bluuebunny/crossref_metadata_2025 huggingface dataset:
A Brassinosteroid-Hypersensitive Mutant of BAK1 Indicates That a Convergence of Photomorphogenic and Hormonal Signaling Modulates Phototropism
Authors: Whippo Craig W. , Hangarter Roger P.
Published: August 2005

The phototropic response of Arabidopsis (Arabidopsis thaliana) is induced by the phototropin photoreceptors and modulated by the cryptochrome and phytochrome photoreceptors. Downstream of these photoreceptors, asymmetric lateral redistribution of auxin underlies the differential growth, which results in phototropism. Historical physiological evidence and recent analysis of hormone-induced gene expression demonstrate that auxin and brassinosteroid signaling function interdependently. Similarly, in this study we report evidence that interactions between brassinosteroids and auxin signaling modulate phototropic responsiveness. We found that elongated, a previously identified photomorphogenesis mutant, enhances high-light phototropism and represents a unique allele of BAK1/SERK3, a receptor kinase implicated in brassinosteroid perception. Altogether, our results support the hypothesis that phototropic responsiveness is modulated by inputs that influence control of auxin response factor-mediated transcription.
Submergence-Induced Morphological, Anatomical, and Biochemical Responses in a Terrestrial Species Affect Gas Diffusion Resistance and Photosynthetic Performance
Authors: Mommer Liesje , Pons Thijs L. , Wolters-Arts Mieke , Venema Jan Henk , Visser Eric J.W.
Published: August 2005

Gas exchange between the plant and the environment is severely hampered when plants are submerged, leading to oxygen and energy deficits. A straightforward way to reduce these shortages of oxygen and carbohydrates would be continued photosynthesis under water, but this possibility has received only little attention. Here, we combine several techniques to investigate the consequences of anatomical and biochemical responses of the terrestrial species Rumex palustris to submergence for different aspects of photosynthesis under water. The orientation of the chloroplasts in submergence-acclimated leaves was toward the epidermis instead of the intercellular spaces, indicating that underwater CO2 diffuses through the cuticle and epidermis. Interestingly, both the cuticle thickness and the epidermal cell wall thickness were significantly reduced upon submergence, suggesting a considerable decrease in diffusion resistance. This decrease in diffusion resistance greatly facilitated underwater photosynthesis, as indicated by higher underwater photosynthesis rates in submergence-acclimated leaves at all CO2 concentrations investigated. The increased availability of internal CO2 in these “aquatic” leaves reduced photorespiration, and furthermore reduced excitation pressure of the electron transport system and, thus, the risk of photodamage. Acclimation to submergence also altered photosynthesis biochemistry as reduced Rubisco contents were observed in aquatic leaves, indicating a lower carboxylation capacity. Electron transport capacity was also reduced in these leaves but not as strongly as the reduction in Rubisco, indicating a substantial increase of the ratio between electron transport and carboxylation capacity upon submergence. This novel finding suggests that this ratio may be less conservative than previously thought.
A Copper Chaperone for Superoxide Dismutase That Confers Three Types of Copper/Zinc Superoxide Dismutase Activity in Arabidopsis
Authors: Chu Chiung-Chih , Lee Wen-Chi , Guo Wen-Yu , Pan Shu-Mei , Chen Lih-Jen , Li Hsou-min , Jinn Tsung-Luo
Published: August 2005

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.
Effects of Water Stress on Respiration in Soybean Leaves
Authors: Ribas-Carbo Miquel , Taylor Nicolas L. , Giles Larry , Busquets Silvia , Finnegan Patrick M. , Day David A. , Lambers Hans , Medrano Hipólito , Berry Joseph A. , Flexas Jaume
Published: August 2005

The effect of water stress on respiration and mitochondrial electron transport has been studied in soybean (Glycine max) leaves, using the oxygen-isotope-fractionation technique. Treatments with three levels of water stress were applied by irrigation to replace 100%, 50%, and 0% of daily water use by transpiration. The levels of water stress were characterized in terms of light-saturated stomatal conductance (gs): well irrigated (gs > 0.2 mol H2O m−2 s−1), mildly water stressed (gs between 0.1 and 0.2 mol H2O m−2 s−1), and severely water stressed (gs < 0.1 mol H2O m−2 s−1). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (Vt) was not significantly different at any water-stress level. However, severe water stress caused a significant shift of electrons from the cytochrome to the alternative pathway. The electron partitioning through the alternative pathway increased from 10% to 12% under well-watered or mild water-stress conditions to near 40% under severe water stress. Consequently, the calculated rate of mitochondrial ATP synthesis decreased by 32% under severe water stress. Unlike many other stresses, water stress did not affect the levels of mitochondrial alternative oxidase protein. This suggests a biochemical regulation (other than protein synthesis) that causes this mitochondrial electron shift.
Cassava Plants with a Depleted Cyanogenic Glucoside Content in Leaves and Tubers. Distribution of Cyanogenic Glucosides, Their Site of Synthesis and Transport, and Blockage of the Biosynthesis by RNA Interference Technology
Authors: Jørgensen Kirsten , Bak Søren , Busk Peter Kamp , Sørensen Charlotte , Olsen Carl Erik , Puonti-Kaerlas Johanna , Møller Birger Lindberg
Published: August 2005

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.
Evidence for In Vitro Binding of Pectin Side Chains to Cellulose
Authors: Zykwinska Agata W. , Ralet Marie-Christine J. , Garnier Catherine D. , Thibault Jean-François J.
Published: August 2005

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.
Genome-Wide Identification of Potential Plant E2F Target Genes
Authors: Vandepoele Klaas , Vlieghe Kobe , Florquin Kobe , Hennig Lars , Beemster Gerrit T.S. , Gruissem Wilhelm , Van de Peer Yves , Inzé Dirk , De Veylder Lieven
Published: August 2005

Entry into the S phase of the cell cycle is controlled by E2F transcription factors that induce the transcription of genes required for cell cycle progression and DNA replication. Although the E2F pathway is highly conserved in higher eukaryotes, only a few E2F target genes have been experimentally validated in plants. We have combined microarray analysis and bioinformatics tools to identify plant E2F-responsive genes. Promoter regions of genes that were induced at the transcriptional level in Arabidopsis (Arabidopsis thaliana) seedlings ectopically expressing genes for the E2Fa and DPa transcription factors were searched for the presence of E2F-binding sites, resulting in the identification of 181 putative E2F target genes. In most cases, the E2F-binding element was located close to the transcription start site, but occasionally could also be localized in the 5′ untranslated region. Comparison of our results with available microarray data sets from synchronized cell suspensions revealed that the E2F target genes were expressed almost exclusively during G1 and S phases and activated upon reentry of quiescent cells into the cell cycle. To test the robustness of the data for the Arabidopsis E2F target genes, we also searched for the presence of E2F-cis-acting elements in the promoters of the putative orthologous rice (Oryza sativa) genes. Using this approach, we identified 70 potential conserved plant E2F target genes. These genes encode proteins involved in cell cycle regulation, DNA replication, and chromatin dynamics. In addition, we identified several genes for potentially novel S phase regulatory proteins.
Ectopic Expression of Constitutively Activated RACB in Barley Enhances Susceptibility to Powdery Mildew and Abiotic Stress
Authors: Schultheiss Holger , Hensel Götz , Imani Jafargholi , Broeders Sylvia , Sonnewald Uwe , Kogel Karl-Heinz , Kumlehn Jochen , Hückelhoven Ralph
Published: August 2005

Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP protein RACB is involved in susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on, downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO2-assimilation rates and transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply, showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common signaling element in response to both biotic and abiotic stress.
Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii
Authors: Nogaj Luiza A. , Srivastava Alaka , van Lis Robert , Beale Samuel I.
Published: August 2005

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNAGlu. Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 μmol photons s−1 m−2), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.
The TomatoAux/IAATranscription FactorIAA9Is Involved in Fruit Development and Leaf Morphogenesis
Authors: Wang Hua , Jones Brian , Li Zhengguo , Frasse Pierre , Delalande Corinne , Regad Farid , Chaabouni Salma , Latché Alain , Pech Jean-Claude , Bouzayen Mondher
Published: August 2005

Auxin/indole-3-acetic acid (Aux/IAA) proteins are transcriptional regulators that mediate many aspects of plant responses to auxin. While functions of most Aux/IAAs have been defined mainly by gain-of-function mutant alleles in Arabidopsis thaliana, phenotypes associated with loss-of-function mutations have been scarce and subtle. We report here that the downregulation of IAA9, a tomato (Solanum lycopersicum) gene from a distinct subfamily of Aux/IAA genes, results in a pleiotropic phenotype, consistent with its ubiquitous expression pattern. IAA9-inhibited lines have simple leaves instead of wild-type compound leaves, and fruit development is triggered before fertilization, giving rise to parthenocarpy. This indicates that IAA9 is a key mediator of leaf morphogenesis and fruit set. In addition, antisense plants displayed auxin-related growth alterations, including enhanced hypocotyl/stem elongation, increased leaf vascularization, and reduced apical dominance. Auxin dose–response assays revealed that IAA9 downregulated lines were hypersensitive to auxin, although the only early auxin-responsive gene that was found to be upregulated in the antisense lines was IAA3. The activity of the IAA3 promoter was stimulated in the IAA9 antisense genetic background, indicating that IAA9 acts in planta as a transcriptional repressor of auxin signaling. While no mutation in any member of subfamily IV has been reported to date, the phenotypes associated with the downregulation of IAA9 reveal distinct and novel roles for members of the Aux/IAA gene family.
Plasmodesmal-Associated Protein Kinase in Tobacco andArabidopsisRecognizes a Subset of Non-Cell-Autonomous Proteins
Authors: Lee Jung-Youn , Taoka Ken-ichiro , Yoo Byung-Chun , Ben-Nissan Gili , Kim Dong-Jin , Lucas William J.
Published: August 2005

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.
Suppression of Intestinal Polyp Development by Nimesulide, a Selective Cyclooxygenase‐2 Inhibitor, in Min Mice
Authors: Nakatsugi Seiichi , Fukutake Masato , Takahashi Mami , Fukuda Kazunori , Isoi Takashi , Taniguchi Yasuaki , Sugimura Takashi , Wakabayashi Keiji
Published: August 2005

Nonsteroidal anti‐inflammatory drugs (NSAIDs) suppress colon carcinogenesis in man and experimental animals. However, conventional NSAIDs inhibit both cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, and cause gastrointestinal side‐effects. Nimesulide, a selective inhibitor of COX‐2, is much less ulcerogenic. We, therefore, examined its influence on the development of intestinal polyps in Min mice. Female Min mice at 4 weeks old were given 400 ppm nimesulide in their diet for 11 weeks. This treatment resulted in a significant reduction of the numbers of both small and large intestinal polyps, the total being 52% of that in untreated control Min mice. The size of the polyps in the nimesulide‐treated group was also significantly decreased. The results suggest that nimesulide is a good candidate as a chemopreventive agent for human colon cancer with low toxicity.
Differential Dose‐dependent Effects of α‐, β‐Carotenes and Lycopene on Gap‐junctional Intercellular Communication in Rat Liver in vivo
Authors: Krutovskikh Vladimir , Asamoto Makoto , Takasuka Nobuo , Murakoshi Michiaki , Nishino Hoyoku , Tsuda Hiroyuki
Published: August 2005

In order to examine the relevance of alteration of gap‐junctional intercellular communication (GJIC) to chemopreventive activity against carcinogenesis, the effects of α andβ ‐carotene as well as lycopene, typical chemopreventive carotenoids, on cell coupling via gap junctions in rat liver in vivo were studied using a direct functional dye‐transfer technique. We found that all three test compounds given at a dose of 50 mg/kg‐body weight (b.w.) daily, 5 times by gavage, inhibited GJIC, while similar treatment with 5 mg/kg b.w. caused enhancement, especially in the β‐carotene and lycopene‐treated groups. At the dose level of 0.5 mg/kg b.w., the three compounds had no effect. The findings show that all three agents differentially modulate GJIC depending on the dose, with beneficial effects on cell communication only detected at the one dose. The result suggests that determination of the dose of chemicals to be used is crucial for human intervention studies.
Inhibition of Angiogenesis by Rhizoxin, a Microbial Metabolite Containing Two Epoxide Groups
Authors: Onozawa Chizuko , Shimamura Mariko , Iwasaki Shigeo , Oikawa Tsutomu
Published: August 2005

Previous studies by our and other groups have shown that microbial products containing more than one epoxide group, including eponemycin, radicicol, depudecin and AGM‐1470, exhibit anti‐angio‐genic activity in an in vivo assay system involving chorioallantoic membranes (CAMs) of growing chick embryos. Based on these findings, rhizoxin, a microbial metabolite that contains two epoxide groups and exhibits anti‐tubulin activity, was tested for anti‐angiogenic activity in a CAM assay system. Rhizoxin caused dose‐dependent inhibition of embryonic angiogenesis, the IDS(1 value being 2 ng (3.2 pmol) per egg. In addition, this compound (2 nig/kg i.p.) significantly suppressed neovascnlarizatlou induced by M5076 mouse tumor cells in a mouse dorsal air sac assay system, compared to the vehicle alone (P<0.05). These results indicate that rhizoxin is a novel inhibitor of angiogenesis, and that it has potential as a new therapeutic agent for cancer.
Impact of Family History on the Risk of Breast Cancer among the Japanese
Authors: Hirose Kaoru , Tajima Kazuo , Hamajima Nobuyuki , Kuroishi Tetsuo , Miura Shigeto , Tokudome Shinkan
Published: August 2005

To assess the impact of family history on the risk of developing breast cancer, a case‐referent study was conducted using data from the Hospital‐based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), Japan. In total, 1,551 breast cancer cases were included and 28,450 women, confirmed as free of cancer, were recruited as the reference group. The odds ratios (ORs) and their 95% confidence intervals (95%CIs) were estimated by using an unconditional logistic regression model. Stratification by age at diagnosis was done to evaluate the impact of family history on early (<40 years of age) vs. later (≫40 years of age) onset. New findings obtained from the present study were as follows. 1) For women having a family member with ovarian cancer, the OR for breast cancer diagnosed under age 40 was 5.04 (95%CI: 1.48–17.1). 2) The effect of family history on the risk of breast cancer was greater when a sister(s) was (were) positive (OR=3.51) than when the mother was positive (OR=1.47). 3) The estimated proportional ratio (PR) for breast cancer was 2.63 (95%CI: 2.05 3.38) among sisters excluding probands, while PR for cancers other than breast cancer was 1.09 (95%CI: 0.94–1.27). This result suggested that a family history of breast cancer, especially among sisters, elevated the risk of developing breast cancer. Furthermore, when women had a sister(s) with breast cancer diagnosed prior to 40 years of age, their family history of breast cancer affected their risk of cancer of the breast and other organs.
Dose‐dependent Promoting Effects of Catechol on Glandular Stomach Carcinogenesis in BALB/c Mice Initiated with N‐Methyl‐N‐nitrosourea
Authors: Kobayashi Kiyoshi , Shimizu Nobuyuki , Tsukamoto Tetsuya , Inada Ken‐ichi , Nakanishi Hayao , Goto Kazuhiro , Mutai Mamoru , Tatematsu Masae
Published: August 2005

The effects of cateehol administration in the diet on stomach carcinogenesis in mice after initiation with N‐methyl‐N‐nitrosourea (MNU) in the drinking water were investigated in a development trial for a new experimental protocol. Male 6‐week‐old BALB/c mice were given MNU in the drinking water intermittently for a total of three one‐week periods, with one‐week intervals, at the concentration of 120 ppm (groups 1 and 2). Groups 3 and 4 served as non initiated controls. From week 7, groups 1 and 3 were divided into three subgroups and the mice were fed on diet containing 0.05% (groups la and 3a), 0.2% (groups Ib and 3b), 0.8% (groups 1c and 3c) or 0% (groups 2 and 4) cateehol for 29 weeks. At week 20, appreciably enhanced development of pepsinogen 1‐altered pyloric glands was noted in all catechol‐treated groups, in a partially dose‐dependent manner (12.8±12.5, 13.8±11.7, and 24.0±12.7/100 pyloric glands respectively, for groups 1, 2 and 3). The incidences of adenomas (groups 1, 2 and 3) were also increased. At week 35, dose‐dependent induction of adenocarcinomas in groups 1 (3/19), 2 (3/19) and 3 (14/20) was evident. In addition, the depth of invasion of the adenocarcinomas was enhanced by cateehol in a dose‐dependent manner, though the histological type was not influenced. Thus, the administration of cateehol in the diet strongly enhanced the pre‐neoplastic and neoplastic lesions in mouse glandular stomach induced by MNU in the drinking water, in a dose‐dependent manner
Promotion of Rat Hepatocarcinogenesis by Dimethylarsinic Acid: Association with Elevated Ornithine Decarboxylase Activity and Formation of 8‐Hydroxydeoxyguanosine in the Liver
Authors: Wanibuchi Hideki , Hori Takaaki , Meenakshi Vijayaraghavan , Ichihara Toshio , Yamamoto Shinji , Yano Yoshihisa , Otani Shuzo , Nakae Dai , Konishi Yoichi , Fukushima Shoji
Published: August 2005

Arsenicals are epidemiologicaUy significant chemicals in relation to induction of liver cancer in man. In the present study, we investigated the dose‐dependent promotion potential of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenicals in mammals, in a rat liver carcinogenesis model. In experiment 1, glutathione‐S‐transferase placental form (GST‐P)‐positive foci, putative preneoplas‐tic lesions, were employed as endpoints of a liver medium‐term bioassay for carcinogens (Ito test). Starting 2 weeks after initiation with diethylnitrosamine, male F344 rats were treated with 0, 25, 50 or 100 ppm of DMAA in the drinking water for 6 weeks. All animals underwent two‐thirds partial hepatectomy at week 3 after initiation. Examination of liver sections after termination at 8 weeks revealed dose‐dependent increases in the numbers and areas of GST‐P‐positive foci in DMAA‐treated rats as compared with controls. In experiment 2, ornithine decarboxylase activity, which is a biomarker of cell proliferation, was found to be significantly increased in the livers of rats treated with DMAA. In experiment 3, formation of 8‐hydroxydeoxyguanosine, which is a marker of oxygen radical‐mediated DNA damage, was significantly increased after administration of DMAA. These results indicate that DMAA has the potential to promote rat liver carcinogenesis, possibly via a mechanism involving stimulation of cell proliferation and DNA damage caused by oxygen radicals
Prohibitin Expression Is Decreased in the Regenerating Liver but Not in Chemically Induced Hepatic Tumors in Rats
Authors: Tanno Satoshi , Fukuda Ikue , Saito Yoshinori , Ogawa Katsuhiro
Published: August 2005

Expression of prohibitin, a growth‐regulatory protein, was immunohistochemically investigated in normal rat tissues, regenerating livers, and chemically induced preneoplastic and neoplastic hepatic lesions. Specific cell types including hepatocytes, striated and smooth muscle cells, cardiac cells, squamous epithelial cells, sebaceous gland cells, hair root outer sheath cells, salivary gland duct epithelial cells, chondrocytes, immature spermatocytes and oocytes were found to be positive. In regenerating livers, prohibitin protein disappeared as early as 3 h after two‐thirds hepatectomy and returned to near the original level by 24 h, while its mRNA level did not markedly vary. The timing of the disappearance was coincident with the expression of c‐myc, suggesting a relation to quiescent hepatocytes entering the cell cycle. However, no pronounced decrease was evident in the most hyperplastic hepatic nodules and hepatocellnlar carcinomas investigated. Examination of 9 rat hepatocellular carcinoma cell lines, 6 hyperplastic hepatic nodules and 5 hepatocellular carcinomas revealed a single case of a base substitution in prohibitin cDNA, identified as a synonymous sense change. The observed abundant expression of prohibitin in quiescent hepatocytes and its rapid loss under conditions of regeneration indicate a growth‐regulatory function, but our results do not suggest any critical role in rat hepatocarcinogenesis.
Increased DNA Methyltransferase Expression Is Associated with an Early Stage of Human Hepatocarcinogenesis
Authors: Sun Lin , Hui Ai‐Min , Kanai Yae , Sakamoto Michiie , Hirohashi Setsuo
Published: August 2005

The present study was designed to determine whether changes in DNA methyltransferase (DNA MTase) expression are involved in hepatocarcinogenesis. We examined DNA MTase expression in normal liver tissue (with no remarkable histological findings), liver tissue showing chronic hepatitis or cirrhosis, which are generally thought to be precancerous conditions, and hepatocellular carcinomas (HCCs) using the reverse‐transcriptase polymerase chain reaction assay. DNA MTase mRNA levels were significantly higher in liver tissue showing chronic hepatitis and cirrhosis (DNA MTase mRNA/β‐actin mRNA ratio=0.30±0.22, n=24, P<0.01) than in normal liver tissue either from patients with liver metastatic lesions of colonic cancer (0.14±0.05, n= 6) or from patients with HCCs (0.16±0.07, n= 3). DNA MTase mRNA levels were even higher in HCC tissue (0.34±0.18, n=29). These results suggest that increased DNA MTase expression may be an early event during hepatocar‐cinogenesis. DNA MTase is a potential target for HCC preventive therapy.
Detection of Altered Adhesion Molecule Expression in Experimental Tumors by a Radiolabeled Monoclonal Antibody
Authors: Saga Tsuneo , Sakahara Harumi , Yao Zhengsheng , Nakamoto Yuji , Sato Noriko , Hattori Noriko , Zhang Meili , Zhao Songji , Aoki Tomokazu , Miyatake Shin–ichi , Namba Yuziro , Konishi Junji
Published: August 2005

Adhesion molecules play a major role in the processes of invasion and metastasis of malignant tumors. Their expression within tumors has been reported to be quantitatively and qualitatively altered according to the invasiveness and metastatic potential of the tumor. The present study tested whether the intratumoral expression of integrin α3 can be detected by a radiolabeled monoclonal antibody. The in vitro hinding study with four different human cancer cells showed that radioiodinated GA17 antibody recognizing integrin “3 bound specifically to these cells to varying degrees, according to the antigen density on each cell. The biodistribution study with 125I– and 111In–labeled antibodies showed specific localization of radiolabeled GA17 to the xenografts. However, the in vivo tumor localization was not proportional to the antigen density calculated in vitro, and antibody metabolism varied among the tumors, as was also confirmed by in vitro radionuclide retention assay. The intratumoral distribution of radioactivities varied reflecting the antigen expression within the tumor. These results indicate that 1) integrin α3 was expressed in various kinds of tumors and could he localized by the radiolabeled antibody, and 2) the expression of integrin “3 and the metabolism of the radiolabeled antibody after binding to the antigen within the tumor were variable among the tumors, which affected the radionuclide distribution characteristics. The expression of adhesion molecules within these tumors was noninvasively detected by a radiolabeled antibody. It may be possible to use integrin a3, when it is overexpressed, as a target of therapy with antibodies radiolabeled with a or β emitters.
Histocompatibility Leukocyte Antigen‐A2‐restricted and Tumor‐specific Cytotoxic T Lymphocytes from Tumor‐infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer
Authors: Suekane Shigetaka , Nakao Masanobu , Inoue Michiro , Noda Shinshi , Itoh Kyogo
Published: August 2005

T lymphocytes play an important role in tumor rejection. To understand T cell‐mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin‐2 (IL‐2)‐activated tumor‐infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)‐class I‐restricted and tumor‐specific cytotoxicity. We established a CD3+CD4‐CD8+ cytotoxic T lymphocyte (CTL) line from the IL‐2‐activated TIL of a 37‐year‐old patient with testicular embryonal cancer. A 6 h 51Cr‐release assay was performed to measure the cytotoxicity of the CTL. The CD3+CD4‐CD8+ CTL line showed cytotoxicity against HLA‐A2+ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA‐A2” tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA‐A2‐restricted and tumor‐specific CTL at the tumor site of testicular embryonal cancer.
Nitric Oxide Synthase Activity in Human Lung Cancer
Authors: Fujimoto Hisao , Ando Yukio , Yamashita Taro , Terazaki Hisayasu , Tanaka Yoshiya , Sasaki Jiichiro , Matsumoto Mitsuhiro , Suga Moritaka , Ando Masayuki
Published: August 2005

Nitric oxide synthases (NOS) exist in human tumor cell lines and solid tumor tissues, and it has been suggested that NO may play important roles in growth, progression or metastasis of tumors. We investigated the activity and distribution of NOS in a series of human lung cancer and normal lung tissues. Seventy‐two primary lung cancer samples (44 cases of adenocarcinoma, 18 of squamous cell carcinoma, 4 of large cell carcinoma, 2 of small cell carcinoma, 2 of adenosquamous carcinoma, and 2 of carcinoids) and corresponding normal lung samples were obtained from surgically treated patients. In normal lung tissues, little NOS activity was observed with no correlation between the patients' age and NOS activity. The total NOS activities in lung adenocarcinoma samples were significantly higher than those in other types of lung cancers or normal lung samples (P<0.05), Analysis by tumor grade of the adenocarcinoma samples revealed no significant difference of NOS activity between grades. TNM classification showed that, although T stage did not correlate with NOS activity, cancer tissues from patients with N2 disease tended to have lower activity than those from patients with NO or Nl disease. Immunohistochemical studies revealed that the intensity of NOS immunoreactivity correlated with NOS activity. These results suggest that NO may play an important role in the metabolism and behavior of lung cancers, especially adenocarcinoma.
Nitric Oxide Generation from Hydroxyurea via Copper‐catalyzed Peroxidation and Implications for Pharmacological Actions of Hydroxyurea
Authors: Sato Keizo , Akaike Takaaki , Sawa Tomohiro , Miyamoto Yoichi , Suga Moritaka , Ando Masayuki , Maeda Hiroshi
Published: August 2005

We investigated the generation of nitric oxide (NO) by H2O2dependent peroxidation of hydroxyurea in the presence of copper‐containing proteins such as Cu, Zn‐snperoxide dismutase (Cu, Zn‐SOD) or ceruloplasmin as a catalyst. In the reaction mixture of hydroxyurea, Cu, Zn‐SOD, and H2O2, “NO generation was identified by measuring the specific electron spin resonance (ESR) signal of 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐l‐oxyl 3‐oxide (PTIO). The ESR signal of the NO‐hemoglobin adduct was also detected in human red blood cells during copper‐catalyzed pcroxidation of hydroxyurea. The‘NO production during peroxidation of hydroxyurea was quantified as NO2 formation, measured by using the Griess assay, and the amount of NO2 was dependent on the concentration of hydroxyurea of the reaction mixture. ESR spin trapping with 5,5‐dimethyl‐l‐pyrroline JV‐oxide (DMPO) showed hydroxy radical COH) generation in the reaction of H2O2 with either Cu, Zn‐SOD or ceruloplasmin. Several “OH scavengers, such as ethanol, thiourea, DMPO, and dimethylsulfoxide, and the metalchelating agent diethylenetriaminepentaacetic acid significantly inhibited‘NO generation from hydroxyurea. This indicates that‘NO release from hydroxyurea may be mediated by‘OH derived from the copper‐catalyzed Fenton‐like reaction. Incubation of hydroxyurea and Cu, Zn‐SOD with xanthine oxidase and hypoxanthine in a system forming O2→ H2O2 also resulted in appreciable‘NO production. These results suggest that‘NO production from hydroxyurea catalyzed by copper‐containing proteins may be the molecular basis of the pharmacological and antitumor action of hydroxyurea.
Anti‐tumor Efficacy of Paclitaxel against Human Lung Cancer Xenografts
Authors: Yamori Takao , Sato Shigeo , Chikazawa Hirotaka , Kadota Toshihito
Published: August 2005

We examined paclitaxel for anti‐tumor activity against human lung cancer xenografts in nude mice and compared its efficacy with that of cisplatin, currently a key drug for lung cancer chemotherapy. Five non‐small cell lung cancers (A549, NCI‐H23, NCI‐H226, NCI‐H460 and NCI‐H522) and 2 small cell lung cancers (DMS114 and DMS273) were chosen for this study, since these cell lines have been well characterized as regards in vitro and in vivo drug sensitivity. These cells were exposed to graded concentrations of paclitaxel (0.1 to 1000 nM) for 48 h. The 50% growth‐inhibitory concentrations (GI50) for the cell lines ranged from 4 to 24 nM, which are much lower than the achievable peak plasma concentration of paclitaxel. In the in vivo study, 4 cell lines (A549, NCI‐H23, NCI‐H460, DMS‐273) were grown as subcutaneous tumor xenografts in nude mice. Paclitaxel was given intravenously as consecutive daily injections for 5 days at the doses of 24 and 12 mg/kg/day. Against every xenograft, paclitaxel produced a statistically significant tumor growth inhibition compared to the saline control. Paclitaxel at 24 mg/kg/day was more effective than cisplatin at 3 mg/kg/day with the same dosing schedule as above, although the toxicity of paclitaxel was similar to or rather lower than that of cisplatin, in terms of body weight loss. In addition, paclitaxel showed potent activity against 2 other lung cancer xenografts (NCI‐H226 and DMS114). Therefore, paclitaxel showed more effective, wider‐spectrum anti‐tumor activity than cisplatin in this panel of 6 lung cancer xenografts. These findings support the potential utility of paclitaxel in the treatment of human lung cancer
The Role of Glucuronidation in 7‐Ethyl‐10‐hydroxycamptothecin Resistance in vitro
Authors: Takahashi Toshiaki , Fujiwara Yasuhiro , Yamakido Michio , Katoh Osamu , Watanabe Hiromitsu , Mackenzie Peter I.
Published: August 2005

Although glucuronidation catalyzed by uridine 5′‐diphosphoglucuronosyltransferase (UGT) is a major pathway of drug inactivation in humans, glucuronidation in malignant cells has received little attention as a cause of anti‐cancer drug resistance. In this study, we tried to elucidate the role of SN‐38 glucuronidation in the CPT‐11‐resistant human lung cancer cell line PC‐7/CPT. PC‐7/CPT cells possessed an increased activity to glucuronidate SN‐38 compared to the parent cells, PC‐7, Furthermore, sensitivity of PC‐7/CPT cells to SN‐38 was improved by inhibiting UGT activity. Western and northern blot analyses demonstrated that this increased activity was due to increased levels of UGT protein and mRNA. These results not only imply that npregulation of UGT activity in PC‐7/CPT cells may contribute in part to SN‐38 resistance, hut also illustrate the importance of drug metabolism within malignant cells themselves, as a cause of drug resistance
Enhanced Topoisomerase I Activity and Increased Topoisomerase IIα Content in Cisplatin‐resistant Cancer Cell Lines
Authors: Minagawa Yukihisa , Kigawa Junzo , Irie Takashi , Kanamori Yasunobu , Itamochi Hiroaki , Cheng Xiusi , Terakawa Naoki
Published: August 2005

Although the combined effects of cisplatin (CDDP) and DNA topoisomerase (Topo) inhibitors have been described in recent literature, little is known about the combined effects and their biological basis in CDDP‐resistant cells. The aim of the present study was to elucidate the combined effect of CDDP and Topo inhibitors on CDDP‐resistant cells as well as to investigate the biological factors involved in the sensitivity to these anti‐cancer agents. We found synergistic actions between CDDP and SN‐38 (a Topo I inhibitor) or VP‐16 (a Topo II inhibitor) in KFr cells, a CDDP‐resistant subline of the KF epithelial ovarian carcinoma cell line, but not in the parent KF cells. We subsequently assayed Topo protein levels and enzymatic activities in two sets of CDDP‐sensitive and ‐resistant cell lines: KF and KFr, and HeLa and HeLa/CDDP. The levels of Topo I protein in the CDDP‐resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to CDDP. Topo I enzymatic activity, however, was 2‐ to 4‐fold higher in the CDDP‐resistant cell lines than in their respective parent cell lines. In contrast, higher levels of Topo lice protein were observed both before and after CDDP exposure in the CDDP‐resistant cells than in their controls. However, no difference in Topo II catalytic activity was observed between the CDDP‐resistant and ‐sensitive cells.
Chronic administration of phosphodiesterase 5 inhibitor improves erectile and endothelial function in a rat model of diabetes
Authors: AHN GOOK JUN , YU JAE YOUNG , CHOI SEUL MIN , KANG KYUNG KOO , AHN BYOUNG OK , KWON JONG WON , KANG SUNG KEUN , LEE BYEONG CHUN , HWANG WOO SUK
Published: August 2005

This study was conducted to determine if the long‐term administration of the phosphodiesterase type 5 (PDE 5) inhibitor, DA‐8159, to diabetic rats can ameliorate the development of erectile dysfunction (ED) and endothelial dysfunction. After inducing diabetes with streptozotocin, DA‐8159 was orally administered at a dose of 3 mg/kg or 10 mg/kg for 8 weeks. To examine the effect on erectile response, electrostimulation of the cavernous nerve with the parameters of 3 V, 5 ms, 5 Hz or 10 Hz, was performed to measure the intracavernous pressure (ICP) and mean arterial pressure (MAP). Thoracic aorta relaxation in vitro was evaluated by adding acetycholine (Ach) cumulatively to the bathing medium. In addition, the plasma endothelin‐1 (ET‐1) levels were measured in order to investigate the effect of DA‐8159 on endothelial dysfunction. The area under the curve (AUC) from the ICP/MAP ratio in the 10 Hz stimulation showed a significantly increased AUC after the 10 mg/kg treatment compared with the diabetic group (8891 ± 619 vs. 6316 ± 1016, respectively, p < 0.05). At the 5 Hz frequency, DA‐8159 10 mg/kg also induced a significant increase in the AUC compared with the diabetic control. The maximum ICP/MAP ratio (%) of the 10 mg/kg treatment group was significantly higher in both the 10 Hz and 5 Hz frequency groups (p < 0.05). A treatment of 3 mg/kg tended to increase the AUC and peak ICP/MAP but was not statistically significant. The Ach EC50 value of the diabetic group was significantly higher than in the normal control (120.50 ± 22.90 nm vs. 86.80 ± 9.30 nm, respectively), and 10 mg/kg treatment group showed a significantly lower EC50 value (88.38 ± 19.7 nm). The ET‐1 level was lower in groups treated with DA‐8159, 3 mg/kg and 10 mg/kg treatment induced a statistical difference compared with the diabetic control (1.15 ± 0.34 fmol/mL vs. 2.51 ± 0.55 fmol/mL, respectively, p < 0.05). These results demonstrate that chronic administration of DA‐8159 could attenuate the development of the ED in diabetes and its effect is associated with an improvement in the endothelial function.
From spermatocytes to spermatozoa in an infertile XYY male
Authors: RIVES NATHALIE , MILAZZO JEAN PIERRE , MIRAUX LUDIVINE , NORTH MARIE‐ODILE , SIBERT LOUIS , MACÉ BERTRAND
Published: August 2005

Sex chromosome distribution and aneuploidy as well as germ cell degeneration were evaluated in meiotic and post‐meiotic cells from an infertile XYY male. Sex chromosome distribution was assessed by multicolour fluorescence in situ hybridization on meiotic preparations. Post‐meiotic cell aneuploidy was characterized by a method combining multicolour fluorescence in situ hybridization and immunocytochemistry using the proacrosin‐specific monoclonal antibody (mAb 4D4). TUNEL assay was carried out on seminiferous tubules to evaluate germ cell degeneration. At the prophase stage of the first meiotic division, 63.64% of cells at the pachytene stage carried three sex chromosomes. The ratio of X‐bearing to Y‐bearing spermatids and spermatozoa differed significantly from 1 : 1 with an excess of Y‐bearing spermatids and spermatozoa. The frequency of hyperhaploid XY spermatids was increased in the XYY male, as well as the incidence of YY, XY and disomic 18 ejaculated spermatozoa. A preferential elimination of germ cells by apoptosis occurred in spermatocytes I. The persistence of the extra Y chromosome during meiosis of an XYY male is associated with a high rate of spermatocyte I degeneration and a low rate of aneuploid spermatozoa.
Molecular evaluation of CFTR sequence variants in male infertility of testicular origin
Authors: LARRIBA S. , BONACHE S. , SARQUELLA J. , RAMOS M. D. , GIMÉNEZ J. , BASSAS L. , CASALS T.
Published: August 2005

Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non‐obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well‐defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population. Specifically, the IVS8‐6(5T) allele carrier frequency was similar in NOb infertile patients when compared with fertile men, but it is noteworthy that, when fertile men were classified into having optimal and suboptimal fertility, no 5T allele was found among the 35 men with optimal fertility parameters. In conclusion, extensive CFTR analysis in infertile individuals and fertile population as adequate control definitively excludes the involvement of the CFTR gene variants in sperm production and stresses the importance of carefully identifying those individuals with obstructive defects, in whom CFTR screening will be beneficial.