bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Osmotically Induced Antifungal Chitinases and Bacterial Expression of an Active Recombinant Isoform
Authors: Yun D. J. , D'Urzo M. P. , Abad L. , Takeda S. , Salzman R. , Chen Z. , Lee H. , Hasegawa P. M. , Bressan R. A. 
Published: July 2002

Abstract
NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14: 357–368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16: 1–10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl β-D-thiogalactopyranoside induction occurred at 18[deg]C but not at 25 or 37[deg]C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells.
Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1
Authors: Whitmer Serap , Canel Camilo , Hallard Didier , Gonçalves Cecilia , Verpoorte Robert 
Published: July 2002

Abstract
We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.
Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings1
Authors: Millar A. Harvey , Atkin Owen K. , Ian Menz R. , Henry Beverley , Farquhar Graham , Day David A. 
Published: July 2002

Abstract
Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.
Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1
Authors: Shpigel Etai , Roiz Levava , Goren Raphael , Shoseyov Oded 
Published: July 2002

Abstract
Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.
Subcellular Localization Studies Indicate That Lipoxygenases 1 to 6 Are Not Involved in Lipid Mobilization during Soybean Germination1
Authors: Wang Cunxi , Croft Kevan P.C. , Järlfors Ulla , Hildebrand David F. 
Published: July 2002

Abstract
Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (&lt;0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.
Accumulation of Soybean Glycinin and Its Assembly with the Glutelins in Rice1
Authors: Katsube Tomoyuki , Kurisaka Nobuyuki , Ogawa Masahiro , Maruyama Nobuyuki , Ohtsuka Reiko , Utsumi Shigeru , Takaiwa Fumio 
Published: July 2002

Abstract
Saline-soluble glycinins and insoluble glutelins are the major storage proteins in soybean (Glycine max) and rice (Oryza sativa), respectively. In spite of their differences in solubility properties, both proteins are members of the 11S globulin gene family based on their similarities in primary sequences and processing of the coded protein. Wild-type and methionine-modified glycinin coding sequences were expressed in transgenic rice plants under the control of the rice glutelin GluB-1 promoter. Glycinins were specifically synthesized in the endosperm tissue and co-localized with glutelins in type II protein bodies. They assembled into 7S and 11S species, similar to what was observed in developing soybean seeds. This pattern was quite different from that displayed by the rice glutelins in untransformed plants, in which processed subunits sedimenting at 2S were apparent. In glycinin-expressing transgenic plants, however, glutelins were observed sedimenting at 7S and 11S with lesser amounts in the 2S region. A portion of the glycinins was also found associated in the insoluble glutelin fraction. Renaturation experiments suggested that the hybrid glycinin-glutelin oligomers were formed through specific interactions. Overall, these results indicate that despite significant differences in the assembly of soybean glycinin and rice glutelin, both proteins can assemble with each other to form soluble hexameric oligomers or insoluble aggregates.
Biochemical and Immunocytochemical Characterization of Two Types of Myosins in Cultured Tobacco Bright Yellow-2 Cells
Authors: Yokota Etsuo , Yukawa Chiharu , Muto Shoshi , Sonobe Seiji , Shimmen Teruo 
Published: July 2002

Abstract
We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 μm/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 μm/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 μm/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca2+ at concentrations higher than 10−6 m. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.
GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter
Authors: Wulff Cristian , Norambuena Lorena , Orellana Ariel 
Published: July 2002

Abstract
The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan α,1–2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls.
Dg93, a Nodule-Abundant mRNA of Datisca glomerata with Homology to a Soybean Early Nodulin Gene
Authors: Okubara Patricia A. , Fujishige Nancy A. , Hirsch Ann M. , Berry Alison M. 
Published: July 2002

Abstract
We have isolated a 590-bp full-length cDNA clone designatedDg93, an mRNA that is highly expressed in symbiotic root nodules of the actinorhizal host Datisca glomerata. Dg93mRNA encodes a deduced polypeptide of 105 amino acids with significant identity (74%) to the soybean (Glycine max) early nodulin (ENOD) gene GmENOD93 (Kouchi and Hata, 1993).Dg93 mRNA is abundant in nodules at 4 weeks post inoculation, the earliest time assayed, and steady-state mRNA levels remain elevated 11 weeks after inoculation. Spatial patterns ofDg93 mRNA expression are complex, with transcript accumulation in the nodule lobe meristem, early infection zone, periderm, and cells of the vascular cylinder, but not in the surrounding uninfected cortical cells. Dg93 is encoded by a small gene family in D. glomerata. To our knowledge, this is the first report of a gene from an actinorhizal host that is expressed in the nodule meristem and that shares sequence homology with an early nodulin gene from a legume.
Purified γ-Glutamyl Transpeptidases from Tomato Exhibit High Affinity for Glutathione and GlutathioneS-Conjugates
Authors: Martin Melinda Neal , Slovin Janet P. 
Published: July 2002

Abstractγ-Glutamyl transpeptidases (γGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (γ-l-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two γGTases (I and II) with K m values for glutathione of 110 and 90 μm were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, γ-linked Glu and the artificial substrates γ-l-glutamyl-p-nitroanilide and γ-l-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, includingl-Met, l-Phe, l-Trp,l-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. γGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 m NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two γGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD.
Cloning, Developmental, and Tissue-Specific Expression of Sucrose:Sucrose 1-Fructosyl Transferase from Taraxacum officinale. Fructan Localization in Roots
Authors: Van den Ende Wim , Michiels An , Van Wonterghem Dominik , Vergauwen Rudy , Van Laere André 
Published: July 2002

Abstract
Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%,Helianthus tuberosus 80%), but homology to 1-SST fromAllium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development ofT. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels.
Potassium-Efflux Channels in Extensor and Flexor Cells of the Motor Organ of Samanea saman Are Not Identical. Effects of Cytosolic Calcium
Authors: Moshelion Menachem , Moran Nava 
Published: July 2002

Abstract
Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells (“flexors” and “extensors”). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K+efflux via depolarization-dependent potassium (KD) channels. To compare between flexor and extensor KDchannels and to test for a possible interaction of these channels with the Ca2+-mobilizing phosphoinositide cascade evoked in these motor cells by the “shrinking signals,” we probed the channels with varying (5 nm–3 mm) cytosolic free-Ca2+ concentration ([Ca2+]cyt) in patch-clamped inside-out excised membrane patches. Ca2+ was not required for KD channel activation. [Ca2+]cytof 600 nm decreased the mean number of open KD channels in flexors, as monitored at −30 mV. Detailed analysis revealed that in flexors millimolar [Ca2+]cyt decreased the maximum number of open channels, but simultaneously increased KD channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca2+]cyt practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor KD channels were affected by [Ca2+]cyt. Irrespective of [Ca2+]cyt, the steady-state gating of extensor KD channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor KD channels were similar and decreased by approximately 20% at millimolar [Ca2+]cyt. It is intriguing that the extensor KD channels were significantly less K+selective than those in flexors.
Involvement of a Nuclear-Encoded Basic Helix-Loop-Helix Protein in Transcription of the Light-Responsive Promoter ofpsbD
Authors: Baba Kyoko , Nakano Takeshi , Yamagishi Kazutoshi , Yoshida Shigeo 
Published: July 2002

Abstract
In the chloroplast psbD light-responsive promoter (LRP), a highly conserved sequence exists upstream from the bacterial −10/−35 elements. Multiple sequence-specific DNA binding proteins are predicted to bind to the conserved sequence as transcription factors. Using yeast one-hybrid screening of an Arabidopsis cDNA library, a possible DNA binding protein of the psbD LRP upstream sequence was identified. The protein, designated PTF1, is a novel protein of 355 amino acids (estimated molecular weight of 39.6) that contains a basic helix-loop-helix DNA binding motif in the predicted N-terminal region of the mature protein. Transient expression assay of PTF1-GFP fusion protein showed that PTF1 was localized in chloroplasts. Using the modified DNA sequence in the one-hybrid system, the ACC repeat was shown to be essential for PTF1 binding. The rate of psbD LRP mRNA accumulation was reduced in a T-DNA-inserted Arabidopsis ptf1 mutant. Compared with wild-type plants, the mutant had pale green cotyledons and its growth was inhibited under short-day conditions. These results suggest that PTF1 is a trans-acting factor of the psbD LRP.
The Internal Propeptide of the Ricin Precursor Carries a Sequence-Specific Determinant for Vacuolar Sorting
Authors: Frigerio Lorenzo , Jolliffe Nicholas A. , Di Cola Alessandra , Felipe Doramys Hernández , Paris Nadine , Neuhaus Jean-Marc , Lord J. Michael , Ceriotti Aldo , Roberts Lynne M. 
Published: July 2002

Abstract
Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin is synthesized as a single polypeptide precursor comprising the catalytic A chain and the Gal-binding B chain joined by a 12-amino acid linker propeptide. Upon arrival in the vacuole, the linker is removed. Here, we replicate these events in transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show that the internal linker propeptide is responsible for vacuolar sorting and is sufficient to redirect the ricin heterodimer to the vacuole when fused to the A or the B chain. This internal peptide can also target two different secretory protein reporters to the vacuole. Moreover, mutation of the isoleucine residue within an NPIR-like motif of the propeptide affects vacuolar sorting in proricin and in the reconstituted A-B heterodimer. This is the first reported example of a sequence-specific vacuolar sorting signal located within an internal propeptide.
Correlated conformational fluctuations during enzymatic catalysis: Implications for catalytic rate enhancement
Authors: Alper Kutay O. , Singla Manish , Stone Jennifer L. , Bagdassarian Carey K. 
Published: July 2002

AbstractCorrelated enzymatic conformational fluctuations are shown to contribute to the rate of enhancement achieved during catalysis. Cytidine deaminase serves as a model system. Crystallographic temperature factor data for this enzyme complexed with substrate analog, transition‐state analog, and product are available, thereby establishing a measure of atomic scale conformational fluctuations along the (approximate) reaction coordinate. First, a neural network‐based algorithm is used to visualize the decreased conformational fluctuations at the transition state. Second, a dynamic diffusion equation along the reaction coordinate is solved and shows that the flux velocity through the associated enzymatic conformation space is greatest at the transition state. These results suggest (1) that there are both dynamic and energetic restrictions to conformational fluctuations at the transition state, (2) that enzymatic catalysis occurs on a fluctuating potential energy surface, and (3) a form for the potential energy. The Michaelis‐Menten equations are modified to describe catalysis on this fluctuating potential energy profile, leading to enhanced catalytic rates when fluctuations along the reaction coordinate are appropriately correlated. This represents a dynamic tuning of the enzyme for maximally effective transformation of the ES complex into EP.