Protein in Adriamycin‐resistant Tumor Cells and the Inhibition of Human Tumor Growth in Athymic Mice by MRK‐20 Monoclonal Antibody against the Protein
Authors: Ishii Shigetaka , Naito Mikihiko , Tsuruo Takashi
Published: August 2005

Treatment of tumors with monoclonal antibodies against tumor antigen is one of the selective modalities for cancer therapy. We examined the therapeutic effect of MRK‐20 (IgG1), a murine monoclonal antibody against resistance‐associated 85‐kDa membrane protein. The 85‐kDa protein is expressed on the surface of multidrug‐resistant cells induced by adriamycin. This protein is also expressed in some multiple‐drug‐resistant cells, including atypical multidrug‐resistant cells. The protein, once lost during long‐term culture without adriamycin, was rapidly induced by treatment with adriamycin but not with vinblastine or etoposide, suggesting a close relationship of the protein with adriamycin resistance but not with multidrug resistance. The antibody MRK‐20 suppressed the growth of subcutaneously implanted tumors expressing the 85‐kDa protein. Adriamycin‐resistant human ovarian tumor 2780AD and innately resistant human erythroleukemia HEL cells in athymic mice were completely cured by treatment with MRK‐20 antibody when the antibody was administered i.v. 2 days after s.c. tumor implantation. On the other hand, MRK‐20 did not show any effect on the growth of the 85‐kDa protein‐negative A2780 human ovarian tumor. These results indicate that the effect of MRK‐20 is highly specific to cells expressing 85‐kDa protein.
Stereo(C7)‐dependent Topoisomerase II Inhibition and Tumor Growth Suppression by a New Quinolone, BO‐2367
Authors: Yoshinari Tomoko , Mano Eiichi , Arakawa Hiroharu , Kurama Masae , Iguchi Tomoko , Nakagawa Susumu , Tanaka Nobuo , Okura Akira
Published: August 2005

A new antimicrobial quinolone (—)BO‐2367, (‐)‐7‐[(1R*,2R*,6R*)‐2‐amino‐8‐azabicyclo[4.3.0.]‐non‐3‐en‐8‐yl]‐l‐cyclopropyl‐6,8‐difluoro‐l,4‐dihydro‐4‐oxo‐3‐quinorinecarboxyric acid, strongly inhibited both mammalian and bacterial topoisomerase II. The IC50 values of (—)BO‐2367 against the DNA relaxation activity of L1210 topoisomerase II and the supercoiling activities of Escherichia coli gyrase and Micrococcus luteus gyrase were 3.8, 0.5, and 1 μM, respectively. This compound enhanced double‐stranded DNA cleavage mediated by topoisomerase II not only with purified enzyme, but also with intact L1210 cells. All these activities of (—)BO‐2367 were more than 2‐fold stronger than those of VP‐16. Intriguingly, (+)BO‐2367, which has an enantiomeric substitnent at the C7 position of (‐)BO‐2367, did not affect the activity of the mammalian topoisomerase II, while it inhibited E. coll gyrase. Intraperitoneal injection of (‐)BO‐2367 at 0.08 mg/kg increased the lifespan of CDF1female mice bearing ascitic L1210 leukemia by 2.4 times, and subcutaneous injection at 1.25 mg/kg completely inhibited the growth of colon 26 carcinoma implanted subcutaneously. These results suggest that (—)BO‐2367 is a potent antitumor agent which targets topoisomerase II. These enantiomers should be a useful tool for studying drug‐topoisomerase II interactions.
Improved Detection Rate of Early Breast Cancer in Mass Screening Combined with Mammography
Authors: Ohuchi Noriaki , Yoshida Koichi , Kimura Michio , Ouchi Akio , Kamioki Shuzo , Shiiba Ken‐ichi , Matoba Naoya , Kojima Seiichi , Takahashi Kiichi , Matsuno Seiki , Fukao Akira , Abe Rikiya , Mori Shozo
Published: August 2005

A cohort study to compare mass screening with and without mammography was conducted in Miyagi Prefecture, Japan in order to establish whether the effectiveness of breast cancer screening would be improved when mammography was combined with physical examination. A trial of mass screening combined with mammography was carried out in 9634 women aged over 50. Lateral imaging of the breast using single‐view film mammography was performed at the first stage of mass screening in addition to physical examination of the breast. Results in the trial were compared with those obtained in 35511 age‐matched subjects without mammography. Thirty breast cancers were found in the trial with a detection rate of 0.31%, which was much higher than that (0.08%) obtained by physical examination without mammography. In 15 of the 30 patients the breast tumor was not palpated at the first screening, but abnormal findings were detected in the mammography. A higher rate (73%) of early breast cancer was obtained in the screening trial with mammography than that (39%) obtained in the screening with physical examination alone. Mass screening combined with mammography is superior to that without mammography for breast cancer screening, especially for the detection of non‐palpable, early breast cancer.
Rapid Diagnosis of Gastric Malignant Lymphoma from Biopsy Specimens: Detection of Immunoglobulin Heavy Chain Rearrangement by Polymerase Chain Reaction
Authors: Ono Hiroyuki , Kondo Hitoshi , Saito Daizo , Yoshida Shigeaki , Shirao Kuniaki , Yamaguchi Hajime , Yokota Toshihiro , Hosokawa Koichi , Fukuda Haruhiko , Hayashi Shuya , Ochiai Atsushi , Oguro Yanao
Published: August 2005

The endoscopic appearances of the gastrointestinal lymphomas differ widely, and it is often difficult to make the distinction between a benign lymphoproliferative disorder and a malignant lymphoma even with a histologic evaluation. Since almost all primary malignant lymphomas of the gastrointestinal tract are of B‐cell origin, the confirmation of monoclonality in immunoglobulin (Ig) is helpful for differential diagnosis. Rearrangements of the Ig heavy chain gene were examined by polymerase chain reaction (PCR) analysis in frozen biopsy specimens of human stomach. The sensitivity of the analysis was sufficient to detect even a 5% clonal B‐cell proliferation and results could be obtained within 17 h. In a clinical investigation, seven of eight cases (88%) of primary gastric malignant lymphoma showed a single band in polyacrylamide gel electrophoresis (PAGE) after PCR, suggesting a monoclonal proliferation of B‐cell lineage. By contrast, all seven cases of reactive lymphoreticnlar hyperplasias showed a broad smear pattern in PAGE, which is thought to reflect polyclonal proliferation. None of the lymphocytes infiltrating around gastritis (7 cases), gastric ulcers (12 cases) and gastric carcinomas (15 cases) showed a monoclonal proliferation pattern. These findings suggest that detection of monoclonality in Ig heavy gene rearrangement by PCR is useful for the differential diagnosis of B‐cell lymphoproliferative diseases in the gastrointestinal tract.