bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines
Authors: Hoffmann Erich , Lipatov Aleksandr S. , Webby Richard J. , Govorkova Elena A. , Webster Robert G.
Published: August 2005

If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N
154
) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S
223
(characteristic of 2004 viruses) was converted to N
223
(as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N
223
, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N
223
residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.
Deleterious genomic mutation rate for viability inDrosophila melanogasterusing concomitant sibling controls
Authors: Gong Yi , Woodruff R.C , Thompson J.N
Published: August 2005

New deleterious mutations may reduce health and fitness and are involved in the evolution and maintenance of numerous biological processes. Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. However, these estimated rates vary widely, mainly because of inadequate experimental controls. Here we describe an experimental design (the Binscy assay) with concomitant sibling controls and estimateUfor viability inDrosophila melanogasterto be 0.31. This estimate, like most published studies, focuses on viability mutations and the overall deleterious genomic mutation rate would therefore be higher.
ENHANCED RELEASE OF ENDOTHELIUM‐DERIVED HYPERPOLARIZING FACTOR IN SMALL CORONARY ARTERIES FROM RATS WITH CONGESTIVE HEART FAILURE
Authors: Ueda Atsunori , Ohyanagi Mitsumasa , Koida Satsuki , Iwasaki Tadaaki
Published: August 2005

1. Previous studies have suggested that the production of nitric oxide (NO) is reduced in coronary vessels of animals with congestive heart failure (CHF). However, the response to endothelium‐derived hyperpolarizing factor (EDHF) in small coronary resistance arteries from CHF rats has not been investigated. The aim of the present study was to determine whether flow‐induced dilation (FID) is altered in small coronary arteries from CHF rats and to characterize the role of EDHF in this process.

2. Small coronary arteries (97 ± 6 µm) were isolated from control rats and from rats in which CHF was induced by left coronary artery ligation. The arteries were cannulated at 60 mmHg with flow. Changes in internal diameter were examined using videomicroscopy.

3. There was no significant difference in FID in small coronary arteries between control and CHF rats (68 ± 6 vs 61 ± 4% (expressed as a percentage of maximal dilation induced by nitroprusside (%MaxDNP)), respectively). Flow‐induced dilation in control rat vessels showed greater attenuation by NG‐monomethyl‐l‐arginine (l‐NMMA) than vessels from CHF rats (%NO‐mediated FID 32 ± 5 vs 16 ± 3% (%MaxDNP), respectively). Pretreatment with indomethacin had no significant effect on FID in vessels from either rat group. Flow‐induced dilation was attenuated by KCl (40 mmol/L) to a greater degree in vessels from CHF rats in the presence of l‐NMMA and indomethacin compared with vessels from control rats (%EDHF‐mediated FID: 36 ± 4 vs 25 ± 5% (%MaxDNP), respectively). Flow‐induced dilation was abolished by removal of the endothelium and was significantly decreased in vessels from CHF rats in response to charybdotoxin plus apamin or tetrabutylamnmonium compared with control rat vessels. 17‐Octadecynoic acid had no significant effect on FID in vesselas from either control or CHF rats.

4. In conclusion, the FID of small coronary arteries is mediated by K+ channels, including the KCa channels. Endothelium‐derived hyperpolarizing factor‐mediated dilation may compensate for the loss of NO‐mediated dilation in CHF.
INHIBITION OF HUMAN CYP1A2 OXIDATION OF 5,6‐DIMETHYL‐XANTHENONE‐4‐ACETIC ACID BY ACRIDINES: A MOLECULAR MODELLING STUDY
Authors: Paxton James W , Kestell Philip , Chiang Daniel , Zhou Shufeng , Lewis David FV
Published: August 2005

1. The aim of the present study was to investigate the structural requirements for the inhibition of 6‐methyl‐hydroxylation of the antitumour agent 5,6‐dimethyl‐xanthenone‐4‐acetic acid (DMXAA) by acridine analogues and use a CYP1A2 homology model to provide some insight into this interaction.

2. Concentrations causing 50% inhibition (IC50) of the 6‐methylhydroxylation of DMXAA were determined in human liver microsomes in the presence of various acridines. Some of the acridines were also tested for their ability to inhibit the CYP1A2‐mediated 7‐ethoxyresorufin O‐de‐ethylation. The molecular modelling studies of human CYP1A2 used the crystal structure of rabbit CYP2C5 as a template based on protein sequence homology and an interactive docking procedure using a dynamic hydrogen bond feature.

3. The in vitro IC50 studies for the inhibition of 6‐methylhydroxylation of DMXAA indicated: (i) the importance of the position of the carboxamide side‐chain on the acridine nucleus (and, to a lesser extent, its composition); (ii) the addition of hydroxyl groups to the 5‐, 6‐ and 7‐position of the acridine nucleus diminished the inhibitory potency; and (iii) amsacrine (acridine nucleus with methansulphonanilide side‐chain at the 9‐position) had no significant inhibitory effect. Similar structural trends were observed for the inhibition of O‐de‐ethylation of 7‐ethoxyresorufin by acridines, supporting the involvement of CYP1A2 in DMXAA 6‐methyl hydroxylation.

4. The molecular modelling studies indicated: (i) both DMXAA and N‐[2‐(dimethylamino)‐ethyl]acridine‐4‐carboxamide (DACA) form two hydrogen bonds plus putative π–π stacking interactions with the CYP1A2‐binding domain, typical of CYP1A2 substrates and inhibitors; (ii) the DMXAA 6‐methyl group is 4.0 Å from the central iron atom of the heme moiety and ideal for oxidation; (iii) the known oxidation sites for DACA are orientated away from the heme iron, supporting the non‐involvement of CYP1A2; and (iv) amsacrine did not fit the putative CYP1A2 site owing to the steric hindrance of the bulky methanesulphonanilide side‐chain.

5. These results suggest that docking studies with this homology model may be useful in the design of further acridine anticancer agents, in particular to identify agents that do not interact either as substrates or inhibitors with the CYP1A2‐binding domain.
ANTI‐OXIDANT MECHANISMS OF KOLAVIRON: STUDIES ON SERUM LIPOPROTEIN OXIDATION, METAL CHELATION AND OXIDATIVE MEMBRANE DAMAGE IN RATS
Authors: Farombi EO , Nwaokeafor IA
Published: August 2005

1. In the present study, we have examined the ability of kolaviron, a natural biflavonoid from Garcinia kola seeds, to prevent the susceptibility of rat serum lipoprotein to undergo oxidative modification in vitro and ex vivo. In addition, its ability to chelate metal ions and mitigate iron/ascorbate‐induced damage to microsomal lipids was investigated.

2. Lipoprotein resistance to copper‐induced oxidation was highly improved in rats treated with kolaviron (100 mg/kg) for 7 days, as demonstrated by a significant increase in lag time compared with control. A significant (P < 0.05) decrease in area under the curve (AUC) and slope of propagation was observed in kolaviron‐treated rats compared with control. Conjugated dienes formed after 240 min of lipoprotein oxidation were markedly decreased in kolaviron‐treated rats compared with controls. Malondialdehyde concentrations were significantly reduced in the serum lipoproteins of kolaviron‐treated rats with an attendant significant increase in the total anti‐oxidant activity compared with control.

3. In vitro, kolaviron (10–60 µmol/L) inhibited the Cu2+‐induced oxidation of rat serum lipoprotein in a concentration‐dependent manner. Kolaviron, at 20 and 60 µmol/L, produced 48 and 87% inhibition of oxidation of lipoprotein, respectively. Compared with control, kolaviron, at 10 and 20 µmol/L, resulted in 29 and 47% decreases in AUC, respectively. In addition, kolaviron (10 µmol/L) elicited a 53% increase in lag time, whereas 40 and 60 µmol/L kolaviron produced 38 and 88% decreases in slope, respectively.

4. Kolaviron effectively prevented microsomal lipid peroxidation induced by iron/ascorbate in a concentration‐dependent manner. Kolaviron at the highest dose tested (90 µmol/L) had a significant chelating effect on Fe2+ (78%).

5. In conclusion, our data demonstrate that kolaviron protects against the oxidation of lipoprotein, presumably by mechanisms involving metal chelation and anti‐oxidant activity, and, as such, may be of importance in relation to the development of atherosclerosis.
Female resistance and male force: context and patterns of copulation in the New Zealand stitchbird Notiomystis cincta
Authors: Low Matthew
Published: August 2005

The New Zealand stitchbird or hihi Notiomystis cincta is unique in that it has two distinct mating positions, in addition to the male standing on the female's back, as is seen in all other birds, it also copulates face‐to‐face. In this study, 43 male stitchbirds first attracted a female to their territory and supplemented their within‐pair matings by intruding into other territories and attempting forced copulations – often resulting in high levels of female harassment. I recorded the temporal variation of both attempted and successful copulations relative to the female's fertile period in order to understand the function of copulation variation in this species. Each of 105 observed copulations were classified according to whether they were: (1) within‐pair or extra‐pair, (2) forced or unforced, and (3) face‐to‐face or standing. Two copulation categories ‘within‐pair unforced standing’ (50%) and ‘extra‐pair forced face‐to‐face’ (27%) accounted for the majority of all observed copulations, and this supported the previous categorisation of face‐to‐face copulation being forced by extra‐pair males in this species. However, in 10% of copulations the female conceded to copulate with an extra‐pair male in a standing position while displaying only subtle signs of resistance, thus, female stitchbirds may show convenience polyandry when the costs of resistance are high. The peak in copulation frequency centred on two days prior to the laying of the first egg and occurred within a period of six days before and seven days after the first egg was laid. Unsuccessful extra‐pair forced copulation attempts closely followed the distribution of all copulations. Male age and morphometrics did not predict whether a female would resist or accept extra‐pair copulation attempts, and female resistance did not appear to depend on male quality. Despite the current trend focussing on female fitness benefits associated with EPCs, it appears that male stitchbirds gain EPCs through forced copulation and females gain no apparent benefit.
Male‐biased sex ratio among unhatched eggs in great tit Parus major, blue tit P. caeruleus and collared flycatcher Ficedula albicollis
Authors: Cichoń Mariusz , Sendecka Joanna , Gustafsson Lars
Published: August 2005

An increasing number of studies have demonstrated that brood sex ratios are frequently unequal, but the proximate mechanisms underlying this deviation are largely unknown. In the current study we analysed deviation from expected 1:1 sex ratio among dead embryos from unhatched eggs collected from partially unhatched clutches of three passerine bird species. We showed that male embryos were significantly overrepresented among unhatched eggs of great tit Parus major, blue tit P. caeruleus and collared flycatcher Ficedula albicollis. Moreover, the bias in sex ratio differed among species and tended to differ among study years. We discuss several hypotheses to explain the observed male bias among unhatched eggs. We conclude that sex specific embryo mortality may contribute to explain the observed variation in sex ratios in several species of wild birds and that sexing unhatched eggs is important in studies of sex ratio allocation.
Differentiation of a Ewing's Sarcoma Cell Line towards Neural and Mesenchymal Cell Lineages
Authors: Kodama Ken , Doi Osamu , Higashiyama Masahiko , Yokouchi Hideoki , Tateishi Ryuhei , Mori Yoichi
Published: August 2005

Two different pathways of differentiation were investigated in Ewing's sarcoma (ES) cell line, designated CADO‐ES1, which has been established in our laboratory. This cell line was induced to differentiate and display a neural phenotype when treated with dibutyryl cyclic adenosine mono‐phosphate or when cultured in serum‐free medium (HB101). In these in vitro differentiation studies, two different phenotypes were demonstrated by light and electron microscopy. One phenotype, present in a major portion of the cell population, had long neurites in which microtubules were ultrastructurally demonstrated. The other one, present in a minor portion of the cell population, consisted of flat cells with many short processes. After differentiation in serum‐free medium, tumorigenicity in nude mice or colony‐forming efficiency in soft agar was strongly depressed. In the cells, N‐myc, c‐fos and c‐src genes were not amplified, and although c‐myc was amplified by up to 2‐fold, depending on the culture conditions, this appeared to be unrelated to the changes of phenotype. When tumor cells were transplanted into nude mice, cartilage was formed. The cartilage was immunoreactive with the antibody for HLA‐ABC, indicating that it was derived from the tumor cells, not from mouse tissue.
Transfection‐induced Tumor Necrosis Factor‐α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine‐activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule‐1 on the Glioma Cells
Authors: Takaoka Toru , Yoshida Jun , Mizuno Masaaki , Sugita Kenichiro
Published: August 2005

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor‐α (TNF‐α) gene into human glioma cells (U251‐SP), which were used as target cells. TNF‐α is known to increase both the expression of intercellular adhesion molecule‐1 (ICAM‐1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine‐activated killer (LAK) cell cytolysis. We compared the expression of ICAM‐1 induced by TNF‐α generated by the TNF‐α gene‐transfected cells with that induced by exogenously added TNF‐α. When the TNF‐α gene was transfected into U251‐SP cells, the expression of ICAM‐1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF‐α. Also, the cytolytic activity of LAK cells induced by transfection‐induced TNF‐α was significantly stronger than that induced by exogenously added TNF‐α. The increased susceptibility was quenched by anti‐ICAM‐1 monoclonal antibody. These data indicated that continuous expression of ICAM‐1 induced by TNF‐α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.
Increased Activity of Insulin‐like Growth Factor‐binding Protein in Human Thyroid Papillary Cancer Tissue
Authors: Yashiro Tohru , Arai Mariko , Shizume Kazuo , Obara Takao , Murakami Hitomi , Hizuka Naomi , Emoto Naoya , Miyakawa Megumi , Ito Kunihiko , Tsushima Toshio
Published: August 2005

It has been shown that both insulin‐like growth factor‐I (IGF‐I) and IGF‐binding proteins (IGFBPs) are produced by thyroid cells in culture and that the cells respond to IGF‐I with increased DNA synthesis, suggesting an autocrine/paracrine role of IGF‐I in the regulation of thyroid cell growth. We investigated the tissue contents of immunoreactive IGF‐I (irIGF‐I) and IGFBPs in human papillary carcinoma and compared them with those of normal thyroid tissue. When irIGF‐I was measured after separation of the IGFBPs by gel‐filtration, its content in carcinoma tissue was not different from that in adjacent normal tissue (566±58 vs. 424±75 pg/mg protein, N = 10). Nor was there any difference in the abundance of IGF‐I mRNA expression determined by slot blot analysis. On the other hand, IGFBP activity measured in terms of 125I‐IGF‐I binding was significantly higher in cancer extracts. Western ligand blot analysis of IGFBPs revealed several species (24–42 kDa) of IGFBPs. The IGF‐I‐binding activity of 38–41 kDa species (corresponding to IGFBP‐3) was not different between extracts of cancer tissue and those of normal tissue, whereas that of 28–32 kDa species was significantly higher in cancer tissue extracts. Since IGFBPs have been reported to modulate cellular responses to IGF‐I, the present data suggest that higher IGFBP activity in cancer tissue is involved in regulating growth of thyroid papillary carcinoma cells.
Characterization of Mice Bearing Subclones of Colon 26 Adenocarcinoma Disqualifies Interleukin‐6 as the Sole Inducer of Cachexia
Authors: Soda Kuniyasu , Kawakami Masanobu , Kashii Akiyoshi , Miyata Michio
Published: August 2005

A snbclone (clone 20) of chemically induced, marine colon adcnocarcinoma with a potent ability to induce cachexia and another subclone (clone 5) without such an activity were transplanted to syngeneic mice (CDFi) and their tissue weights, blood components and cytokine levels in sera were compared. Mice transplanted with clone 20 showed a profound body‐weight loss by 15 days after inoculation when the tumor accounted for less than 1% of the body weight, along with marked reduction of food and water intakes. Thereafter, they transiently gained in body weight with restoration of food and water intakes. Thus, the change in body weight was biphasic and not proportional to the tumor size. Body fat was lost preferentially, accompanied with a decrease in plasma triglyceride levels. The thymus contracted remarkably, and the peripheral lymphocyte count decreased extensively. Mice transplanted with clone 5, in contrast, did not show any of these changes characteristic of cachexia. Serum concentration of interleukin‐6, which has been proposed as the principal inducer of cachexia in mice with colon 26, increased in mice with clone 5 to levels comparable to those in mice with clone 20. The changes in mice hearing clone 20 could not all be explained in terms of known biological activities of interleukin‐6. Additional unknown factors, therefore, are presumed to contribute to cachexia in mice with clone 20. Identification of them should be helpful in the care of cachectic patients.
Intravenous Administration of Polyethylene Glycol‐modified Tumor Necrosis Factor‐α Completely Regressed Solid Tumor in Meth‐A Murine Sarcoma Model
Authors: Tsutsumi Yasuo , Kihira Tetsunari , Tsunoda Shin‐ichi , Kubo Kazuyoshi , Miyake Masaharu , Kanamori Toshhiori , Nakagawa Shinsaku , Mayumi Tadanori
Published: August 2005

Complete regression of solid tumors was achieved by plural intravenous (i.v.) administrations of polyethylene glycol (PEG)‐modified tumor necrosis factor‐a (TNF‐α), prepared by covalently modifying natural human TNF‐α with N‐succinimidyl succinate PEG. The anti‐tumor efficacy of PEG‐modifled TNF‐α (MPEG‐TNF‐α), in which 56% of the TNF‐α‐lysine residues were coupled with PEG, was compared with that of native TNF‐α in the Meth‐A murine fibrosarcoma model. MPEG‐TNF‐α and native TNF‐α were given as i.v. injections twice a week for 2 weeks. The anti‐tumor activity of MPEG‐TNF‐α was dose‐dependent and was far superior to that of native TNF‐α. Complete regression was observed in 3 of the 8 mice administered native TNF‐α at the dose of 10,000 JRU (Japan reference unit), but 4 of the 5 remaining mice died during the therapeutic period. At 5,000 JRU of native TNF‐α, no case of complete regression was observed. By contrast, complete regression was obtained in all 10 mice given 200 JRU of MPEG‐TNF‐α. No side‐effects were observed at the dose of 500 JRU of MPEG‐TNF‐α, which was 2.5 times the minimal dose (200 JRU) of MPEG‐TNF‐α required for complete regression in all treated mice. MPEG‐TNF‐α appears to have potential as a candidate anti‐tumor therapeutic agent.
HTLV‐II Non‐integrated Malignant Lymphoma Induction in Japanese White Rabbits Following Intravenous Inoculation of HTLV‐II‐infected Simian Leukocyte Cell Line (Si‐IIA)
Authors: Hayashi Kazuhiko , Ohara Nobuya , Koirala Tirtha Raj , Ino Hideo , Chen Hong Li , Teramoto Norihiro , Kondo Eisaku , Yoshino Tadashi , Takahashi Kiyoshi , Yamada Masao , Tomita Noriko , Miyamoto Kanji , Fujimoto Koji , Yoshikawa Yasuhiro , Akagi Tadaatsu
Published: August 2005

Lymphoma induction in rabbits by an unknown factor derived from an HTLV‐II‐producing simian (Cynomolgus) leukocyte cell line (Si‐IIA) is reported. Thirteen of 17 male Japanese white rabbits (76%) inoculated intravenously with Si‐IIA cells developed malignant lymphoma including Hodgkin‐like lymphoma between 62 and 167 days after inoculation. Historically, there was extensive diffuse or nodular infiltration of either large cell type or mixed type lymphoma cells in many organs, frequently involving the spleen, liver, lymph nodes and kidneys, and less frequently the thymus, bone marrow, lungs, heart, skin and gastrointestinal tract. Hodgkin‐like lymphoma was also observed in two rabbits. Chromosomal analysis of five cell lines established from tumor‐bearing rabbits revealed the male rabbit karyotype. The immunophenotype of these tumor cells was usually T‐cell (CD5+or, r RT1+, RT2+or‐, CD45+, CD4−, RABELA− and MHC class II‐DQ+) except for Hodgkin‐like lymphoma cells which expressed only CD45. However, integration of the HTLV‐II provirus genome could not be demonstrated in the tumor tissues or any of the rabbit cell lines by polymerase chain reaction or Southern blot analysis. Moreover, no lymphoma was induced by inoculation of HTLV‐IIC, MOT (other HTLV‐II‐producing human cell lines) or TALL‐1 (control). Two of four rabbits injected with cell‐free pellets from Si‐IIA cultures died of malignant lymphoma (15‐20 days). Five irradiated rabbit cell lines were inoculated but only one (Ra‐SLN) induced lymphoma in 1 of 3 rabbits at 27 days. Neither Herpesvirus saimiri nor Herpesvirus ateles (simian oncogenic viruses) was detected in Si‐IIA cells by immunofluorescence testing. These data suggest that the high rate of lymphoma induction in rabbits may be caused not by only HTLV‐II or well known simian oncogenic viruses, but rather by an unknown passenger agent derived from Si‐IIA or HTLV‐IIA, with which Si‐IIA was established.
Lack of Any Positive Effect of Intestinal Metaplasia on Induction of Gastric Tumors in Wistar Rats Treated with N‐Methyl‐N‐nitrosourea in Their Drinking Water
Authors: Watanabe Hiromitsu , Ando Yasumi , Yamada Kazumasa , Okamoto Taro , Ito Akihiro
Published: August 2005

The influence of intestinal metaplasia on gastric cancer induction was examined in five‐week‐old male Wistan:Crj rats. The animals were first treated with two 10 Gy doses of X‐rays to the gastric region at a 3‐day interval (total 20 Gy) and then, starting two months after the irradiation, received 100 ppm N‐methyl‐N‐nitrosourea (MNU) in their drinking water for 15 weeks. Thereafter they were maintained for 37 weeks with or without a dietary 1% sodium chloride (NaCl) supplement. The incidences of gastric adenocarcinomas in the MNU or MNU plus NaCl groups were significantly higher than in animals receiving X‐rays plus MNU with or without NaCl. Intestinal metaplasias and the numbers of alkaline phosphatase(ALP)‐positive foci were significantly increased in the X‐ray irradiation groups but the numbers of ALP‐positive foci were not increased with or without 1% NaCl. An inverse relationship between incidences of gastric tumors and intestinal metaplasias was apparent. The present experiment thus showed that the presence of intestinal metaplasia does not exert a positive influence on induction of gastric neoplasia by MNU in the rat.
Immunohistochemical Demonstration of Intestinal‐type Alkaline Phosphatase in Stomach Tumors Induced by N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine in Rats
Authors: Yuasa Hirofumi , Hirano Kazuyuki , Kodama Hirohiko , Nakanishi Hayao , Imai Toshio , Tsuda Hiroyuki , Imaida Katsumi , Tatematsu Masae
Published: August 2005

A potyclonal antibody against rat intestinal‐type alkaline phosphatase (I‐ALP) was generated and proven to be applicable immunohistochemically to paraffin‐embedded sections. Expression of I‐ALP in normal tissues, intestinal metaplasia and stomach tumors induced by N‐methyl‐N′‐nitro‐N‐nitroso‐guanidine (MNNG) was then investigated in five different strains of rats. Male SD (Crj:CD), Lewis (LEW/Crj), WKY (WKY/NCrj), Wistar (CrjrWistar) and F344 (F344/DuCrj) animals were given drinking water containing 100/μ/ml of MNNG for 30 weeks and were killed at week 50. Among the 5 strains, stomach adenocarcinomas were found most frequently in the SD case. The susceptibility of rats to induction of stomach carcinoma did not correlate with the development of intestinal metaplasias in each strain. Histochemical staining for mucin demonstrated all stomach tumors (adeno‐matous hyperplasias and well‐differentiated adenocarcinomas) to consist mainly of gastric type cells (pyloric gland cell and surface mucous cell types), with intestinal‐type tumor cells (goblet cell and intestinal absorptive cell types) being only occasional findings. Immunohistochemically, I‐ALP was strongly positive on the striated cell borders of small intestinal absorptive cells of the villus and on brush borders of epithelial cells of kidney proximal tubules. I‐ALP was also detected in the normal stomach, limited to the striated cell borders of absorptive cells of the upper one‐fourth of intestinal metaplastic glands. I‐ALP may thus be a useful marker for stomach tumor cells of intestinal absorptive cell type, indicative of maturation and differentiation. No stomach tumors consisting mainly of intestinal‐type cells were found, and therefore there was no suggestion of any derivation from intestinal metaplasias.
CPT‐11: Population Pharmacokinetic Model and Estimation of Pharmacokinetics Using the Bayesian Method in Patients with Lung Cancer
Authors: Yamamoto Nobuyuki , Tamura Tomohide , Karato Atsuya , Uenaka Kazunori , Eguchi Kenji , Shinkai Tetsu , Ohe Yuichiro , Oshita Fumihiro , Arioka Hitoshi , Nakashima Hajime , Shiraishi Jun‐ichi , Fukuda Minoru , Higuchi Shun , Saijo Nagahiro
Published: August 2005

In this study, we aimed to develop a population pharmacokinetic model for CPT‐11 and to use the Bayesian method to estimate CPT‐11 pharmacokinetic parameters in each of 43 patients who received combined therapy consisting of CPT‐11 and etoposide. The group was divided into first and second data sets of 30 and 13 patients, respectively. We developed a population pharmacokinetic model of CPT‐11 based on the first data set. The individual pharmacokinetic parameters [area under the concentration curve (AUC) and clearance (CD] were subsequently estimated by using the Bayesian method on the second data set. Plasma CPT‐11 concentrations were measured by high‐performance liquid chromatography, and compartmental pharmacokinetic models were fitted by the Bayesian method. The population pharmacokinetic model was developed by using the nonlinear mixed effect model. We selected the volume of the central compartment (Vc), CL, and distribution rate constants (K12, K21) as population pharmacokinetic parameters. The population mean values (CV%) of Vc, CL, K12, and K21 were, respectively, 31.8 (15.7%) liter/m2,14.1 (27.8%) liter/h/m2,1.1 (8.4%)/h, and 0.41 (30.3%)/h. Residual intraindivirtual variability was 22.9%. The optimal sampling regime for estimation of the AUC and CL in using the Bayesian method was the two time points of 1 and 8 h post infusion. The mean predictive error, the mean absolute predictive error, and the root mean squared error were ‐3.3, 9.4, 3.2% (AUC) and 6.3, 10.0, 3.5% (CL), respectively. We concluded that the AUC and CL of CPT‐11 could be estimated from plasma concentrations at two times by using the Bayesian method.
Cytokine Production in Five Tumor Cell Lines with Activity to Induce Cancer Cachexia Syndrome in Nude Mice
Authors: Iseki Hideaki , Kajimura Naoko , Ohue Chiharu , Tanaka Rie , Akiyama Yasuto , Yamaguchi Ken
Published: August 2005

To identify the so‐called toxohormone, which is a tumor‐derived factor with activity to induce cancer cachexla syndrome in tumor‐bearing animals, 5 human cancer cell lines with this activity were studied for cytokine production. Tumor cell products with activity to inhibit lipoprotein lipase (LPL) were shown to play an important role in the development of the cancer cachexia syndrome. All culture media conditioned by the 5 cell lines possessed LPL‐inhibitory activity. However, the activity differed with the cell line. In order to characterize the activity, we examined whether the cultured cells produced cytokines with activity to inhibit LPL. A melanoma cell line, SEKI, and a neuroepithelioma cell line, NAGAI, were found to express a large amount of leukemia inhibitory factor (LIF) mRNA. Furthermore, both of these cell lines were demonstrated to produce a large amount of LIF protein, and plasma levels of LIF were extremely elevated in SEKI‐ and NAGAI‐bearing nude mice, indicating that LIF produced by the tumor cells induced cancer cachexia syndrome in the animals. Thus, LIF fulfills the requirements for a toxohormone, except for suppressive activity on liver catalase. In contrast, the mechanisms responsible for cachexia in the MKN‐1‐, LX‐1‐ and LS180‐bearing mice remain unknown. These findings suggest that various types of bioactive substances produced by cancer cells could be toxohormones.
Allelic Frequency of p53 Gene Codon 72 Polymorphism in Urologic Cancers
Authors: Wu Wen‐Jeng , Kakehi Yoshiyuki , Habuchi Tomonori , Kinoshita Hidefumi , Ogawa Osamu , Terachi Toshiro , Huang Chun‐Hsiung , Chiang Chin‐Pei , Yoshida Osamu
Published: August 2005

Alterations in the p53 tumor suppressor gene appear to be important in the development of many human tumors. The wild‐type p53 gene has a polymorphism at codon 72 that presents the arginine (CGC) or proline (CCC) genotype, which recently has been reported to be associated with genetically determined susceptibility to smoking‐related lung cancers. To determine whether this p53 genotype influences individual risk of urologic cancer and/or its progression, we used polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis to assay the allelic frequencies of this polymorphism in 85 renal cell carcinoma patients, 151 urothelial cancer patients, 33 testicular cancer patients, 28 prostatic cancer patients and 56 patients without neoplastic disease. The allelic distributions of the three genotypes (Arg/Arg, Arg/Pro, Pro/Pro) in patients with renal cell carcinoma (29.4%, 55.3%, 15.3%), urothelial cancers (45.7%, 39.7%, 14.6%), testicular cancer (45.4%, 48.5%, 6.1%) or prostate cancer (42,9%, 50.0%, 7.1%) did not differ significantly from those in the normal controls. However, Pro/Pro genotype in renal cell carcinoma and urothelial cancer (smoking‐related cancers) was more frequent than that in prostate cancer and testicular cancer (smoking‐unrelated cancers) with borderline significance (P=0.0881). There was no particular correlation between frequency of the three genotypes and grade or stage of each type of tumor. The association of genetic predisposition to urologic cancers with p53 gene codon 72 polymorphism is not so clear as the previous study of Japanese lung cancer patients, but this polymorphism may play some role in urothelial cancers and renal cell carcinoma, in which smoking is an epidemiological risk factor.
Characterization of a Novel Human Tumor Necrosis Factor‐α Mutant with Increased Cytotoxic Activity
Authors: Masegi Tsukio , Kato Arata , Kitai Kazuo , Fukuoka Masami , Ogawa Hiroko , Ichikawa Yataro , Nakamura Satoshi , Watanabe Naoki , Niitsu Yoshiro
Published: August 2005

Various novel recombinant human tumor necrosis factor‐α (TNF) mutants were prepared using protein engineering techniques, and their cytotoxic activity was compared with that of the intact form of TNF (intact TNF). Mutant 471 (a TNF mutant molecule with the deletion of 7 amino acids at the amino‐terminal and the substitution of Pro8Ser9Asp10 by ArgLysArg) had a 6‐fold higher cytotoxic activity against murine L929 cells. The mutant TNF had an increased ability to bind to TNF receptor on murine L929 fibroblasts cells. A cross‐linking study revealed that mutant 471 had an increased ability to form an active trimer. Mutant 471 also showed higher cytotoxic activity against human KYM myosarcoma cells and human MIA PaCa‐2 pancreatic carcinoma cells. The possible cachectin activity of the mutant was almost the same as that of intact TNF. These results suggest that mutant 471 might be a more promising candidate as an anticancer agent than intact TNF.
Expression of Sialylparagloboside in a Case of Liposarcoma: Aberrant Glycosylation in Tumors Arising in Adipose Tissues
Authors: Kubo Yoshitugu , Hamanaka Sumiko , Kawashima Ikuo , Tai Tadashi , Kawamura Susumu , Okita Kiwamu
Published: August 2005

Gangliosides of liposarcoma, lipoma and lipids from omental tissues were analyzed. By immunostaining after thin layer chromatography, gangliosides of liposarcoma were identified as GM3, sialyl‐paragloboside and GD3, whereas those of lipoma were GM3 and GD3, and those of fat in omental tissues were GM3, GD1a, GD3 and some unknown ones. Expression of sialylparagloboside is thought to be very rare.
1,2‐Dibromoethane as an Initiating Agent for Cell Transformation
Authors: Colacci Annamaria , Perocco Paolo , Vaccari Monica , Vià Claudia Da , Silingardi Paola , Manzini Elena , Horn Wolfango , Bartoli Silvana , Grilli Sandro
Published: August 2005

The two‐stage transformation assay increases the sensitivity of cells to chemicals and permits detection of carcinogens acting as initiating agents. 1,2‐Dibromoethane, a representative halogenated aliphatic, has been tested in the two‐stage BALB/c 3T3 cells transformation test at dosages from 16 μM to 128 μM. This dose range is much lower than those previously found efficient in transforming BALB/c 3T3 cells. Apart from the lowest dose, which induced borderline effects, all the other assayed dosages appeared to induce heritable changes in the traget cells. The initiated cells were revealed as fully transformed foci both in the combination with a chronic promoting treatment and also by allowing cells to perform more rounds of cell replication. The results clearly show that 1,2‐dibromo‐ethane can act as an initiator of cell transformation.
Tyrosine Phosphorylation of the Proto‐oncogene Product Vav and Its Association with the Adapter Grb2/Ash in a Human Leukemia Cell Line UT‐7
Authors: Hanazono Yutaka , Sasaki Ko , Odai Hideharu , Mimura Toshihide , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru
Published: August 2005

The vav proto‐oncogene product (Vav) is expressed exclusively in hematopoietic cells and is reported to have guanine nucleotide exchange activity. Here we report that granulocyte‐macrophage colonystimulating factor, interleukin‐3, and erythropoietin induce tyrosine phosphorylation of Vav in a human leukemia cell line UT‐7. Tyrosine phosphorylation of Vav is rapid and transient; it occurs within 1 min of the stimulation and at physiological concentrations of the factors. Furthermore, we show that Vav is constitutively associated with the adapter molecule Grb2/Ash in UT‐7. These data suggest that tryosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav are members of a signaling pathway leading to Ras activation in hematopoietic cells.
Reversal of Tumor Necrosis Factor Resistance in Tumor Cells by Adriamycin via Suppression of Intracellular Resistance Factors
Authors: Watanabe Naoki , Okamoto Tetsuro , Tsuji Naoki , Sasaki Hiroyoshi , Akiyama Shinichiro , Kobayashi Daisuke , Sato Tsutomu , Yamauchi Naofumi , Niitsu Yoshiro
Published: August 2005

Tumor necrosis factor (TNF) and various chemotherapeutic drugs show synergistic antitumor effects in vitro and in vivo, though the mechanism is not clear. Based on our previous finding that endogenous TNF (enTNF) acts as an intracellular resistance factor against exogenous TNF by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD), we examined the suppression of these resistance factors by chemotherapeutic drugs and the resulting increase in TNF cytotoxicity. Pretreatment of HeLa cells, which produce an appreciable amount of enTNF and show apparent TNF resistance, with TNF followed by adriamycin (ADM) resulted in an additive effect, whereas pretreatment with ADM followed by TNF resulted in a synergistic effect. After treatment of HeLa cells with ADM, the expression of enTNF was remarkably suppressed and MnSOD activity was decreased by one‐half. These results indicate that suppression of the intracellular resistance factors, i.e., enTNF and MnSOD, by ADM plays an important role in the mechanism of the synergistic antitumor effect of TNF in combination with ADM.
Contribution of Chromosome 9p21‐22 Deletion to the Progression of Human Renal Cell Carcinoma
Authors: Kinoshita Hidefumi , Yamada Hitoshi , Ogawa Osamu , Kakehi Yoshiyuki , Osaka Mitsuhiko , Nakamura Eijiro , Mishina Mutsuki , Habuchi Tomonori , Takahashi Rei , Sugiyama Taketoshi , Yoshida Osamu
Published: August 2005

To investigate the possible role of genomic aberrations of chromosome 9p21‐22 in the tumorigenesis of human renal cell carcinoma (RCC), 10 RCC cell lines, 55 primary RCCs and 5 metastatic lesions were studied by Southern blotting and polymerase chain reaction‐based analysis. Nine of 10 RCC cell lines showed a homozygous deletion of MTS1/CDKN2/(p16), while only 1 in 55 primary tumors had this deletion. Loss of heterozygosity on 9p21‐22 was observed in 5 of 10 informative primary RCCs from patients with metastasis, but in only 4 of 31 informative tumors (13%) without metastasis (P= 0.025). Furthermore, 3 of 5 metastatic tumors (60%) showed hemi‐ or homozygous deletion of MTS1/CDKN2. These results indicate that the 9p21‐22 deletion may be a relatively late event in RCC tumorigenesis and could be associated with RCC metastasis.
Effects of Nine Active Ingredients in Chinese Herbal Medicine Sho‐saiko‐to on 2‐(2‐Furyl)‐3‐(5‐nitro‐2‐furyl)acryIamide Mutagenicity
Authors: Ohtsuka Masanori , Fukuda Kazunori , Yano Hirohisa , Kojiro Masamichi
Published: August 2005

The antimutagenic effects of nine active compounds in the Chinese herbal medicine “sho‐saiko‐to” on mutagenesis induced by a direct‐acting mutagen, 2‐(2‐furyI)‐3‐(5‐nitro‐2‐furyl)acrylamide (AF‐2) were investigated in Salmonella typhimurium, strain TA100. The active compounds examined were classified into two major groups, saponins and flavonoids, the former comprising glycyrrhizin, saikosaponins a, c and d, and ginsenosides Rbl and Rgl, and the latter, baicalin, baicalein and wogonin. Saikosaponin a and ginsenoside Rbl were found to reduce the mutagenicity of AF‐2 significantly when applied post‐AF‐2‐treatment in the Salmonella mutagenicity assay. Ginsenoside Rbl also decreased the mutagenic activity of AF‐2 in a simultaneous treatment protocol. The results indicate that saikosaponin a and ginsenoside Rbl may enhance DNA repair, and ginsenoside Rbl may also have the ability to inactivate the mutagenic activity of AF‐2 directly. On the other hand, saikosaponin d and baicalin showed a slight enhancing effect. None of the compounds, except baicalein, showed any toxic effect on the test strain. These findings may be useful for the development of chemopreventive agents.
Prevention by Methionine of Enhancement of Hepatocarcinogenesis by Coadministration of a Choline‐deficient L‐Amino Acid‐defined Diet and Ethionine in Rats
Authors: Tsujiuchi Toshifumi , Kobayashi Eisaku , Nakae Dai , Mizumoto Yasushi , Andoh Nobuaki , Kitada Hiromichi , Ohashi Kazuo , Fukuda Tomokazu , Kido Akira , Tsutsumi Masahiro , Denda Ayumi , Konishi Yoichi
Published: August 2005

The effects of methionine on hepatocarcinogenesis induced by Coadministration of a choline‐deflcient L‐amino acid‐defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline‐supplemented L‐amino acid‐defined (CSAA) diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by Coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c‐myc and c‐Ha‐ras expression, as well as generation of 8‐hydroxyguanine (8‐OHGua) and 2‐thiobarbituric acid‐reacting substances (TBARS), by Coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8 or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c‐myc and c‐Ha‐ras, 8‐OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocked these actions. These results indicate that addition of methionine prevents the induction of c‐myc and c‐Ha‐ras expression, 8‐OHGua formation and TBARS generation, as well as hepatocellular lesions, by Coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.
Expression of CD44 Variant Exons 8‐10 in Gastric Cancer
Authors: Yamaguchi Akio , Saito Mitsugu , Goi Takanori , Iida Atsushi , Takeuchi Kazuo , Hirose Kazuo , Nakagawara Gizo , Urano Takeshi , Furukawa Koichi , Shiku Hiroshi
Published: August 2005

The expression of CD44 variant containing variant exons 8‐10 product (CD44v8‐10) was studied by western blot analysis and immunohistochemistry in gastric cancers using a monoclonal antibody, 44‐IV. On western blots, a single band of 130 kD was recognized in stomach cancer cell lines. CD44v8‐10 expression, with reactivity localized in the cell membrane, was found in 65 (33.5%) of the 194 advanced gastric cancers. There was no correlation between CD44v8‐10 immunoreactivity and serosal, lymphatic, or lymph node invasion. However, there was significant correlation with CD44v8‐10 immunoreactivity and venous invasion. CD44v8‐10‐positive cancers were more frequently associated with hematogenous metastasis than those which were immunonegative. There was an inverse association between CD44v8‐10 immunoreactivity and peritoneal dissemination, especially in diffuse type adenocarcinomas. These observations indicate that CD44v8‐10 may play a role in the metastasis of gastric cancer.
Modifying Influence of Swine Serum‐induced Liver Fibrosis on Development of Preneoplastic Lesions in Rat Liver
Authors: Wada Shigetsugu , Kato Toshio , Mutai Mamoru , Ozaki Keisuke , Yamaguchi Shuji , Kim Dae Joong , Baba‐Toriyama Hiroyasu , Asamoto Makoto , Ito Nobuyuki , Tsuda Hiroyuki
Published: August 2005

Modifying effects of fibrosis or a cirrhotic state, caused by treatment with swine serum (SS), on the induction of preneoplastic focal lesions were assessed in a rat medium‐term liver bioassay model for the detection of environmental carcinogens, in which the test compound is administered during the promotion phase after initiation with diethylnitrosamine. In experiment I, repeated intraperitoneal administration of SS concomitantly with the hepatopromoting agent deoxycholic acid (DCA) or phenobarbital (PB) resulted in a cirrhotic state and a significant increase in the number or size of preneoplastic glutathione S‐transferase placental form (GST‐P)‐positive liver cell foci as compared to the corresponding DCA or PB alone groups. In experiment II, SS was given prior to commencement of tbe same medium‐term bioassay system, in which a known hepatopromoting agent, DCA, 17‐α‐ethynylestradiol, or 2‐acetyIaminofluorene, was applied. In this case, the liver did not show obvious cirrhotic change and, rather than any enhancement, slight inhibition of promotion occurred. The results indicate that a coexisting, but not a pre‐existing, cirrhotic condition acts to increase growth pressure on GST‐P+ preneoplastic foci, and suggest that concomitant administration of SS with the promoting agent could be applied to improve the sensitivity of the assay protocol.
Analysis of Early Initiating Event(s) in Radiation‐induced Thymic Lymphomagenesis
Authors: Muto Masahiro , Chen Ying , Kubo Eiko , Mita Kazuei
Published: August 2005

Since the T cell receptor rearrangement is a sequential process and unique to the progeny of each clone, we investigated the early initiating events in radiation‐induced thymic lymphomagenesis by comparing the oncogenic alterations with the pattern of γ T cell receptor (TCR) rearrangements. We reported previously that after leukemogenic irradiation, preneoplastic cells developed, albeit infrequently, from thymic leukemia antigen‐2+ (TL‐2+) thymocytes. Limited numbers of TL‐2+ cells from individual irradiated B10.Thy 1.1 mice were injected into B10.Thy 1.2 mice intrathymically, and the common genetic changes among the donor‐type T cell lymphomas were investigated with regard to p53 gene and chromosome aberrations. The results indicated that some mutations in the p53 gene had taken place in these lymphomas, hut there was no common mutation among the donor‐type lymphomas from individual irradiated mice, suggesting that these mutations were late‐occurring events in the process of oncogenesis. On the other hand, there were common chromosome aberrations or translocations such as trisomy 15, t(7F;10C), t(1A;13D) or t(6A;XB) among the donor‐type lymphomas derived from half of the individual irradiated mice. This indicated that the aberrations/translocations, which occurred in single progenitor cells at the early T cell differentiation either just before or after γ T cell receptor rearrangements, might he important candidates for initiating events. In the donor‐type lymphomas from the other half of the individual irradiated mice, microgenetic changes were suggested to be initial events and also might take place in single progenitor cells just before or right after γ TCR rearrangements.