bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomes form into seven groups in hexaploid and tetraploid wheat as a prelude to meiosis
Authors: Martinez‐Perez Enrique , Shaw Peter , Aragon‐Alcaide Luis , Moore Graham
Published: September 2003

Hexaploid wheat possesses 42 chromosomes derived from its three ancestral genomes. The 21 pairs of chromosomes can be further divided into seven groups of six chromosomes (one chromosome pair being derived from each of the three ancestral genomes), based on the similarity of their gene order. Previous studies have revealed that, during anther development, the chromosomes associate in 21 pairs via their centromeres. The present study reveals that, as a prelude to meiosis, these 21 chromosome pairs in hexaploid (and tetraploid) wheat associate via the centromeres into seven groups as the telomeres begin to cluster. This results in the association of multiple chromosomes, which then need to be resolved as meiosis progresses. The formation of the seven chromosome clusters now explains the occasional occurrence of remnants of multiple associations, which have been reported at later stages of meiosis in hexaploid (and tetraploid) wheat. Importantly, the chromosomes have the opportunity to be resorted via these multiple interactions. As meiosis progresses, such interactions are resolved through the action of loci such as Ph1, leaving chromosomes as homologous pairs.
The YORE‐YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis
Authors: Kurata Tetsuya , Kawabata‐Awai Chie , Sakuradani Eiji , Shimizu Sakayu , Okada Kiyotaka , Wada Takuji
Published: September 2003

We have identified a new Arabidopsis mutant, yore‐yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double‐stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre‐1 was approximately one‐sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome‐morphology mutants, glabra 2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax‐requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.
Towards deciphering phloem: a transcriptome analysis of the phloem of Apium graveolens
Authors: Vilaine F. , Palauqui J.‐C. , Amselem J. , Kusiak C. , Lemoine R. , Dinant S.
Published: September 2003

Events occurring in the phloem tissue are key to understanding a wide range of developmental and physiological processes in vascular plants. While a considerable amount of molecular information on phloem proteins has emerged in the past decade, a unified picture of the molecular mechanisms involved in phloem differentiation and function is still lacking. New models to increase our understanding of this complex tissue can be created by the development of global approaches such as genomic analysis. In order to obtain a comprehensive overview of the molecular biology of the phloem tissue, we developed a genomic approach using Apium graveolens as a model. cDNA libraries were constructed from mRNAs extracted from isolated phloem of petioles. Expression data obtained from the analysis of 989 expressed sequence tags (ESTs) and the transcript profile deduced from a cDNA macroarray of 1326 clones were combined to identify genes showing distinct expression patterns in the vascular tissues. Comparisons of expression profiles obtained from the phloem, xylem and storage parenchyma tissues uncovered tissue‐specific differential expression patterns for given sets of genes. The major classes of mRNAs predominantly found in the phloem encode proteins related to phloem structure, metal homeostasis or distribution, stress responses and degradation or turnover of proteins. Of great interest for future studies are the genes we found to be specifically expressed in the phloem but for which the function is still unknown, and also those genes described in previous reports to be up or downregulated by specific interactions. From a broader prospective, our results also clearly demonstrate that cDNA macroarray technology can be used to identify the key genes involved in various physiological and developmental processes in the phloem.
Transgene‐induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss‐of‐function mutations
Authors: Lawrence Richard J. , Pikaard Craig S.
Published: September 2003

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss‐of‐function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium‐mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double‐stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana‐ and A. arenosa‐derived centromere repeats. These results indicate that a single RNAi‐inducing transgene can dominantly repress multiple orthologs.
Characterization and functional analysis of three wheat genes with homology to the CONSTANS flowering time gene in transgenic rice
Authors: Nemoto Yasue , Kisaka Mayumi , Fuse Takuichi , Yano Masahiro , Ogihara Yasunari
Published: September 2003

The CONSTANS (CO) gene of Arabidopsis plays a key role in the photoperiodic flowering pathway. To investigate photoperiod responses in cereals in more detail, we isolated three kinds of CO/Hd1 (rice ortholog of CO) homolog from hexaploid wheat, derived from the A, B, and D genomes and designated as wheat ortholog of CO from A genome (TaHd1‐1), TaHd1‐2, and TaHd1‐3, respectively. They were highly similar to each other and to Hd1, and in addition harbored two conserved regions: two zinc finger motifs and CONSTANS, CONSTANS‐LIKE and TIMING OF CAB EXPRESSION 1 (CCT) domain like CO/Hd1. They were located on the long arm of the homoeologous chromosome 6. TaHd1‐2 harbored a 63‐bp deletion at the promoter region containing the GATA‐1 box, and consequently, we detected no subsequent transcript. The TaHd1‐1 genomic clone was introduced to a rice line deficient in Hd1 function. Transgenic plants complemented the functions of rice Hd1: they promoted heading under short‐day (SD) conditions and delayed it under long‐day (LD)/natural conditions, indicating that Hd1 proteins from SD and LD plants share common structures and functions.
Distribution and characterization of over 1000 T‐DNA tags in rice genome
Authors: Chen Shuangyan , Jin Weizheng , Wang Mingyi , Zhang Fan , Zhou Jie , Jia Qiaojun , Wu Yunrong , Liu Feiyan , Wu Ping
Published: September 2003

We generated T‐DNA insertions throughout the rice genome for saturation mutagenesis. More than 1000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T‐DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene‐rich regions. Few insertions (2.4%) were found in repetitive regions. T‐DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5′‐ and 3′‐regulatory regions outside the coding regions both at 500‐bp size and in introns rather than in exons. Such distribution patterns and biases for T‐DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T‐DNA integration mechanism itself. Rice will require approximately the same number of T‐DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T‐DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).
Establishment of a high‐efficiency SNP‐based framework marker set for Arabidopsis
Authors: Törjék O. , Berger D. , Meyer R. C. , Müssig C. , Schmid K. J. , Rosleff Sörensen T. , Weisshaar B. , Mitchell‐Olds T. , Altmann T.
Published: September 2003

The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)‐based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col‐0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single‐tube/single‐capillary format. In addition, for 110 framework markers matrix‐assisted laser desorption/ionization time of flight (MALDI‐ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col‐0 and C24. Subsets of 10–62 of the established markers discriminate between various combinations of the accessions Col‐0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col‐0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) × Col‐0 cross.
The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis
Authors: Kim Jeong Hoe , Choi Dongsu , Kende Hans
Published: September 2003

Previously, we identified a novel rice gene, GROWTH‐REGULATING FACTOR1 (OsGRF1), which encodes a putative transcription factor that appears to play a regulatory role in stem elongation. We now describe the GRF gene family of Arabidopsis thaliana (AtGRF), which comprises nine members. The deduced AtGRF proteins contain the same characteristic regions—the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains—as do OsGRF1 and related proteins in rice, as well as features indicating a function in transcriptional regulation. Most of the AtGRF genes are strongly expressed in actively growing and developing tissues, such as shoot tips, flower buds, and roots, but weakly in mature stem and leaf tissues. Overexpression of AtGRF1 and AtGRF2 resulted in larger leaves and cotyledons, as well as in delayed bolting of the inflorescence stem when compared to wild‐type plants. In contrast, triple insertional null mutants of AtGRF1–AtGRF3 had smaller leaves and cotyledons, whereas single mutants displayed no changes in phenotype and double mutants displayed only minor ones. The alteration of leaf growth in overexpressors and triple mutants was based on an increase or decrease in cell size, respectively. These results indicate that AtGRF proteins play a role in the regulation of cell expansion in leaf and cotyledon tissues.
Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role
Authors: Fujii Tsuneko , Fujii Junichi , Taniguchi Naoyuki
Published: September 2003

A family of proteins with thioredoxin (TRx)‐dependent peroxidase activity, referred to as peroxiredoxins (PRx), has been identified in many species. The sixth member of this family, PRxVI, contains only one conserved cysteine residue, while other members contain additional cysteines. We have isolated a cDNA for rat PRxVI and constructed a large scale baculovirus system to produce the recombinant protein. The protein was purified by a simple two‐step procedure utilizing ion‐exchange and gel‐filtration chromatography. The purified PRxVI exhibited a low level of glutathione‐dependent peroxidase but not TRx‐dependent activity. PRxVI expression was the highest in lung, followed by brain, kidney, heart, testis, etc. as judged by Northern and Western blot analyses using a rabbit antibody to the purified PRxVI. Immunohistochemical analyses showed strong staining in the epithelium of the bronchus and bronchioles in lung and in the epithelial cells of kidney tubules. In addition, Sertoli cells in testis and islet of Langerhans cells in pancreas were strongly stained. The developmental changes of PRxVI expression in lung and kidney were low in the prenatal stage but induced postnatally. Moreover, intraperitoneal administration of chloroform induced PRxVI mRNA in kidney. When the distribution and the induced expression of PRxVI under conditions of oxidative stress are considered, a physiological role of it as an antioxidative enzyme is indicated.
Characterization of a proximal element in the rat preadipocyte factor‐1 (Pref‐1) gene promoter
Authors: Takemori Hiroshi , Doi Junko , Katoh Yoshiko , Halder Sunil K , Lin Xing‐zi , Horike Nanao , Hatano Osamu , Okamoto Mitsuhiro
Published: September 2003

Preadipocyte factor‐1 (Pref‐1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref‐1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref‐1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down‐regulation of Pref‐1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from −76 to −47) of the rat Pref‐1 gene was essential for its expression in adrenocortical carcinoma‐derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC‐box), seemed to contain three elements, two early growth response (Egr) elements and one GC‐box, overlapping each other. Mutations of four or five nucleotides in a 7‐nucleotides‐stretch in the midst of the Egr/GC‐box eliminated the binding of Sp1/3, abolished the activation by Egr‐factor(s) and diminished the Pref‐1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC‐box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7‐nucleotides‐stretch in the midst of the Egr/GC‐box might be important for the Pref‐1 promoter activity, and this proximal element was possibly activated by a still‐unidentified nuclear factor(s). This element would function as the promoter of the Pref‐1 gene in H295R cells, but not in HeLa cells.
Identification and characterization of novel chromogranin B‐derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS
Authors: Wang Zesheng , Vandenberghe Isabel , Depreitere Jan , Devreese Bart , Clerens Stefan , Nouwen Etienne J , Van Beeumen Jozef , De Potter Werner
Published: September 2003

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size‐exclusion, immunoaffinity, and reversed‐phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586–R602 (SR‐17) and is phosphorylated at one or two serine residues. Another novel peptide H603–Q636 (HQ‐34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin‐like peptide fragment (KR‐11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C‐terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.
Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam
Authors: Yoshioka Shiro , Takahashi Satoshi , Hori Hiroshi , Ishimori Koichiro , Morishima Isao
Published: September 2003

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous‐CO form exhibited the Soret peak at 420 nm and the Fe‐CO stretching line at 498 cm−1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate‐binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O–O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O–O bond.
Functional interactions of an Escherichia coli ribosomal ATPase
Authors: Kiel Michael C. , Ganoza M. Clelia
Published: September 2003

The gene encoding ribosome‐bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino‐acid sequence of RbbA reveals the presence of two ATP‐binding domains in the N‐terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF‐Tu and EF‐G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF‐Tu but not for EF‐G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein‐chain elongation is proposed.
Time‐resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29
Authors: Crimi Massimo , Dorra Dieter , Bösinger Carola S. , Giuffra Elisabetta , Holzwarth Alfred R. , Bassi Roberto
Published: September 2003

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over‐expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady‐state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a ≈ 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin‐free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (τ) as well as relative fluorescence quantum yields (rfqy) of the two long‐lived components (τ3 and τ4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of τ3 and in the average lifetime (≈ 2.4 ns with zeaxanthin and 3.2–3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2–5.3) is observed on chlorophyll a fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein‐bound zeaxanthin induces a significant quenching effect.
Phosphorylation of 1‐deoxy‐D‐xylulose by D‐xylulokinase of Escherichia coli
Authors: Wungsintaweekul Juraithip , Herz Stefan , Hecht Stefan , Eisenreich Wolfgang , Feicht Richard , Rohdich Felix , Bacher Adelbert , Zenk Meinhart H.
Published: September 2003

1‐deoxy‐d‐xylulose 5‐phosphate serves as a precursor for the biosynthesis of the vitamins thiamine and pyridoxal and for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate via the nonmevalonate pathway of terpenoid biosynthesis. Earlier studies had shown that Escherichia coli incorporates unphosphorylated 1‐deoxy‐d‐xylulose into the terpenoid side chain of ubiquinones with high efficacy. We show that d‐xylulokinase of E. coli (EC 2.7.1.17) catalyzes the phosphorylation of 1‐deoxy‐d‐xylulose at the hydroxy group of C‐5 at a rate of 1.6 µmol·mg min−1. This reaction constitutes a potential salvage pathway for the generation of 1‐deoxy‐d‐xylulose 5‐phosphate from exogenous or endogenous 1‐deoxy‐d‐xylulose as starting material for the biosynthesis of terpenoids, thiamine and pyridoxal.
Aggresome formation by anti‐Ras intracellular scFv fragments
Authors: Cardinale Alessio , Filesi Ilaria , Biocca Silvia
Published: September 2003

Diverting the antigen from its normal intracellular location to other compartments in an antibody‐mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett.,439, 197–202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem.267, 1196–205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen–antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti‐Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated‐scFv fragments in which p21Ras is sequestered and degraded in an antibody‐mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP‐scFv with a myc‐tagged ubiquitin and colocalization with specific anti‐proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen–antibody complex is naturally addressed to the ubiquitin–proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.
Interaction of mastoparan with membranes studied by 1H‐NMR spectroscopy in detergent micelles and by solid‐state 2H‐NMR and 15N‐NMR spectroscopy in oriented lipid bilayers
Authors: Hori Yumiko , Demura Makoto , Iwadate Mitsuo , Ulrich Anne S , Niidome Takuro , Aoyagi Haruhiko , Asakura Tetsuo
Published: September 2003

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14‐residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H‐NMR spectroscopy in perdeuterated (SDS‐d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic α‐helix with high‐order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3–4 residues. Secondly, solid‐state 2H‐NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup‐deuterated dimyristoylglycerophosphocholine (DMPC‐d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the α‐segments and β‐segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG‐rich phase and a DMPG‐depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N‐labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid‐state 15N‐NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in‐plane and a trans‐membrane alignment. The two populations of 90% in‐plane and 10% trans‐membrane helices are characterized by a mosaic spread of ± 30° and ± 10°, respectively. The biological activity of mastoparan is discussed in terms of a pore‐forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip‐flop between the monolayers.
Structure of the 5′ sequences of the human γ‐glutamyltransferase gene
Authors: Visvikis Athanase , Pawlak André , Accaoui Marie José , Ichino Kazuhiko , Leh Hervé , Guellaen Georges , Wellman Maria
Published: September 2003

In humans, five distinct mRNAs code for γ‐glutamyltransferase (GGT). Their coding regions are identical and their 5′ untranslated regions exhibit both common and type‐specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5′ region of the human GGT gene. The common regions of the 5′ UTR are encoded by five exons, localized within a 2.4‐kb region of the genomic DNA. Three of them are separated only by intron‐donor or intron‐acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5′ UTRs in a tissue‐specific manner. In addition, we have isolated a genomic fragment containing the most distal 5′ sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5′ RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell‐type‐specific manners.
Existence of a tightly regulated water channel in Saccharomyces cerevisiae
Authors: Meyrial Valérie , Laizé Vincent , Gobin Renée , Ripoche Pierre , Hohmann Stefan , Tacnet Frédérique
Published: September 2003

The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1‐1p and Aqy2‐1p. Previous work demonstrated that Aqy1‐1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2‐1p in this system. Specific antibodies were used to follow the expression of Aqy1‐1p and Aqy2‐1p in the yeast. Aqy1‐1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2‐1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2‐1p expression was repressed by hyper‐osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2‐1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2‐1p was detected by stopped‐flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin‐mediated water transport in yeast is discussed.
Evaluation of the mechanism of aromatase cytochrome P450
Authors: Kao Yeh‐Chih , Korzekwa Kenneth R. , Laughton Charles A. , Chen Shiuan
Published: September 2003

Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4‐hydroxyandrostenedione (4‐OHA), 7α‐(4′‐amino)phenylthio‐1,4‐androstandiene‐3,17‐dione (7α‐APTADD), and bridge (2,19‐methyleneoxy) androstene‐3,17‐dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I‐helix region and located near the C‐19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild‐type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10‐fold increase in the Ki value of 7α‐APTADD for S478T over that for the wild‐type enzyme, suggesting that Ser478 might be near the C‐7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19‐ol intermediate was generated by both S478A and S478T than the wild‐type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4‐OHA for H480Q was found to be three times that of the wild‐type enzyme. In addition, significantly more 19‐ol and 19‐al intermediates were detected for both mutants H480K and H480Q than for the wild‐type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C‐3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.
Binding of two mono‐acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling
Authors: Douliez Jean‐Paul , Jégou Sandrine , Pato Christine , Mollé Daniel , Tran Vinh , Marion Didier
Published: September 2003

The binding of two mono‐acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C‐terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1‐myristoylglycerophosphatidylcholine (MyrGro‐PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 µm and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro‐PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen‐addad, C. & Pebay‐Peyroula, E. (1999) Eur. J. Biochem.264, 562–568.]. Results from isothermal titration calorimetry experiments indicated non‐classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two ω‐hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.
Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK‐N‐SH neuroblastoma cell invasion
Authors: Farina Antonietta R. , Tacconelli Antonella , Cappabianca Lucia , Masciulli Maria‐Paola , Holmgren Arne , Beckett Geoffery J. , Gulino Alberto , Mackay Andrew R.
Published: September 2003

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)‐1 and TIMP‐2 activity with an approximate IC50 of 0.3 µm, matrix metalloproteinase (MMP)‐2 activity with an approximate IC50 of 2 µm but did not inhibit MMP‐9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP‐2 and MMP‐9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP‐2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP‐2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK‐N‐SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP‐2 activity in the presence of Trx. Trx stimulated SK‐N‐SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.
Evaluating low level sequence identities
Authors: Nicholas Hugh B. , Arst Herbert N. , Caddick Mark X.
Published: September 2003

A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7–18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene136, 49–54]. We tested this hypothesis by a sensitive combination of position‐specific log‐odds scoring matrix methods. The position‐specific log‐odds scoring matrices were derived from a large number of 3‐dehydroquinate synthase and 5‐enolpyruvylshikimate‐3‐phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position‐specific log‐odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA–AROM homology even more problematic.
Solution structure of human immunodeficiency virus type 1 Vpr(13–33) peptide in micelles
Authors: Engler Andrea , Stangler Thomas , Willbold Dieter
Published: September 2003

Human immunodeficiency virus type 1 protein R (HIV‐1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G2 cell cycle arrest and is involved in cellular differentiation and cell death. Also, Vpr subcellular localization is as variable as its functions. It is known that, consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr obviously is dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 13–33 of HIV‐1 Vpr in micelles. Employing nuclear magnetic resonance and circular dichroism spectroscopy we found this part of Vpr, known to be essential for nuclear localization, to be almost completely α helical. Our results provide structural data suggesting residues 13–33 of Vpr to form an amphipathic, leucine‐zipper‐like α helix that serves as a basis for interactions with a variety of viral and cellular factors.
The C‐terminal cysteine‐rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2
Authors: Heine Petra , Braun Norbert , Sévigny Jean , Robson Simon C. , Servos Jörg , Zimmermann Herbert
Published: September 2003

Ecto‐nucleoside triphosphate diphosphohydrolases (E‐NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) share a common membrane topography with a transmembrane domain at both the N‐ and C‐terminus, an extensive extracellular loop with five ‘apyrase conserved regions’ (ACR1 to ACR5), and a cysteine‐rich C‐terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N‐terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino‐acid residues between ACR3 and ACR5 and in particular the cysteine‐rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild‐type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild‐type enzymes.
A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O‐glycosylated mutant of human serum albumin with two additional disulfide bridges
Authors: Minchiotti Lorenzo , Campagnoli Monica , Rossi Antonio , Cosulich Maria E. , Monti Maria , Pucci Piero , Kragh‐Hansen Ulrich , Granel Brigitte , Disdier Patrick , Weiller Pierre‐Jean , Galliano Monica
Published: September 2003

Albumin Kénitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kénitra (Morocco). The slow‐migrating variant and the normal protein were isolated by anion‐exchange chromatography and, after treatment with CNBr, the digests were analyzed by two‐dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse‐phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kénitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single‐base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S–S bridges and showed that albumin Kénitra is partially O‐glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino‐acid sequence analysis of the tryptic fragment 592–597.
Identification of tyrosine‐phosphorylated proteins associated with the nuclear envelope
Authors: Otto Henning , Dreger Mathias , Bengtsson Luiza , Hucho Ferdinand
Published: September 2003

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation‐dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine‐phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine‐phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine‐specific antibodies, tryptic in‐gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2β, the heat shock protein hsc70, and the DNA/RNA‐binding proteins PSF, hypothetical 16‐kDa protein, and NonO, which copurify with the nuclear envelope.
The C‐terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2α) against thermal aggregation
Authors: Roher Nerea , Miró Francesc , Boldyreff Brigitte , Llorens Franc , Plana Maria , Issinger Olaf‐Georg , Itarte Emilio
Published: September 2003

The C‐terminal domain (residues 518–803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6‐N‐terminal tag (grp94‐CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol. Grp94‐CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2α), although it did not protect against thermal inactivation. This anti‐aggregation effect of grp94‐CT was concentration dependent, with full protection achieved at grp94‐CT/CK2α molar ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti‐aggregation effects of grp94‐CT on CK2α without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C‐terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.
Phosphatidyl inositol‐4,5‐bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes
Authors: Asteggiano Carla , Berberián Graciela , Beaugé Luis
Published: September 2003

This work shows the existence of a phosphatidylinositol 4,5‐bisphosphate (PtdIns‐4,5‐P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross‐immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns‐4,5‐P2. In addition, PtdIns‐4,5‐P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100–130% when membrane vesicles are incubated (15–20 s at 37 °C) with 1 mm MgATP and 1 µm Ca2+ (b) in the presence of 100 µm Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns‐4,5‐P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns‐4,5‐P2 is greatly reduced compared with that in the presence of 1 µm Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns‐4,5‐P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP‐stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+‐dependent levels of PtdIns‐4,5‐P2 bound to the exchanger molecule.
Involvement of Rel factors in the expression of antimicrobial peptide genes in amphibia
Authors: Miele Rossella , Björklund Gunnel , Barra Donatella , Simmaco Maurizio , Engström Ylva
Published: September 2003

Genes coding for antimicrobial peptides in amphibia reveal a remarkably high number of structural motifs for response elements, previously identified in the genes of insect antimicrobial peptides and in those of the mammalian acute phase response. This study focuses on the functional analysis of the bombinin gene promoter in a Drosophila blood cell line, and the identification of κB‐binding factors in skin secretions of the frog Bombina orientalis. Transfection experiments demonstrated that the bombinin gene promoter was activated in a lipopolysaccharide‐dependent manner, and that insect Rel factors target specific sequences in the amphibian gene promoter. After bathing frogs in bacteria, their skin secretions contained κB‐specific binding complexes, indicating that Rel factors are crucial components in the response against gram‐negative bacteria in this species. These results suggest that a common ancestral control mechanism governs the expression of the first line host‐defence from insects to vertebrates.