bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Aspergillus pH‐responsive transcription factor PacC regulates virulence
Authors: Bignell Elaine , Negrete‐Urtasun Susana , Calcagno Ana Maria , Haynes Ken , Arst Herbert N. , Rogers Tom 
Published: February 2005

SummaryThe ability of a pathogen to adapt to the host environment is usually required for the initiation of disease. Here we have investigated the importance of the Aspergillus nidulans PacC‐mediated pH response in the pathogenesis of pulmonary aspergillosis. Using mutational analysis, we demonstrate that, in neutropenic mice, elimination of the A. nidulans pH‐responsive transcription factor PacC, blocking the ambient pH signal transduction pathway or prevention of PacC proteolytic processing acutely attenuates virulence. Infections caused by these alkali‐sensitive mutants are characterized by limited growth in vivo and a reduction of inflammatory cell infiltration. In stark contrast, constitutive activation of PacC causes increased mortality marked by extensive fungal invasive growth. PacC action is therefore required for, and able to enhance virulence, demonstrating that the A. nidulans pH‐responsive transcription factor PacC plays a pivotal role in pulmonary pathogenesis.
Supportive and inhibitory elements of a putative PrfA‐dependent promoter inListeria monocytogenes
Authors: Luo Qin , Herler Michael , Müller‐Altrock Stefanie , Goebel Werner 
Published: February 2005

SummaryElements essential for PrfA‐dependent transcription were analysed on two promoters ofListeria monocytogenes, the PrfA‐dependent promoter of the phospholipase geneplcA(PplcA) and a putative promoter of thearoAgene (ParoA2) which contains a similar PrfA‐binding site and a similar −10 box as PplcAbut does not function as PrfA‐dependent promoter. We constructed a series of hybridplcA‐aroApromoters by exchanging corresponding sequence elements of these two ‘promoters’. The results showed that the two critical elements of PrfA‐dependent promoters, the PrfA‐box and the −10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the −10 box of ParoA2were strongly inhibitory for PrfA‐dependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actualin vivoandin vitrousedaroApromoter (ParoA1) and a sequence immediately downstream of the putative −10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA‐dependent transcription from ParoA2. Taking into consideration the lessons learned from this study we were able to construct a functional PrfA‐dependentaroApromoter.
Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2‐Cys peroxiredoxin
Authors: Dombrecht Bruno , Heusdens Christophe , Beullens Serge , Verreth Christel , Mulkers Esther , Proost Paul , Vanderleyden Jos , Michiels Jan 
Published: February 2005

SummaryIn general, oxidative stress, the consequence of an aerobic lifestyle, induces bacterial antioxidant defence enzymes. Here we report on a peroxiredoxin of Rhizobium etli, prxS, strongly expressed under microaerobic conditions and during the symbiotic interaction with Phaseolus vulgaris. The microaerobic induction of the prxS‐rpoN2 operon is mediated by the alternative sigma factor RpoN and the enhancer‐binding protein NifA. The RpoN‐dependent promoter is also active under low‐nitrogen conditions through the enhancer‐binding protein NtrC. An additional symbiosis‐specific weak promoter is located between prxS and rpoN2. Constitutive expression of prxS confers enhanced survival and growth to R. etli in the presence of H2O2. Single prxS mutants are not affected in their symbiotic abilities or defence response against oxidative stress under free‐living conditions. In contrast, a prxS katG double mutant has a significantly reduced (>40%) nitrogen fixation capacity, suggesting a functional redundancy between PrxS and KatG, a bifunctional catalase‐peroxidase. In vitro assays demonstrate  the  reduction  of  PrxS  protein  by  DTT and thioredoxin. PrxS displays substrate specificity towards H2O2 (Km = 62 µM) over alkyl hydroperoxides (Km > 1 mM). Peroxidase activity is abolished in both the peroxidatic (C56) and resolving (C156) cysteine PrxS mutants, while the conserved C81 residue is required for proper folding of the protein. Resolving of the R. etli PrxS peroxidatic cysteine is probably an intramolecular process and intra‐ and intersubunit associations were observed. Taken together, our data support, for the first time, a role for an atypical 2‐Cys peroxiredoxin against oxidative stress in R. etli bacteroids.
Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control
Authors: Menges Margit , De Jager Sarah M. , Gruissem Wilhelm , Murray James A.H. 
Published: February 2005

SummaryArabidopsis has over 80 genes encoding conserved and plant‐specific core cell cycle regulators, but in most cases neither their timing of expression in the cell cycle is known nor whether they represent redundant and/or tissue‐specific functions. Here we identify novel cell cycle regulators, including new cyclin‐dependent kinases related to the mammalian galactosyltransferase‐associated protein kinase p58, and new classes of cyclin‐like and CDK‐like proteins showing strong tissue specificity of expression. We analyse expression of all cell cycle regulators in synchronized Arabidopsis cell cultures using multiple approaches including Affymetrix microarrays, massively parallel signature sequencing and real‐time reverse transcriptase polymerase chain reaction, and in plant material using the results of over 320 microarray experiments. These global analyses reveal that most core cell cycle regulators are expressed across almost all tissues and more than 85% are expressed at detectable levels in the cell suspension culture, allowing us to present a unified model of transcriptional regulation of the plant cell cycle. Characteristic patterns of D‐cyclin expression in early and late G1 phase, either limited to the re‐entry cycle or continuously oscillating, suggest that several CYCD genes with strong oscillatory regulation in late G1 may play the role of cyclin E in plants. Alone amongst the six groups of A and B type cyclins, members of CYCA3 peak in S‐phase suggest it is a major component of S‐phase kinases, whereas others show a peak in G2/M. 82 genes share this G2/M regulatory pattern, about half being new candidate mitotic genes of previously unknown function.
A novel isoform of glucan, water dikinase phosphorylates pre‐phosphorylated α‐glucans and is involved in starch degradation in Arabidopsis
Authors: Baunsgaard Lone , Lütken Henrik , Mikkelsen René , Glaring Mikkel A. , Pham Tam T. , Blennow Andreas 
Published: February 2005

SummaryAn Arabidopsis thaliana gene encoding a homologue of the potato α‐glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C‐3 position of the glucose unit of preferably pre‐phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild‐type leaf starch, the level of C‐3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C‐3 linked phospho‐ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.
ROS perception in Arabidopsis thaliana: the ozone‐induced calcium response
Authors: Evans Nicola H. , McAinsh Martin R. , Hetherington Alistair M. , Knight Marc R. 
Published: February 2005

SummaryOzone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic‐free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre‐treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.
Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors
Authors: Milosevic Javorina , Schwarz Sigrid C. , Krohn Knut , Poppe Monika , Storch Alexander , Schwarz Johannes 
Published: February 2005

AbstractThe efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O2, whereas exposing these cells to 21% oxygen prohibited long‐term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain‐derived precursors cultured in 3 versus 21% oxygen after 1–2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self‐renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons.
Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase
Authors: Jin Hong , Sha Di , Wei Jianning , Davis Kathleen M. , Wu Heng , Jin Ying , Wu Jang‐Yen 
Published: February 2005

AbstractIn this work, we report that the recombinant glutathione S‐transferase (GST)‐human l‐glutamic acid decarboxylase (HGAD) isoforms, 65‐kDa l‐glutamic acid decarboxylase (GAD) (GST‐HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Δ1–70) and HGAD67(Δ1–90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST‐HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the Vmax of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin‐agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti‐GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.
Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis
Authors: Dumitrescu Pene Teodora , Rosé Sergio D. , Lejen Tatiana , Marcu Monica G. , Trifaró José‐Maria 
Published: February 2005

AbstractStimulation‐induced chromaffin cell cortical F‐actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+‐dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+‐binding sites. F‐actin severing activity of Sc is Ca2+‐dependent, whereas Sc‐evoked actin nucleation is Ca2+‐independent. Sc domain role in secretion was studied by co‐transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)‐fusion Sc constructs. Cells over‐expressing actin severing Sc1‐6 or Sc1‐2 (first and second actin binding sites) constructs, increased F‐actin disassembly and hGH release upon depolarization. Over‐expression of nucleating Sc5‐6, Sc5 or ScABP3 (third actin site) constructs decreased F‐actin disassembly and hGH release upon stimulation. Over‐expression of ScL5‐6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F‐actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly ⇆ assembly) and release (facilitation ⇆ inhibition). The position of the switch (severing ⇆ nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation‐induced Ca2+ entry would increase Sc‐evoked cortical F‐actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc‐evoked actin nucleation.
Conformational changes at benzodiazepine binding sites of GABAA receptors detected with a novel technique
Authors: Berezhnoy Dmytro , Baur Roland , Gonthier Anne , Foucaud Bernard , Goeldner Maurice , Sigel Erwin 
Published: February 2005

AbstractBenzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABAA receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, α1H101Cβ2γ2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the α/γ subunit interface, or if it occurs in both α1 subunits, exclusively reaction at the α/γ subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15–1788 efficiently inhibited the covalent reaction in the presence of 100 µm GABA but only partially in its absence or in the presence of 10 µm GABA. It is concluded that Ro15–1788 efficiently protects activated and desensitized states, but not the resting state.
The expression of GLP‐1 receptor mRNA and protein allows the effect of GLP‐1 on glucose metabolism in the human hypothalamus and brainstem
Authors: Alvarez Elvira , Martínez M. Dolores , Roncero Isabel , Chowen Julie A. , García‐Cuartero Beatriz , Gispert Juan D. , Sanz Carmen , Vázquez Patricia , Maldonado Antonio , De Cáceres Javier , Desco Manuel , Pozo Miguel Angel , Blázquez Enrique 
Published: February 2005

AbstractIn the present work, several experimental approaches were used to determine the presence of the glucagon‐like peptide‐1 receptor (GLP‐1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP‐1 receptor mRNA in several brain areas. In addition, GLP‐1R, glucose transporter isoform (GLUT‐2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP‐1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross‐linking assays. Specific binding of 125I‐GLP‐1(7–36) amide to the GLP‐1R was detected in several brain areas and was inhibited by unlabelled GLP‐1(7–36) amide, exendin‐4 and exendin (9–39). A further aim of this work was to evaluate cerebral‐glucose metabolism in control subjects by positron emission tomography (PET), using 2‐[F‐18] deoxy‐d‐glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP‐1(7–36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG‐6‐phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT‐2‐ and GK‐containing cells.
A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant‐negative activity
Authors: Chávez‐Gutiérrez Lucia , Bourdais Julie , Aranda Gonzalo , Vargas Miguel Angel , Matta‐Camacho Edna , Ducancel Frédéric , Segovia Lorenzo , Joseph‐Bravo Patricia , Charli Jean‐Louis 
Published: February 2005

AbstractThyrotropin‐releasing hormone is inactivated in the extracellular space by a membrane‐bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14–intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C‐terminal domain. PPII* was expressed in COS‐7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co‐transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose‐dependent reduction in PPII activity and the formation of covalent PPII–PPII* heterodimers. PPII* is therefore a powerful dominant‐negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.
Distinct roles of PKC isoforms in NCAM‐mediated neurite outgrowth
Authors: Kolkova Kateryna , Stensman Helena , Berezin Vladimir , Bock Elisabeth , Larsson Christer 
Published: February 2005

AbstractThe role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)‐mediated neurite outgrowth was tested using a co‐culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12‐E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM‐stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM‐mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase‐deficient forms of PKCα, βI and βII blocked NCAM‐mediated neurite extension, but had no effect on nerve growth factor (NGF)‐mediated neurite outgrowth. Expression of two PKCɛ constructs: (i) a fragment from PKCɛ encompassing the pseudosubstrate, the C1a domain (including the actin‐binding site, ABS), and parts of the V3 region, or (ii) the PKCɛ‐specific ABS blocked NCAM‐mediated neurite extension in both cases. These two constructs also partially inhibited NGF‐stimulated neuritogenesis indicating that PKCɛ is a positive regulator of both NCAM‐ and NGF‐mediated differentiation. We suggest that PKCɛ is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM‐stimulated neurite outgrowth.
Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase β and DNA‐ligase
Authors: Krishna T. Hari , Mahipal S. , Sudhakar A. , Sugimoto H. , Kalluri Raghu , Rao K. Subba 
Published: February 2005

AbstractSynthetic deoxy‐oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase β. High levels of DNA polymerase β supplementation resulted in gap‐filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32‐mer). However, at lower concentrations of DNA polymerase β, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5′‐PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA‐ligase in addition to recombinant DNA polymerase β. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase β and DNA‐ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.