bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pre‐synaptic and post‐synaptic localization of EphA4 and EphB2 in adult mouse forebrain
Authors: Bouvier David , Corera Amadou T. , Tremblay Marie‐Ève , Riad Mustapha , Chagnon Miguel , Murai Keith K. , Pasquale Elena B. , Fon Edward A. , Doucet Guy 
Published: April 2008

AbstractThe ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long‐term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post‐synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD‐95‐enriched post‐synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre‐synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.
Altered expression of key dopaminergic regulatory proteins in the postnatal brain following perinatal n‐3 fatty acid dietary deficiency
Authors: Kuperstein Faina , Eilam Raya , Yavin Ephrain 
Published: April 2008

AbstractThe consequences of maternal linolenic acid (LNA, 18:3n‐3) dietary deficiency on key dopamine (DA)‐associated regulatory proteins in mesolimbic and mesocortical structures of the postnatal rat brain have been investigated. A marked (4.5‐fold) decrease of the DA‐synthesizing enzyme tyrosine hydroxylase accompanied by a down‐regulation (approx 7.5‐fold) of the vesicular monoamine transporter (VMAT‐2) and a depletion of VMAT‐associated vesicles in the hippocampus were observed in deficient offspring compared with adequately fed controls. The DA transporter (DAT) was not affected by the LNA deficiency indicative of a DAT/VMAT‐2 ratio increase that may enhance the risk of damage of the dopaminergic (DAergic) terminal. A robust increase in DA receptor (DAR1 and DAR2) levels was noticed in the cortex and striatum structures possibly to compensate for the low levels of DA in synaptic clefts. Microglia activation characterized by enhanced levels of ED1 antibody and nuclear internalization of p65 NFκB was noticed following LNA deficiency. Diminished levels of 22:6n‐3 docosahexaenoic acid (Schiefermeier and Yavin 2002), the most ubiquitous metabolite generated by LNA is proposed to reduce the anti‐oxidant arsenal in the developing brain and cause microglia activation and enhanced oxidative stress to increase the risk of certain DA‐associated neurological disorders.
Intra‐neural administration of fractalkine attenuates neuropathic pain‐related behaviour
Authors: Holmes Fiona E. , Arnott Nighat , Vanderplank Penny , Kerr Niall C. H. , Longbrake Erin E. , Popovich Philip G. , Imai Toshio , Combadière Christophe , Murphy Philip M. , Wynick David 
Published: April 2008

AbstractThere is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of neuropathic pain states. Here we demonstrate that levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA in lumbar dorsal root ganglia (DRG) increase 5.8‐fold 7 days after sciatic nerve axotomy, and 1.7‐ and 2.9‐fold, 3 and 7 days respectively, after the spared nerve injury (SNI) model of neuropathic pain. In contrast, no significant change in the levels of fractalkine mRNA is apparent in the DRG after axotomy or SNI. The increase in CX3CR1 mRNA is paralleled by a 3.9‐ and 2.1‐fold increase in the number of CX3CR1‐positive macrophages in the DRG 7 days after axotomy and SNI, respectively. Expression of CX3CR1 in macrophages is also markedly increased in the sciatic nerve proximal to site of injury, by 25.7‐fold after axotomy and 16.2‐fold after SNI, 7 days after injury. Intra‐neural injection into the sciatic nerve of 400 ng or 100 ng of fractalkine in adult 129OlaHsd mice significantly delayed the development of allodynia for 3 days following SNI. Further, CX3CR1 knockout (KO) mice display an increase in allodynia for three weeks after SNI compared to strain‐matched Balb/c controls. Taken together, these results suggest an anti‐allodynic role for fractalkine and its receptor in the mouse.
Enhanced expression of Harvey ras induced by serum deprivation in cultured astrocytes
Authors: Messina Samantha , Molinaro Gemma , Bruno Valeria , Battaglia Giuseppe , Spinsanti Paola , Di Pardo Alba , Nicoletti Ferdinando , Frati Luigi , Porcellini Antonio 
Published: April 2008

AbstractTrophic deprivation contributes to astrocyte damage that occurs in acute and chronic neurodegenerative disorders. Unraveling the underlying mechanisms may pave way to novel cytoprotective strategies. Cultured mouse astrocytes responded to trophic deprivation with a large and transient increase in the expression of p21ras, which was secondary to an enhanced formation of reactive oxygen species (ROS) detected by cytofluorimetric analysis after preloading with 2′,7′‐dichlorofluorescein diacetate. The increase in p21ras levels was largely attenuated by the reducing agent, N‐acetylcysteine, which was proven to reduce ROS formation in astrocytes subjected to serum deprivation. We extended the analysis to the Ha‐Ras isoform, which has been implicated in mechanisms of cytotoxicity. We found that serum deprivation enhanced the expression and activity of Ha‐Ras without changing Ha‐Ras mRNA levels. The increase in Ha‐Ras levels was sensitive to the protein synthesis inhibitor, cycloheximide, suggesting that serum deprivation increases translation of preformed Ha‐Ras mRNA. The late decline in Ha‐Ras levels observed after 60 min was prevented by the proteasome inhibitor, MG132, as well as by the selective mitogen‐activated protein kinase (MAPK) inhibitor, PD98059. Serum deprivation led to the activation of the MAPK pathway in cultured astrocytes, as shown by an increase in phosphorylated extracellular signal‐regulated kinase 1/2 levels after 5 and 30 min. Finally, using the siRNA technology, we found that an acute knock‐down of Ha‐Ras was protective against astrocyte damage induced by serum deprivation. We conclude that cultured astrocytes respond to trophic deprivation with an increased expression in Ha‐Ras, which is limited by the concomitant activation of the MAPK pathway, but is nevertheless involved in the pathophysiology of cell damage.
Methamphetamine‐induced neurotoxicity and microglial activation are not mediated by fractalkine receptor signaling
Authors: Thomas David M. , Francescutti‐Verbeem Dina M. , Kuhn Donald M. 
Published: April 2008

AbstractMethamphetamine (METH) damages dopamine (DA) nerve endings by a process that has been linked to microglial activation but the signaling pathways that mediate this response have not yet been delineated. Cardona et al. [Nat. Neurosci. 9 (2006), 917] recently identified the microglial‐specific fractalkine receptor (CX3CR1) as an important mediator of MPTP‐induced neurodegeneration of DA neurons. Because the CNS damage caused by METH and MPTP is highly selective for the DA neuronal system in mouse models of neurotoxicity, we hypothesized that the CX3CR1 plays a role in METH‐induced neurotoxicity and microglial activation. Mice in which the CX3CR1 gene has been deleted and replaced with a cDNA encoding enhanced green fluorescent protein (eGFP) were treated with METH and examined for striatal neurotoxicity. METH depleted DA, caused microglial activation, and increased body temperature in CX3CR1 knockout mice to the same extent and over the same time course seen in wild‐type controls. The effects of METH in CX3CR1 knockout mice were not gender‐dependent and did not extend beyond the striatum. Striatal microglia expressing eGFP constitutively show morphological changes after METH that are characteristic of activation. This response was restricted to the striatum and contrasted sharply with unresponsive eGFP‐microglia in surrounding brain areas that are not damaged by METH. We conclude from these studies that CX3CR1 signaling does not modulate METH neurotoxicity or microglial activation. Furthermore, it appears that striatal‐resident microglia respond to METH with an activation cascade and then return to a surveying state without undergoing apoptosis or migration.
Detection of Active Butyrate-Degrading Microorganisms in Methanogenic Sludges by RNA-Based Stable Isotope Probing
Authors: Hatamoto Masashi , Imachi Hiroyuki , Yashiro Yuto , Ohashi Akiyoshi , Harada Hideki 
Published: April 2008

ABSTRACT

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the
13
C-labeled rRNA fractions were distinct from unlabeled fractions, and
Syntrophaceae
species,
Tepidanaerobacter
sp., and
Clostridium
spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.
The Supernumerary Chromosome of
Nectria haematococca
That Carries Pea-Pathogenicity-Related Genes Also Carries a Trait for Pea Rhizosphere Competitiveness
Authors: Rodriguez-Carres M. , White G. , Tsuchiya D. , Taga M. , VanEtten H. D. 
Published: April 2008

ABSTRACT

Fungi are found in a wide range of environments, and the ecological and host diversity of the fungus
Nectria haematococca
has been shown to be due in part to unique genes on different supernumerary chromosomes. These chromosomes have been called “conditionally dispensable” (CD) since they are not needed for axenic growth but are important for expanding the host range of individual isolates. From a biological perspective, the CD chromosomes can be compared to bacterial plasmids that carry unique genes that can define the habits of these microorganisms. The current study establishes that the
N. haematococca PDA1
-CD chromosome, which contains the genes for pea pathogenicity (
PEP
cluster) on pea roots, also carries a gene(s) for the utilization of homoserine, a compound found in large amounts in pea root exudates. Competition studies demonstrate that an isolate that lacks the
PEP
cluster but carries a portion of the CD chromosome which includes the homoserine utilization (
HUT
) gene(s) is more competitive in the pea rhizosphere than an isolate without the CD chromosome.
Synthesis and Accumulation of Cyanophycin in Transgenic Strains of
Saccharomyces cerevisiae
Authors: Steinle Anna , Oppermann-Sanio Fred Bernd , Reichelt Rudolf , Steinbüchel Alexander 
Published: April 2008

ABSTRACT

Cyanophycin [multi-
l
-arginyl-poly(
l
-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of
Saccharomyces cerevisiae
. The episomal vector systems pESC (with the galactose-inducible promoter
GAL1
) and pYEX-BX (with the copper ion-inducible promoter
CUP1
) were chosen to express the cyanophycin synthetase gene from the cyanobacterium
Synechocystis
sp. strain PCC 6308 (
cphA
6308
) in yeast. Expression experiments with transgenic yeasts revealed that the use of the
CUP1
promoter is much more efficient for CGP production than the
GAL1
promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from
Escherichia coli
TOP10 harboring
cphA
6308
reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.
In Vitro Mutagenesis of
Bacillus subtilis
by Using a Modified Tn
7
Transposon with an Outward-Facing Inducible Promoter
Authors: Bordi Christophe , Butcher Bronwyn G. , Shi Qiaojuan , Hachmann Anna-Barbara , Peters Joseph E. , Helmann John D. 
Published: April 2008

ABSTRACT

A Tn
7
donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium
Bacillus subtilis
. This new mini-Tn
7
, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the
B. subtilis
genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.
Free-Living Tube Worm Endosymbionts Found at Deep-Sea Vents
Authors: Harmer Tara L. , Rotjan Randi D. , Nussbaumer Andrea D. , Bright Monika , Ng Andrew W. , DeChaine Eric G. , Cavanaugh Colleen M. 
Published: April 2008

ABSTRACT
Recent evidence suggests that deep-sea vestimentiferan tube worms acquire their endosymbiotic bacteria from the environment each generation; thus, free-living symbionts should exist. Here, free-living tube worm symbiont phylotypes were detected in vent seawater and in biofilms at multiple deep-sea vent habitats by PCR amplification, DNA sequence analysis, and fluorescence in situ hybridization. These findings support environmental transmission as a means of symbiont acquisition for deep-sea tube worms.
Isolation of Fungal Cellobiohydrolase I Genes from Sporocarps and Forest Soils by PCR
Authors: Edwards Ivan P. , Upchurch Rima A. , Zak Donald R. 
Published: April 2008

ABSTRACT

Cellulose is the major component of plant biomass, and microbial cellulose utilization is a key step in the decomposition of plant detritus. Despite this, little is known about the diversity of cellulolytic microbial communities in soil. Fungi are well known for their cellulolytic activity and mediate key functions during the decomposition of plant detritus in terrestrial ecosystems. We developed new oligonucleotide primers for fungal exocellulase genes (cellobiohydrolase,
cbhI
) and used these to isolate distinct
cbhI
homologues from four species of litter-decomposing basidiomycete fungi (
Clitocybe nuda
,
Clitocybe gibba
,
Clitopilus prunulus
, and
Chlorophyllum molybdites
) and two species of ascomycete fungi (
Xylaria polymorpha
and
Sarcoscypha occidentalis
). Evidence for
cbhI
gene families was found in three of the four basidiomycete species. Additionally, we isolated and cloned
cbhI
genes from the forest floor and mineral soil of two upland forests in northern lower Michigan, one dominated by oak (
Quercus velutina
,
Q. alba
) and the other dominated by sugar maple (
Acer saccharum
) and American basswood (
Tilia americana
). Phylogenetic analysis demonstrated that cellobiohydrolase genes recovered from the floor of both forests tended to cluster with
Xylaria
or in one of two unidentified groups, whereas cellobiohydrolase genes recovered from soil tended to cluster with
Trichoderma
,
Alternaria
, Eurotiales, and basidiomycete sequences. The ability to amplify a key fungal gene involved in plant litter decomposition has the potential to unlock the identity and dynamics of the cellulolytic fungal community in situ.
Contributions of the Response Regulators Ssk1p and Skn7p in the Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast
Candida lusitaniae
Authors: Ruprich-Robert Gwenaël , Chapeland-Leclerc Florence , Boisnard Stéphanie , Florent Martine , Bories Gaël , Papon Nicolas 
Published: April 2008

ABSTRACT

We recently characterized the histidine kinase receptor genes of
Candida lusitaniae
. For the present study, we have further investigated the role of
SSK1
and
SKN7
, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.
Inactivation of
lsr2
Results in a Hypermotile Phenotype in
Mycobacterium smegmatis
Authors: Arora Kriti , Whiteford Danelle C. , Lau-Bonilla Dalia , Davitt Christine M. , Dahl John L. 
Published: April 2008

ABSTRACT

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of
Mycobacterium smegmatis
, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (
M. smegmatis
Δ

rel
Msm

strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the
lsr2
gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated

rel
Msm

and
lsr2
genes, and so a single
lsr2
mutant was created to analyze the gene's function. The
lsr2
gene was inactivated in the wild-type
M. smegmatis
mc
2
155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and Δ

rel
Msm

strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as “hypermotility”), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing
lsr2
(pLSR2) reverts the strain to the mc
2
155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.
Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy
Authors: Vadillo-Rodriguez Virginia , Beveridge Terry J. , Dutcher John R. 
Published: April 2008

ABSTRACT

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium,
Pseudomonas aeruginosa
PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant,
k
1
, and an effective time constant, τ, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in
k
1
together with a significant decrease in τ. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.
Protective Action of ppGpp in Microcin J25-Sensitive Strains
Authors: Pomares María Fernanda , Vincent Paula A. , Farías Ricardo N. , Salomón Raúl A. 
Published: April 2008

ABSTRACTAsEscherichia colistrains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3′,5′-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level.
Single Gene Deletions of
mrpA
to
mrpG
and
mrpE
Point Mutations Affect Activity of the Mrp Na
+
/H
+
Antiporter of Alkaliphilic
Bacillus
and Formation of Hetero-Oligomeric Mrp Complexes
Authors: Morino Masato , Natsui Shinsuke , Swartz Talia H. , Krulwich Terry A. , Ito Masahiro 
Published: April 2008

ABSTRACT

Mrp antiporters catalyze secondary Na
+
(Li
+
)/H
+
antiport and/or K
+
/H
+
antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the
mrp
operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned
mrp
operon from alkaliphilic
Bacillus
pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient
Escherichia coli
strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar
mrp
gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na
+
/H
+
antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent

K
m

value for Na
+
. The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.