bluuebunny/crossref_metadata_2025 huggingface dataset:
Maturation of Arabidopsis Seeds Is Dependent on Glutathione Biosynthesis within the Embryo
Authors: Cairns Narelle G. , Pasternak Maciej , Wachter Andreas , Cobbett Christopher S. , Meyer Andreas J.
Published: March 2006

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, γ-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoter∷β-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.
Rice HYDROPEROXIDE LYASES with Unique Expression Patterns Generate Distinct Aldehyde Signatures in Arabidopsis
Authors: Chehab E.W. , Raman G. , Walley J.W. , Perea J.V. , Banu G. , Theg S. , Dehesh K.
Published: March 2006

HYDROPEROXIDE LYASE (HPL) genes encode enzymes that catalyze the cleavage of fatty acid hydroperoxides into aldehydes and oxoacids. There are three HPLs in rice (Oryza sativa), designated OsHPL1 through OsHPL3. To explore the possibility of differential functional activities among these genes, we have examined their expression patterns and biochemical properties of their encoded products. Transcript analysis indicates that these genes have distinct patterns and levels of expression. OsHPL1 is ubiquitously expressed, OsHPL2 is expressed in the leaves and leaf sheaths, whereas OsHPL3 is wound inducible and expressed exclusively in leaves. OsHPLs also differ in their substrate preference as determined by in vitro enzyme assays using 9-/13-hydroperoxy linolenic and 9-/13-hydroperoxy linoleic acids as substrates. OsHPL1 and OsHPL2 metabolize 9-/13-hydroperoxides, whereas OsHPL3 metabolizes 13-hydroperoxy linolenic acid exclusively. Sequence alignments of the HPL enzymes have identified signature residues potentially responsible for the substrate specificity/preference of these enzymes. All three OsHPLs are chloroplast localized as determined by chloroplast import assays and green fluorescent protein (GFP) fusion studies. Aldehyde measurements in transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing individual OsHPL-GFP fusions indicate that all rice HPLs are functional in a heterologous system, and each of them generates a distinct signature of the metabolites. Interestingly, these aldehydes were only detectable in leaves, but not in roots, despite similar levels of OsHPL-GFP proteins in both tissues. Similarly, there were undetectable levels of aldehydes in rice roots, in spite of the presence of OsHPL1 transcripts. Together, these data suggest that additional tissue-specific mechanism(s) beyond transcript and HPL enzyme abundance, regulate the levels of HPL-derived metabolites.
Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death
Authors: Vacca Rosa Anna , Valenti Daniela , Bobba Antonella , Merafina Riccardo Sandro , Passarella Salvatore , Marra Ersilia
Published: March 2006

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.
Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis
Authors: Bancos Simona , Szatmári Anna-Mária , Castle Julie , Kozma-Bognár László , Shibata Kyomi , Yokota Takao , Bishop Gerard J. , Nagy Ferenc , Szekeres Miklós
Published: March 2006

Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.
Plastid Cues Posttranscriptionally Regulate the Accumulation of Key Enzymes of the Methylerythritol Phosphate Pathway in Arabidopsis
Authors: Sauret-Güeto Susanna , Botella-Pavía Patricia , Flores-Pérez Úrsula , Martínez-García Jaime F. , San Román Carolina , León Patricia , Boronat Albert , Rodríguez-Concepción Manuel
Published: March 2006

Plastid isoprenoids (including hormones and photosynthetic pigments) are essential for plant growth and development, but relatively little is known of how the production of their metabolic precursors via the recently elucidated methylerythritol phosphate (MEP) pathway is regulated. We have identified an Arabidopsis (Arabidopsis thaliana) mutant that survives an otherwise lethal block of the MEP pathway with fosmidomycin (FSM). In rif10 (resistant to inhibition with FSM 10) plants, the accumulation of flux-controlling enzymes of the pathway is posttranscriptionally up-regulated. Strikingly, this phenotype is linked to a lower accumulation of plastidial isoprenoid pigments such as chlorophylls and carotenoids, resulting in mutant plants that are paler and smaller than the wild type. The rif10 mutant is impaired in plastid RNA processing due to a T-DNA insertion in the coding region of the At3g03710 gene encoding the chloroplast-targeted exoribonuclease polyribonucleotide phosphorylase. FSM resistance and other rif10-like phenotypes were also observed in wild-type Arabidopsis, tomato (Lycopersicon esculentum), and rice (Oryza sativa) seedlings grown in the presence of sublethal concentrations of chloramphenicol (an inhibitor of protein synthesis in plastids). By contrast, treatment with norflurazon (an inhibitor of carotenoid biosynthesis causing a similar pale cotyledon phenotype) did not result in FSM resistance. Together, the results support that plastome-encoded proteins are involved in negatively regulating the posttranscriptional accumulation of specific nuclear-encoded MEP pathway enzymes in chloroplasts. Regulation of the MEP pathway by a mechanism dependent on plastid cues might function under physiological conditions to finely adjust plastidial isoprenoid biosynthesis to the metabolic capabilities or requirements of plastids.
Cytochrome P450CYP710AEncodes the Sterol C-22 Desaturase inArabidopsisand Tomato
Authors: Morikawa Tomomi , Mizutani Masaharu , Aoki Nozomu , Watanabe Bunta , Saga Hirohisa , Saito Shigeki , Oikawa Akira , Suzuki Hideyuki , Sakurai Nozomu , Shibata Daisuke , Wadano Akira , Sakata Kanzo , Ohta Daisaku
Published: March 2006

Δ22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with β-sitosterol to produce stigmasterol (CYP710A1, K  m = 1.0 μM and kinetic constant [k  cat] = 0.53 min−1; CYP710A11, K  m = 3.7 μM and k  cat = 10 min−1). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and β-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:β-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Δ22-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.
Calcium-Dependent Protein Kinase Isoforms inPetuniaHave Distinct Functions in Pollen Tube Growth, Including Regulating Polarity
Authors: Yoon Gyeong Mee , Dowd Peter E. , Gilroy Simon , McCubbin Andrew G.
Published: March 2006

Calcium is a key regulator of pollen tube growth, but little is known concerning the downstream components of the signaling pathways involved. We identified two pollen-expressed calmodulin-like domain protein kinases from Petunia inflata, CALMODULIN-LIKE DOMAIN PROTEIN KINASE1 (Pi CDPK1) and Pi CDPK2. Transient overexpression or expression of catalytically modified Pi CDPK1 disrupted pollen tube growth polarity, whereas expression of Pi CDPK2 constructs inhibited tube growth but not polarity. Pi CDPK1 exhibited plasma membrane localization most likely mediated by acylation, and we present evidence that suggests this localization is critical to the biological function of this kinase. Pi CDPK2 substantially localized to as yet unidentified internal membrane compartments, and this localization was again, at least partially, mediated by acylation. In contrast with Pi CDPK1, altering the localization of Pi CDPK2 did not noticeably alter the effect of overexpressing this isoform on pollen tube growth. Ca2+ requirements for Pi CDPK1 activation correlated closely with Ca2+ concentrations measured in the growth zone at the pollen tube apex. Interestingly, loss of polarity associated with overexpression of Pi CDPK1 was associated with elevated cytosolic Ca2+ throughout the bulging tube tip, suggesting that Pi CDPK1 may participate in maintaining Ca2+ homeostasis. These results are discussed in relation to previous models for Ca2+ regulation of pollen tube growth.
LOW PSII ACCUMULATION1 Is Involved in Efficient Assembly of Photosystem II inArabidopsis thaliana
Authors: Peng Lianwei , Ma Jinfang , Chi Wei , Guo Jinkui , Zhu Shuyong , Lu Qingtao , Lu Congming , Zhang Lixin
Published: March 2006

To gain insight into the processes involved in photosystem II (PSII) biogenesis and maintenance, we characterized the low psii accumulation1 (lpa1) mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant, while other plastid-encoded proteins were translated at rates similar to the wild type. In addition, turnover rates of the PSII core proteins CP47, CP43, D1, and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant. LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1 but not with D2, cytochrome b6, or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.
The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins
Authors: Ma Xueyan , Panjikar Santosh , Koepke Juergen , Loris Elke , Stöckigt Joachim
Published: March 2006

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.
Selective Mobility and Sensitivity to SNAREs Is Exhibited by theArabidopsisKAT1 K+ Channel at the Plasma Membrane
Authors: Sutter Jens-Uwe , Campanoni Prisca , Tyrrell Matthew , Blatt Michael R.
Published: March 2006

Recent findings indicate that proteins in the SNARE superfamily are essential for cell signaling, in addition to facilitating vesicle traffic in plant cell homeostasis, growth, and development. We previously identified SNAREs SYP121/Syr1 from tobacco (Nicotiana tabacum) and the Arabidopsis thaliana homolog SYP121 associated with abscisic acid and drought stress. Disrupting tobacco SYP121 function by expressing a dominant-negative Sp2 fragment had severe effects on growth, development, and traffic to the plasma membrane, and it blocked K+ and Cl− channel responses to abscisic acid in guard cells. These observations raise questions about SNARE control in exocytosis and endocytosis of ion channel proteins and their organization within the plane of the membrane. We have used a dual, in vivo tagging strategy with a photoactivatable green fluorescent protein and externally exposed hemagglutinin epitopes to monitor the distribution and trafficking dynamics of the KAT1 K+ channel transiently expressed in tobacco leaves. KAT1 is localized to the plasma membrane within positionally stable microdomains of ∼0.5 μm in diameter; delivery of the K+ channel, but not of the PMA2 H+-ATPase, to the plasma membrane is suppressed by Sp2 fragments of tobacco and Arabidopsis SYP121, and Sp2 expression leads to profound changes in KAT1 distribution and mobility within the plane of the plasma membrane. These results offer direct evidence for SNARE-mediated traffic of the K+ channel and a role in its distribution within subdomains of the plasma membrane, and they implicate a role for SNAREs in positional anchoring of the K+ channel protein.
A Small Family ofMYB-Regulatory Genes Controls Floral Pigmentation Intensity and Patterning in the GenusAntirrhinum
Authors: Schwinn Kathy , Venail Julien , Shang Yongjin , Mackay Steve , Alm Vibeke , Butelli Eugenio , Oyama Ryan , Bailey Paul , Davies Kevin , Martin Cathie
Published: March 2006

The Rosea1, Rosea2, and Venosa genes encode MYB-related transcription factors active in the flowers of Antirrhinum majus. Analysis of mutant phenotypes shows that these genes control the intensity and pattern of magenta anthocyanin pigmentation in flowers. Despite the structural similarity of these regulatory proteins, they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not equivalent biochemically in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. Differences in anthocyanin pigmentation between at least six species are attributable to variations in the activity of the Rosea and Venosa loci. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in anthocyanin pigmentation in plants.
Highly Specific Gene Silencing by Artificial MicroRNAs inArabidopsis
Authors: Schwab Rebecca , Ossowski Stephan , Riester Markus , Warthmann Norman , Weigel Detlef
Published: March 2006

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).
Salicylic Acid–Independent ENHANCED DISEASE SUSCEPTIBILITY1 Signaling inArabidopsisImmunity and Cell Death Is Regulated by the MonooxygenaseFMO1and the Nudix HydrolaseNUDT7
Authors: Bartsch Michael , Gobbato Enrico , Bednarek Pawel , Debey Svenja , Schultze Joachim L. , Bautor Jaqueline , Parker Jane E.
Published: March 2006

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants.
TheTORNADO1andTORNADO2Genes Function in Several Patterning Processes during Early Leaf Development inArabidopsis thaliana
Authors: Cnops Gerda , Neyt Pia , Raes Jeroen , Petrarulo Marica , Nelissen Hilde , Malenica Nenad , Luschnig Christian , Tietz Olaf , Ditengou Franck , Palme Klaus , Azmi Abdelkrim , Prinsen Els , Van Lijsebettens Mieke
Published: March 2006

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana  TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.
The U-Box Protein CMPG1 Is Required for Efficient Activation of Defense Mechanisms Triggered by Multiple Resistance Genes in Tobacco and Tomato
Authors: González-Lamothe Rocío , Tsitsigiannis Dimitrios I. , Ludwig Andrea A. , Panicot Mireia , Shirasu Ken , Jones Jonathan D.G.
Published: March 2006

We previously identified three Avr9/Cf-9 Rapidly Elicited (ACRE) genes essential for Cf-9– and Cf-4–dependent hypersensitive response (HR) production in Nicotiana benthamiana. Two of them encode putative E3 ubiquitin ligase components. This led us to investigate other ACRE genes associated with the ubiquitination pathway. ACRE74 encodes a U-box E3 ligase homolog, highly related to parsley (Petroselinum crispum) CMPG1 and Arabidopsis thaliana PLANT U-BOX20 (PUB20) and PUB21 proteins, and was called Nt CMPG1. Transcript levels of Nt CMPG1 and the homologous tomato (Solanum lycopersicum) Cmpg1 are induced in Cf9 tobacco (Nicotiana tabacum) and Cf9 tomato after Avr9 elicitation. Tobacco CMPG1 possesses in vitro E3 ligase activity. N. benthamiana plants silenced for Nt CMPG1 show reduced HR after Cf-9/Avr9 elicitation, while overexpression of Nt CMPG1 induces a stronger HR in Cf9 tobacco plants after Avr9 infiltration. In tomato, silencing of Cmpg1 decreased resistance to Cladosporium fulvum. Overexpression of epitope-tagged tobacco CMPG1 mutated in the U-box domain confers a dominant-negative phenotype. We also show that Nt CMPG1 is involved in the Pto/AvrPto and Inf1 responses. In summary, we show that the E3 ligase Nt CMPG1 is essential for plant defense and disease resistance.
The identity of Batasio tengana(Hamilton, 1822), with the description of two new species of Batasio from north‐eastern India (Teleostei: Bagridae)
Authors: Ng H. H.
Published: March 2006

Batasio tengana is redescribed on the basis of fresh material and a neotype designated. Two new species are described: Batasio fasciolatus n. sp. is described from material previously identified as B. tengana from the Brahmaputra River drainage and can be distinguished from congeners in having a combination of five to six vertical dark brown bars on a light brown body, dorsal‐spine length 13·6–16·8% standard length (LS), pectoral‐spine length 12·7–14·3%LS, and eye diameter 16·5–18·8% head length (LH). Batasio spilurus n. sp. is described from the Brahmaputra River drainage in Assam and can be distinguished from congeners in having a short adipose‐fin base (12·6–12·8 v. 14·5–33·3%LS) and more slender caudal peduncle (5·7–6·2 v. 6·7–11·8%LS).
Genetic and morphological characterization of a Lake Ohrid endemic, Salmo (Acantholingua) ohridanus with a comparison to sympatric Salmo trutta
Authors: Sušnik S. , Knizhin I. , Snoj A. , Weiss S.
Published: March 2006

Analysis of both uni‐(two mtDNA gene sequences) and bi‐parentally (seven microsatellite loci) inherited genetic markers, together with analysis of 40 morphological characters, described Salmo ohridanus as a highly divergent member of the genus Salmo. Based on comparative substitution rate differences at the cytochrome b gene, and a rough estimated age of the Salmo trutta complex (i.e. at least 2 million years), the S. ohridanus and Salmo obtusirostris clade probably split from a common ancestor of brown trout Salmo trutta >4 million years ago, overlapping with minimum age estimates of the formation of Europe's oldest freshwater habitat, Lake Ohrid. Comparative analysis with Lake Ohrid brown trout (known regionally as Salmo letnica), supported the notion that these fish have more recently colonized the lake and phylogenetically belong to the Adriatic lineage of brown trout. It is further suggested that species‐specific saturation in the mtDNA control region underestimated the divergence between S. ohridanus and S. trutta. Evidence of rare hybridization between S. ohridanus and Lake Ohrid brown trout was seen at both mtDNA and microsatellite markers, but there was no support for extensive introgression.
Phylogenetic relationships among Thunnus species inferred from rDNA ITS1 sequence
Authors: Chow S. , Nakagawa T. , Suzuki N. , Takeyama H. , Matsunaga T.
Published: March 2006

Intra and interspecific nucleotide sequence variation of rDNA first internal transcribed spacer (ITS1) was analysed using all eight species of the genus Thunnus plus two out‐group species within the same family, skipjack tuna Katsuwonus pelamis and striped bonito Sarda orientalis. Intraspecific nucleotide sequence variation in ITS1, including intra‐genomic variation, was low, ranging from 0·003 to 0·014 [Kimura's two parameter distance (K2P)], whereas variation between species within the genus Thunnus ranged from 0·009 to 0·05. The Atlantic and Pacific northern bluefin tunas Thunnus thynnus thynnus and Thunnus thynnus orientalis, recently proposed to be distinct species, were found to share nearly identical ITS1 sequences (mean K2P = 0·006) well within the range of intraspecific variation. The northern bluefin tuna appeared to be a sister group to albacore Thunnus alalunga, with all other Thunnus species in a distinct clade. The ITS1 phylogeny was consistent with mtDNA phylogeny in clustering the three tropical Thunnus species (T. albacares, T. atlanticus and T. tonggol). Southern bluefin Thunnus maccoyii and bigeye Thunnus obesus tunas showed a closer affinity to this tropical tuna group than to the northern bluefin tuna and albacore. The molecular data supported mitochondrial introgression between species and contradicted morphological subdivision of the genus into two subgenera Neothunnus and Thunnus.
The novel application of an immunological technique reveals the immunosuppressive effect of phytoestrogens in Betta splendens
Authors: Ardia D. R. , Clotfelter E. D.
Published: March 2006

The novel application of a standard technique to assess cell‐mediated immune response to phytohaemagglutinin (PHA) injected in the caudal peduncle in a small fish (<2 g) to test the immunosuppressive effect of three phytoestrogens: genistein, equol and β‐sitosterol is described. Individual Betta splendens exposed to these oestrogenic chemicals produced weaker inflammatory responses to PHA than did control individuals, suggesting that phytoestrogens are immunosuppressive. This technique should enable immune function in fish species, too small to provide sufficient blood for conventional immunological measures, to be assessed.
Trematochromis schreyeniPoll, 1987, a junior synonym of ‘Ctenochromis’ benthicola(Matthes, 1962)(Perciformes: Cichlidae) from Lake Tanganyika
Authors: Takahashi T. , Snoeks J. , Nakaya K.
Published: March 2006

A detailed morphological comparison of Trematochromis schreyeni only known from its holotype, collected at the north‐western end of Lake Tanganyika, and ‘Ctenochromis’benthicola described from the same region, and some additional material led to the conclusion that the two species were conspecifics. Consequently, T. schreyeni is considered to be a junior synonym of ‘C.’benthicola, an endemic species to the lake. A redescription of ‘C.’benthicola is also given. A statistical test comparing a single specimen and several specimens is difficult to carry out, because no variation is known in T. schreyeni. The present study, therefore, calculated Mahalanobis’ distance between the holotype of T. schreyeni and specimens of ‘C.’benthicola, and applied the χ2 test.
Genetic variation in skipjack tuna Katsuwonus pelamis(L.) using PCR‐RFLP analysis of the mitochondrial DNA D‐loop region
Authors: Menezes M. R. , Ikeda M. , Taniguchi N.
Published: March 2006

A high level of genetic diversity was observed in Katsuwonus pelamis populations from India (h = 0·952, ne = 14·3) and Japan (h = 0·897, ne = 8·9). The log‐likelihood (G)‐based exact test revealed significant heterogeneity in the distribution of haplotypes between the two populations (P < 0·01, s.e. = 0·001). This result suggests that the two populations should now be treated as demographically independent and managed separately.
Introgressive hybridization between Dolly Varden Salvelinus malma and white‐spotted charr Salvelinus leucomaenis on Hokkaido Island, Japan
Authors: Yamamoto S. , Kitano S. , Maekawa K. , Koizumi I. , Morita K.
Published: March 2006

Intra‐ and interspecific phylogenetic analysis of Dolly Varden Salvelinus malma and white‐spotted charr Salvelinus leucomaenis throughout Hokkaido Island was conducted using nucleotide sequences of the mitochondrial DNA control region and three nuclear DNA markers [ribosomal DNA (ITS‐1), microsatellite (u‐85), SINE (Fok‐223)]. A total of 19 mtDNA haplotypes from 271 individuals of Dolly Varden, nine haplotypes from 134 white‐spotted charr were defined, and identified two well‐supported monophyletic clades for each species. Two haplotypes of Dolly Varden, however, were clustered together in the white‐spotted charr clade. Those haplotypes were found only from several rivers in the Shiretoko Peninsula, and a neighbouring river of that region. Analyses of ribosomal DNA and Fok‐223 loci revealed that both species are characterized by having completely species‐specific diagnostic sequence and fragment patterns. Dolly Varden of the Shiretoko populations are typically allopatric in distribution where white‐spotted charr do not currently occur. In addition, incongruence in genetic relationships between mtDNA and nuclear DNA markers give strong evidence of historical mtDNA introgression between Dolly Varden and white‐spotted charr. Some white‐spotted charr diagnostic alleles in a nuclear microsatellite locus (u‐85) were found in some Shiretoko Dolly Varden populations, suggesting that introgressive hybridization might have also occurred in relatively recent contact and potentially ongoing evolutionary event. The present study presents an example of historical hybridization and introgression at the southernmost distribution limits of Dolly Varden.
A comparative immunohistochemical study on a galanin‐like peptide in the neuroendocrine system of the alimentary canal of three species of siluriform catfishes
Authors: Bosi G. , Shinn A. P. , Simoni E. , Arrighi S. , Domeneghini C.
Published: March 2006

Immunohistochemical examination of the galaninergic and the diffuse endocrine systems of the alimentary canal of three catfish species, Ameiurus melas, Silurus glanis and Clarias gariepinus showed the presence of galanin‐like immunoreactive endocrine cells in the stomachs and the proximal intestines and a galanin‐like immunoreactivity within the enteric intramural neurons. The three species differed notably in the relative abundance of galaninergic neurons of the digestive system. A galanin‐like peptide was detected in a high number of nerve bundles and nerve fibres running through the exocrine parenchyma and the endocrine islets of the pancreas of all three species. It is conceivable that the galaninergic system in these species plays a role in regulating gut muscle activity, in modulating mucosal secretive and absorptive processes, and in controlling pancreatic hormone secretion.
Significant increase of colonic mutated crypts correlates with age in sporadic cancer and diverticulosis cases, with higher frequency in the left‐ than right‐side colorectum
Authors: Okayasu Isao , Hana Kiyomi , Tsuruta Tomoko , Okamura Nobuyoshi , Ogawa Taishi , Tokuyama Wataru , Kajita Sabine , Yoshida Tsutomu , Mikami Tetuo
Published: March 2006

Mild periodic acid–Schiff (mPAS) staining can discriminate non‐O‐acetylated (mPAS‐positive) from O‐acetylated (mPAS‐negative) epithelial sialoglycoproteins in human colonic mucosa, allowing the three haplotypes expressed from a single polymorphic autosomal gene (oat) to be distinguished. In heterozygotes, we previously demonstrated wholly mPAS‐positive (stem cell mutated) crypts and clusters of two or more mPAS‐positive crypts to be significantly increased with duration of ulcerative colitis. To establish whether such an increase in the number of mutated crypts with age also occurs in normal individuals or in cases with diverticulosis, the O‐acetylation phenotype in the non‐cancerous colonic mucosa of 47 sporadic colorectal cancer patients who were heterozygotes for oat was tested with mild‐PAS staining. PAS‐positive crypts were assessed histologically in relation to age and compared between the left (sigmoid colon and rectum) and right (cecum and ascending colon) sides of the colorectum. Wholly mPAS‐positive (stem cell mutated) crypts and foci in heterozygotes were found to be increased significantly (P < 0.0001) in the left side with aging (r = 0.598 and 0.643, respectively). Such a positive correlation with aging was also confirmed in 19 diverticulosis cases without cancer (r = 0.797 and 0.793, respectively). The frequency of mutated crypts and foci on the right side was significantly lower than on the left side in both spontaneous colorectal cancer and diverticulosis cases. The results provide support for an intimate relationship between accumulation of mutated crypts with aging, possibly with significance for colorectal cancer development. Furthermore, the environment in the right side of the colon may be different from that in the left side in this regard. (Cancer Sci 2006; 97)
Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients
Authors: Usami Noriyasu , Fukui Takayuki , Kondo Masashi , Taniguchi Tetsuo , Yokoyama Toshihiko , Mori Shoichi , Yokoi Kohei , Horio Yoshitsugu , Shimokata Kaoru , Sekido Yoshitaka , Hida Toyoaki
Published: March 2006

Malignant pleural mesothelioma (MPM) is an asbestos‐related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC‐MESO‐1, ACC‐MESO‐4, Y‐MESO‐8A and Y‐MESO‐8D) from Japanese patients, with the latter two from the same patient with biphasic‐like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI‐1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC‐MESO‐1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y‐MESO‐8A and Y‐MESO‐8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies. (Cancer Sci 2006; 97)
Plasminogen activator inhibitor‐1 as a potential marker for the malignancy of gastric cancer
Authors: Sakakibara Takumi , Hibi Kenji , Koike Masahiko , Fujiwara Michitaka , Kodera Yasuhiro , Ito Katsuki , Nakao Akimasa
Published: March 2006

To test the hypothesis that plasminogen activator inhibitor‐1 (PAI‐1) may serve as a candidate marker for the malignancy of gastric cancer, we carried out quantitative reverse transcription–polymerase chain reaction for the PAI‐1 gene and evaluated the possible relationship between PAI‐1 gene expression levels and clinicopathological findings in gastric cancer. A significant increase in PAI‐1 expression scores was observed in lymph node metastasis‐positive gastric cancers (2.11 ± 0.35) compared to metastasis‐negative cancers (0.33 ± 0.49) (P = 0.0048), as well as in distant metastasis‐positive gastric cancers (3.68 ± 0.58) compared to metastasis‐negative cancers (1.20 ± 0.32). The PAI‐1 expression score markedly increased with tumor stage (P < 0.0001; ANOVA test). Moreover, multivariate analysis revealed the PAI‐1 expression score to be a strong and independent prognostic factor for gastric cancer (P = 0.0450). These results suggested that PAI‐1 might serve as a new and promising parameter for the prediction of prognosis in gastric cancer. (Cancer Sci 2006; 97)
A laboratory analysis of gutta‐percha‐filled area obtained using Thermafil, System B and lateral condensation
Authors: De‐Deus G. , Gurgel‐Filho E. D. , Magalhães K. M. , Coutinho‐Filho T.
Published: March 2006

Aim  To determine the percentage of gutta‐percha‐filled area (PGFA) in the apical third of root canals when filled with either Thermafil, System B or lateral condensation.

Methodology  Sixty extracted human maxillary central incisor teeth were root filled as following: G1: lateral condensation (n = 20), G2: System B (n = 20) and G3: Thermafil system (n = 20). A horizontal section was cut 2 and 4 mm from the apical foramen of each tooth. The samples were prepared for microscopic analysis and photomicrographs of each apical surface were taken at a magnification of 50×. Through digital image analysis, the cross‐sectional area of the canal and the gutta‐percha was measured. The PGFA was calculated. The PGFA data obtained in the three groups were analysed using a nonparametric Friedman and Wilcoxon signed‐rank tests.

Results  Significant differences in PGFA were found between the Thermafil System (G3) and both System B (G2) and lateral condensation (G1) (P < 0.01). The greatest PGFA occurred in the Thermafil group. No significant statistical difference was found between System B and lateral condensation (P > 0.05).

Conclusions  The coated carrier gutta‐percha system Thermafil produced significantly higher PGFAs than lateral condensation and System B techniques.
A finite element analysis of ferrule design on restoration resistance and distribution of stress within a root
Authors: Ichim I. , Kuzmanovic D. V. , Love R. M.
Published: March 2006

Aim  To analyse the effect of ferrule height upon the mechanical resistance and stress distribution within a root to explain variations in the pattern of root fracture.

Methodology  An extracted, intact, caries free, maxillary right central incisor was scanned by laser and then reconstructed on a computer to produce a model of the tooth and associated periodontal ligament. A simulated post/core/crown restoration was constructed on conventional tooth preparations with various ferrules. The crown was loaded with a simulated 500 N force and the simulated displacement of components and the tensile and compressive stress within the tooth structure were recorded.

Results  Without a ferrule preparation, the simulated crown tilted to the labial and rotated distally. With increasing ferrule height the displacement and rotation of the crown reduced in conventional and crown‐lengthening models with maximum reduction occurring when the ferrule height reached 1.5 mm. In ferrule models, higher levels of tensile stress developed in internal (by a factor of 8) and mid‐root palatal (by a factor of 90) dentine at the cervical margin of the preparation. With an increase in ferrule height, the area of tensile stress within the palatal mid‐dentine expanded towards the cervical margin. Similar patterns and stress values were recorded for the crown‐lengthening models.

Conclusion  The study confirms that a ferrule increases the mechanical resistance of a post/core/crown restoration. However a ferrule creates a larger area of palatal dentine under tensile stress that may be a favourable condition for a crack to develop. Crown‐lengthening did not alter the levels or pattern of stress within compared with conventional ferrule preparations.
Quantification of neuropeptides (calcitonin gene‐related peptide, substance P, neurokinin A, neuropeptide Y and vasoactive intestinal polypeptide) expressed in healthy and inflamed human dental pulp
Authors: Caviedes‐Bucheli J. , Lombana N. , Azuero‐Holguín M. M. , Munoz H. R.
Published: March 2006

Aim  To quantify the expression of calcitonin gene‐related peptide (CGRP), substance P (SP), neurokinin A (NKA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in healthy and inflamed human dental pulp tissue.

Methodology  Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 12 pulp samples were obtained from premolars where extraction was indicated for orthodontic purposes. In six of these premolar teeth inflammation was induced by mechanical pulp exposure prior to sample collection. All samples were processed and 125I‐labelled; neuropeptides were quantified by competition assays. anova and Mann–Whitney's (post hoc) tests were used to establish statistically significant differences between the groups.

Results  Expression of five neuropeptides was found in all human pulp samples. Statistical analysis revealed a significantly higher (P < 0.05) expression of CGRP, SP, NKA and NPY in both inflammatory conditions compared with healthy pulp control values. VIP expression remained stable during the inflammatory conditions.

Conclusion  Expression of CGRP, SP and NKA released from C‐fibres and NPY released from sympathetic fibres is significantly higher in the inflamed human pulp compared with healthy pulp. Expression of VIP released from parasympathetic fibres is not increased during the inflammatory conditions of human dental pulp.
Error and uncertainty in habitat models
Authors: BARRY SIMON , ELITH JANE
Published: March 2006

Species distribution models (habitat models) relate the occurrence or abundance of a species to environmental and/or geographical predictors that then allow predictions to be mapped across an entire region. These models are used in a range of policy settings such as managing greenhouse gases, biosecurity threats and conservation planning. Prediction errors are almost ubiquitous in habitat models. An understanding of the source, magnitude and pattern of these errors is essential if the models are to be used transparently in decision making.

This study considered the sources of errors in habitat models. It divided them into two main classes, error resulting from data deficiencies and error introduced by the specification of the model. Common and important data errors included missing covariates, and samples of species’ occurrences that were small, biased or lack absences. These affected the types of models that could be developed and the probable errors that would occur. Almost all models had missing covariates, and this introduced significant spatial correlation in the errors of the analysis.

A challenging aspect of modelling is that species’ distributions are affected by processes operating in both environmental and geographical space. We differentiated between global (aspatial) and local (spatial) errors, and discussed how they arise and what can be done to alleviate their effects.

Synthesis and applications. This study brings together statistical and ecological thinking to consider the appropriate techniques for habitat modelling. Ecological theory suggests models capable of defining optima, while allowing for interactions between variables. Statistical considerations, including impacts of data errors, suggest models that deal with multimodality and discontinuity in response surfaces. Models are typically simple approximations of the true probability surface. We suggest the use of flexible regression techniques, and explain what makes such methods superior for ecological modelling. The most robust modelling approaches are likely to be those in which care is taken to match the model with knowledge of ecology, and in which each is allowed to inform the other.