bluuebunny/crossref_metadata_2025_split huggingface dataset:
GTP Promotes the Formation of Early-Import Intermediates But Is Not Required during the Translocation Step of Protein Import into Chloroplasts
Authors: Young Michael E. , Keegstra Kenneth , Froehlich John E. 
Published: July 2002

Abstract
Protein import into chloroplasts is an energy-requiring process mediated by a proteinaceous import apparatus. Although previous work has shown that low levels of ATP or GTP can support precursor binding, the role of GTP during the import process remains unclear. Specifically, it is unknown whether GTP plays a separate role from ATP during the early stages of protein import and whether GTP has any role in the later stages of transport. We investigated the role of GTP during the various stages of protein import into chloroplasts by using purified GTP analogs and an in vitro import assay. GTP, GDP, the nonhydrolyzable analog GMP-PNP, and the slowly hydrolyzable analogs guanosine 5′-O-(2-thiodiphosphate) and guanosine 5′-O-(3-thiotriphosphate) were used in this study. Chromatographically purified 5′-guanylyl-imido-diphosphate and guanosine 5′-O-(3-thiotriphosphate) were found to inhibit the formation of early-import intermediates, even in the presence of ATP. We also observed that GTP does not play a role during the translocation of precursors from the intermediate state. We conclude that GTP hydrolysis influences events leading to the formation of early-import intermediates, but not subsequent steps such as precursor translocation.
Water Deficit Effects on Raffinose Family Oligosaccharide Metabolism in Coleus
Authors: Pattanagul Wattana , Madore Monica A. 
Published: July 2002

AbstractVariegated coleus (Coleus blumei Benth.) plants were exposed to a restricted water supply for 21 d. The relative water content in leaf tissues was reduced from 80% (control) to 60% (drought-stressed). Under drought conditions, the stomatal conductance and leaf photosynthetic rate were reduced. In green leaf tissues, drought stress also greatly decreased the diurnal light-period levels of the raffinose family oligosaccharides (RFOs) stachyose and raffinose, as well as those of other non-structural carbohydrates (galactinol, sucrose, hexoses, and starch). However, drought had little effect on soluble carbohydrate content of white, non-photosynthetic leaf tissues. In green tissues, galactinol synthase activity was depressed by drought stress. An accumulation of O-methyl-inositol was also observed, which is consistent with the induction of myoinositol-6-O-methyltransferase activity seen in the stressed green tissues. In source tissues, RFO metabolism is apparently reduced by drought stress through a combined effect of decreased photosynthesis and reduced galactinol synthase activity. Moreover, a further reduction in RFO biosynthesis may have been due to a switch in carbon partitioning to O-methyl-inositol biosynthesis, creating competition for myoinositol, a metabolite shared by both biochemical pathways.
The PsbY Protein Is Not Essential for Oxygenic Photosynthesis in the Cyanobacterium Synechocystis sp. PCC 6803
Authors: Meetam Metha , Keren Nir , Ohad Itzhak , Pakrasi Himadri B. 
Published: July 2002

Abstract
A tetra-manganese cluster in the photosystem II (PSII) pigment-protein complex plays a critical role in the photosynthetic oxygen evolution process. PsbY, a small membrane-spanning polypeptide, has recently been suggested to provide a ligand for manganese in PSII (A.E. Gau, H.H. Thole, A. Sokolenko, L. Altschmied, R.G. Herrmann, E.K. Pistorius [1998] Mol Gen Genet 260: 56–68). We have constructed a mutant strain of the cyanobacteriumSynechocystis sp. PCC 6803 with an inactivatedpsbY gene (sml0007). Southern-blot and polymerase chain reaction analysis showed that the mutant had completely segregated. However, the ΔpsbY mutant cells grew normally under photoautotrophic conditions. Moreover, growth of the wild-type and mutant cells were similar under high-light photoinhibition conditions, as well as in media without any added manganese, calcium, or chloride, three required inorganic cofactors for the oxygen-evolving complex of PSII. Analysis of steady-state and flash-induced oxygen evolution, fluorescence induction, and decay kinetics, and thermoluminescence profiles demonstrated that the ΔpsbY mutant cells have normal photosynthetic activities. We conclude that the PsbY protein inSynechocystis 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the oxygen-evolving complex of PSII.
CO2-Responsive Transcriptional Regulation ofCAH1 Encoding Carbonic Anhydrase Is Mediated by Enhancer and Silencer Regions in Chlamydomonas reinhardtii
Authors: Kucho Ken-ichi , Ohyama Kanji , Fukuzawa Hideya 
Published: July 2002

Abstract
Chlamydomonas reinhardtii adapts to the stress of CO2-limiting conditions through the induction of a set of genes includingCAH1, which encodes a periplasmic carbonic anhydrase.CAH1 is up-regulated under low-CO2conditions (air containing 0.04% [v/v] CO2) in the presence of light, whereas it is down-regulated under high-CO2 conditions (5% [v/v] CO2) or in the dark. In an effort to identify cis-elements involved in the transcriptional regulation of CAH1, a series of 5′-nested deletions of the region upstream of CAH1 were fused to a promoterless arylsulfatase reporter gene (ARS). The upstream region from −651 to +41 relative to the transcription start site was sufficient to regulate the expression of ARS with kinetics similar to those of endogenousCAH1. Deletion of the region between −651 and −294 resulted in a significant decrease in the level of arylsulfatase activity expressed under low-CO2 conditions. The 543-bp upstream region from −651 to −109, without any promoter elements, CAAT-box, or TATA-box, could confer CO2 and light responsiveness on the β2-tubulin minimal promoter. This 543-bp region was divided into two parts: a 358-bp silencer region from −651 to −294, which represses the minimal promoter activity under high-CO2 conditions, and a 185-bp enhancer region from −293 to −109, which activates the promoter under low-CO2conditions in the presence of light.
The Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene Expression during the Transition from System-1 to System-2 Ethylene Synthesis in Tomato
Authors: Barry Cornelius S. , Llop-Tous M. Immaculada , Grierson Donald 
Published: July 2002

Abstract1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the key regulatory enzymes involved in the synthesis of the hormone ethylene and is encoded by a multigene family containing at least eight members in tomato (Lycopersicon esculentum). Increased ethylene production accompanies ripening in tomato, and this coincides with a change in the regulation of ethylene synthesis from auto-inhibitory to autostimulatory. The signaling pathways that operate to bring about this transition from so-called system-1 to system-2 ethylene production are unknown, and we have begun to address these by investigating the regulation of ACS expression during ripening. Transcripts corresponding to four ACS genes,LEACS1A, LEACS2, LEACS4, and LEACS6, were detected in tomato fruit, and expression analysis using the ripening inhibitor(rin) mutant in combination with ethylene treatments and the Never-ripe (Nr) mutant has demonstrated that each is regulated in a unique way. A proposed model suggests that system-1 ethylene is regulated by the expression ofLEACS1A and LEACS6. In fruit a transition period occurs in which the RIN gene plays a pivotal role leading to increased expression of LEACS1A and induction of LEACS4. System-2 ethylene synthesis is subsequently initiated and maintained by ethylene-dependent induction ofLEACS2.
A Minimal Serine/Threonine Protein Kinase Circadianly Regulates Phosphoenolpyruvate Carboxylase Activity in Crassulacean Acid Metabolism-Induced Leaves of the Common Ice Plant
Authors: Taybi Tahar , Patil Shameekumar , Chollet Raymond , Cushman John C. 
Published: July 2002

Abstract
Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca2+-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C4 species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C4 PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca2+-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to l-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.
Trivalent Ions Activate Abscisic Acid-Inducible Promoters through an ABI1-Dependent Pathway in Rice Protoplasts
Authors: Hagenbeek Dik , Quatrano Ralph S. , Rock Christopher D. 
Published: July 2002

Abstract
The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays)VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 andabi1-1 in transactivation of the ABA-inducibleHVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (β-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.
Molecular Evolution of Receptor-Like Kinase Genes in Hexaploid Wheat. Independent Evolution of Orthologs after Polyploidization and Mechanisms of Local Rearrangements at Paralogous Loci
Authors: Feuillet Catherine , Penger Anja , Gellner Klaus , Mast Austin , Keller Beat 
Published: July 2002

Abstract
Hexaploid wheat is a young polyploid species and represents a good model to study mechanisms of gene evolution after polyploidization. Recent studies at the scale of the whole genome have suggested rapid genomic changes after polyploidization but so far the rearrangements that have occurred in terms of gene content and organization have not been analyzed at the microlevel in wheat. Here, we have isolated members of a receptor kinase (Lrk) gene family in hexaploid and diploid wheat, Aegilops tauschii, and barley (Hordeum vulgare). Phylogenetic analysis has allowed us to establish evolutionary relationships (orthology versus paralogy) between the different members of this gene family in wheat as well as with Lrk genes from barley. It also demonstrated that the sequences of the homoeologous Lrk genes evolved independently after polyploidization. In addition, we found evidence for gene loss during the evolution of wheat and barley. Analysis of large genomic fragments isolated from nonorthologous Lrkloci showed a high conservation of the gene content and gene organization at these loci on the homoeologous group 1 chromosomes of wheat and barley. Finally, sequence comparison of two paralogous fragments of chromosome 1B showed a large number of local events (sequence duplications, deletions, and insertions), which reveal rearrangements and mechanisms for genome enlargement at the microlevel.
Maize Non-Photosynthetic Ferredoxin Precursor Is Mis-Sorted to the Intermembrane Space of Chloroplasts in the Presence of Light
Authors: Hirohashi Toshiya , Hase Toshiharu , Nakai Masato 
Published: July 2002

Abstract
Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.
Cryocrystallography of a Kunitz-type serine protease inhibitor: the 90 K structure of winged bean chymotrypsin inhibitor (WCI) at 2.13 Å resolution
Authors: Ravichandran S. , Sen U. , Chakrabarti C. , Dattagupta J. K. 
Published: July 2002

The crystal structure of a Kunitz-type double-headed α-chymotrypsin inhibitor from winged bean seeds has been refined at 2.13 Å resolution using data collected from cryo-cooled (90 K) crystals which belong to the hexagonal space group P6122 with unit-cell parameters a = b = 60.84, c = 207.91 Å. The volume of the unit cell is reduced by 5.3% on cooling. The refinement converged to an R value of 20.0% (R
free = 25.8%) for 11100 unique reflections and the model shows good stereochemistry, with r.m.s. deviations from ideal values for bond lengths and bond angles of 0.011 Å and 1.4°, respectively. The structural architecture of the protein consists of 12 antiparallel β-strands joined in the form of a characteristic β-trefoil fold, with the two reactive-site regions, Asn38–Leu43 and Gln63–Phe68, situated on two external loops. Although the overall protein fold is the same as that of the room-temperature model, some conformational changes are observed in the loop regions and in the side chains of a few surface residues. A total of 176 ordered water molecules and five sulfate ions are included in the model.
Crystallization and preliminary X-ray diffraction studies of Streptococcus pyogenes plasmid pSM19035-encoded ω transcriptional repressor
Authors: Murayama Kazutaka , de la Hoz Ana B. , Alings Claudia , López Gema , Orth Peter , Alonso Juan C. , Saenger Wolfram 
Published: July 2002

The transcriptional repressor, ω protein, from the Streptococcus pyogenes broad-host-range plasmid pSM19035 was crystallized at pH 7.5 and 8.5 by the vapour-diffusion method using PEG 4000 as precipitant. Two crystal forms were obtained; the first belongs to the tetragonal space group P41212 or P43212 and the second to the hexagonal space group P61 or P65. The crystals are most likely to contain one ω protein in the asymmetric unit, with Vm
values of 3.2 and 3.5 Å3 Da−1, respectively. The crystals diffract X-rays to 2.4 and 2.9 Å resolution for the tetragonal and hexagonal systems, respectively.
Solution structure and backbone dynamics of an ω‐conotoxin precursor
Authors: Goldenberg David P. , Koehn Rachel E. , Gilbert Dara E. , Wagner Gerhard 
Published: July 2002

AbstractNuclear magnetic resonance spectroscopy was used to characterize the solution structure and backbone dynamics of a putative precursor form of ω‐conotoxin MVIIA, a 25‐amino‐acid residue peptide antagonist of voltage‐gated Ca2+ channels. The mature peptide is found in the venom of a fish‐hunting marine snail Conus magus and contains an amidated carboxyl terminus that is generated by oxidative cleavage of a Gly residue. The form examined in this study is identical to the mature peptide except for the presence of the unmodified carboxy‐terminal Gly. This form, referred to as ω‐MVIIA–Gly, has previously been shown to refold and form its disulfides more efficiently than the mature form, suggesting that the presence of the terminal Gly may favor folding in vivo. The nuclear magnetic resonance (NMR) structure determination indicated that the fold of ω‐MVIIA–Gly is very similar to that previously determined for the mature form, but revealed that the terminal Gly residue participates in a network of hydrogen bonds involving both backbone and side chain atoms, very likely accounting for the enhanced stability and folding efficiency. 15N relaxation experiments indicated that the backbone is well ordered on the nanosecond time scale but that residues 9–15 undergo a conformational exchange processes with a time constant of ∼35 microseconds. Other studies have implicated this segment in the binding of the peptide to its physiological target, and the observed motions may play a role in allowing the peptide to enter the binding site
Role of water in the catalytic cycle of E. coli dihydrofolate reductase
Authors: Shrimpton Paul , Allemann Rudolf K. 
Published: July 2002

AbstractDihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of 7,8‐dihydrofolate (H2F) to 5,6,7,8‐tetrahydrofolate (H4F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X‐ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 Å. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near‐attack conformations, in which the distance between the pro‐R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 Å and the angle between C4 and the pro‐R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP+ and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.
Applications of an Approximation to Molecular Flow in Cylindrical Tubes
Authors: Helmer John C. 
Published: July 2002

A simple approximation is developed for the molecular flow of gas through a cylindrical tube. The pressure distribution along the wall of the tube is derived and is shown to be a good approximation to the short-tube formula of Clausing. Using these results, the problem of measuring the speed of a pump at the end of a tube is examined. If the pressure gauge is located a distance of one tube radius from the pump, then the intrinsic speed is directly measured in the plane of the gauge.
To determine the capture coefficient of an open-ended tube whose inner surface has a uniform sticking coefficient, we employ a differential equation which reproduces the Monte Carlo results of Smith and Lewin with an error of less than 5%. For tubes of infinite length the capture coefficient is given by k = 2(σ1/2)/(1 + σ1/2), where σ is the sticking coefficient of the inner-tube surface. We repeat the calculation for tubes which have the exit end closed by a surface of the same sticking factor.
The Bcl-2 Protein Family: Arbiters of Cell Survival
Authors: Adams Jerry M. , Cory Suzanne 
Published: July 2002

REVIEW
Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, the cell suicide program critical for development, tissue homeostasis, and protection against pathogens. Those most similar to Bcl-2 promote cell survival by inhibiting adapters needed for activation of the proteases (caspases) that dismantle the cell. More distant relatives instead promote apoptosis, apparently through mechanisms that include displacing the adapters from the pro-survival proteins. Thus, for many but not all apoptotic signals, the balance between these competing activities determines cell fate. Bcl-2 family members are essential for maintenance of major organ systems, and mutations affecting them are implicated in cancer.