bluuebunny/crossref_metadata_2025 huggingface dataset:
Computational and experimental approaches double the number of known introns in the pathogenic yeast Candida albicans
Authors: Mitrovich Quinn M. , Tuch Brian B. , Guthrie Christine , Johnson Alexander D.
Published: March 2007

Candida albicans is the most common fungal pathogen of humans. Frequently found as a commensal within the digestive tracts of healthy individuals, C. albicans is an opportunistic pathogen that causes a wide variety of clinical syndromes in immuno-compromised individuals. A comprehensive annotation of the C. albicans genome sequence was recently published. Because many C. albicans coding sequences are interrupted by introns, proper intron annotation is essential for the accurate definition of genes in this pathogen. Intron annotation is also important for identifying potential targets of splicing regulation, a common mechanism of gene control in eukaryotes. In this study, we report an improved annotation of C. albicans introns. In addition to correcting the existing intron annotations, 25% of which were incorrect, we have used novel computational and experimental approaches to identify new introns, bringing the total to 415, almost double the number previously known. Our identification methods focus primarily on intron features rather than protein-coding features, overcoming biases of traditional intron annotation methods. Introns are not randomly distributed in C. albicans, and are over-represented in genes involved in specific cellular processes, such as splicing, translation, and mitochondrial respiration. This nonrandom distribution suggests functional roles for these introns, and we demonstrate that splicing of two transcripts whose introns have unusual sequence features is responsive to environmental factors.
Evolution and multilevel optimization of the genetic code
Authors: Bollenbach Tobias , Vetsigian Kalin , Kishony Roy
Published: March 2007

The discovery of the genetic code was one of the most important advances of modern biology. But there is more to a DNA code than protein sequence; DNA carries signals for splicing, localization, folding, and regulation that are often embedded within the protein-coding sequence. In this issue, Itzkovitz and Alon show that the specific 64-to-20 mapping found in the genetic code may have been optimized for permitting protein-coding regions to carry this extra information and suggest that this property may have evolved as a side benefit of selection to minimize the negative effects of frameshift errors.
Lexico-semantic structure and the word-frequency effect in recognition memory
Authors: Monaco Joseph D. , Abbott L. F. , Kahana Michael J.
Published: March 2007

The word-frequency effect (WFE) in recognition memory refers to the finding that more rare words are better recognized than more common words. We demonstrate that a familiarity-discrimination model operating on data from a semantic word-association space yields a robust WFE in data on both hit rates and false-alarm rates. Our modeling results suggest that word frequency is encoded in the semantic structure of language, and that this encoding contributes to the WFE observed in item-recognition experiments.
Long-term memory for place learning is facilitated by expression of cAMP response element-binding protein in the dorsal hippocampus
Authors: Brightwell Jennifer J. , Smith Clayton A. , Neve Rachael L. , Colombo Paul J.
Published: March 2007

Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in long-term memory formation. In the current study, we used viral vector-mediated gene transfer to test the hypothesis that formation of long-term memory for place learning can be facilitated by increasing levels of CREB in the dorsal hippocampus. Three days after intrahippocampus infusion, experimental (HSV-CREB) and control (HSV-LacZ; saline) rats were trained during a single session on a place task in a water cross maze. Rats were tested for memory 5 d later. Rats in all groups showed short-term memory, and there were no significant differences among treatment groups during acquisition. However, only HSV-CREB-infused rats showed significant savings between training and test, while HSV-LacZ- and saline-treated rats did not. Quantitative Western blotting confirmed that levels of dorsal hippocampus CREB were increased during training in rats infused with HSV-CREB in comparisons with controls. The present results show that formation of long-term memory can be facilitated by increasing levels of hippocampus CREB protein.
Synapse specificity of long-term potentiation breaks down with aging
Authors: Ris Laurence , Godaux Emile
Published: March 2007

Memory shows age-related decline. According to the current prevailing theoretical model, encoding of memories relies on modifications in the strength of the synapses connecting the different cells within a neuronal network. The selective increases in synaptic weight are thought to be biologically implemented by long-term potentiation (LTP). Here, we report that tetanic stimulation of afferent fibers in slices from 12-mo-old mice triggers an LTP not restricted to the activated synapses. This phenomenon, which can be anticipated to hinder memory encoding, is suppressed by blocking either L-type Ca++ channels or Ca++-induced Ca++ release, both well known to become disregulated with aging.
Rats depend on habit memory for discrimination learning and retention
Authors: Broadbent Nicola J. , Squire Larry R. , Clark Robert E.
Published: March 2007

We explored the circumstances in which rats engage either declarative memory (and the hippocampus) or habit memory (and the dorsal striatum). Rats with damage to the hippocampus or dorsal striatum were given three different two-choice discrimination tasks (odor, object, and pattern). These tasks differed in the number of trials required for learning (∼10, 60, and 220 trials). Dorsal striatum lesions impaired discrimination performance to a greater extent than hippocampal lesions. Strikingly, performance on the task learned most rapidly (the odor discrimination) was severely impaired by dorsal striatum lesions and entirely spared by hippocampal lesions. These findings suggest that discrimination learning in the rat is primarily supported by the dorsal striatum (and habit memory) and that rats engage a habit-based memory system even for a task that takes only a few trials to acquire. Considered together with related studies of humans and nonhuman primates, the findings suggest that different species will approach the same task in different ways.
Hormonal and monoamine signaling during reinforcement of hippocampal long-term potentiation and memory retrieval
Authors: Korz Volker , Frey Julietta U.
Published: March 2007

Recently it was shown that holeboard training can reinforce, i.e., transform early-LTP into late-LTP in the dentate gyrus during the initial formation of a long-term spatial reference memory in rats. The consolidation of LTP as well as of the reference memory was dependent on protein synthesis. We have now investigated the transmitter systems involved in this reinforcement and found that LTP-consolidation and memory retrieval were dependent on β-adrenergic, dopaminergic, and mineralocorticoid receptor (MR) activation, whereas glucocorticoid receptors (GRs) were not involved. Blockade of the β-adrenergic signaling pathway significantly increased the number of reference memory errors compared with MR and dopamine receptor inhibition. In addition, β-adrenergic blockade impaired the working memory. Therefore, we suggest that β-adrenergic receptor activation is the main signaling system required for the retrieval of spatial memory. In addition, other modulatory interactions such as dopaminergic as well as MR systems are involved. This result points to specific roles of different modulatory systems during the retrieval of specific components of spatial memory. The data provide evidence for similar integrative interactions between different signaling systems during cellular memory processes.
A Genomic Approach to Suberin Biosynthesis and Cork Differentiation
Authors: Soler Marçal , Serra Olga , Molinas Marisa , Huguet Gemma , Fluch Silvia , Figueras Mercè
Published: March 2007

Cork (phellem) is a multilayered dead tissue protecting plant mature stems and roots and plant healing tissues from water loss and injuries. Cork cells are made impervious by the deposition of suberin onto cell walls. Although suberin deposition and cork formation are essential for survival of land plants, molecular studies have rarely been conducted on this tissue. Here, we address this question by combining suppression subtractive hybridization together with cDNA microarrays, using as a model the external bark of the cork tree (Quercus suber), from which bottle cork is obtained. A suppression subtractive hybridization library from cork tree bark was prepared containing 236 independent sequences; 69% showed significant homology to database sequences and they corresponded to 135 unique genes. Out of these genes, 43.5% were classified as the main pathways needed for cork biosynthesis. Furthermore, 19% could be related to regulatory functions. To identify genes more specifically required for suberin biosynthesis, cork expressed sequence tags were printed on a microarray and subsequently used to compare cork (phellem) to a non-suberin-producing tissue such as wood (xylem). Based on the results, a list of candidate genes relevant for cork was obtained. This list includes genes for the synthesis, transport, and polymerization of suberin monomers such as components of the fatty acid elongase complexes, ATP-binding cassette transporters, and acyltransferases, among others. Moreover, a number of regulatory genes induced in cork have been identified, including MYB, No-Apical-Meristem, and WRKY transcription factors with putative functions in meristem identity and cork differentiation.
E2F RegulatesFASCIATA1, a Chromatin Assembly Gene Whose Loss Switches on the Endocycle and Activates Gene Expression by Changing the Epigenetic Status
Authors: Ramirez-Parra Elena , Gutierrez Crisanto
Published: March 2007

Maintenance of genome integrity depends on histone chaperone-mediated chromatin reorganization. DNA replication-associated nucleosome deposition relies on chromatin assembly factor-1 (CAF-1). Depletion of CAF-1 in human cells leads to cell death, whereas in Arabidopsis (Arabidopsis thaliana), where it is involved in heterochromatin compaction and homologous recombination, plants are viable. The mechanism that makes the lack of CAF-1 activity compatible with development is not known. Here, we show that the FASCIATA1 (FAS1) gene, which encodes the CAF-1 large subunit, is a target of E2F transcription factors. Mutational studies demonstrate that one of the two E2F binding sites in its promoter has an activator role, whereas the other has a repressor function. Loss of FAS1 results in reduced type A cyclin-dependent kinase activity, inhibits mitotic progression, and promotes a precocious and systemic switch to the endocycle program. Selective up-regulation of the expression of a subset of genes, including those involved in activation of the G2 DNA damage checkpoint, also occurs upon FAS1 loss. This activation is not the result of a global change in chromatin structure, but depends on selective epigenetic changes in histone acetylation and methylation within a small region in their promoters. This suggests that correct chromatin assembly during the S-phase is required to prevent unscheduled changes in the epigenetic marks of target genes. Interestingly, activation of the endocycle switch as well as introduction of activating histone marks in the same set of G2 checkpoint genes are detected upon treatment of wild-type plants with DNA-damaging treatments. Our results are consistent with a model in which defects in chromatin assembly during the S-phase and DNA damage signaling share part of a pathway, which ultimately leads to mitotic arrest and triggers the endocycle program. Together, this might be a bypass mechanism that makes development compatible with cell division arrest induced by DNA damage stress.
AWUSCHEL-LIKE HOMEOBOXGene Represses aYABBYGene Expression Required for Rice Leaf Development
Authors: Dai Mingqiu , Hu Yongfeng , Zhao Yu , Liu Huifang , Zhou Dao-Xiu
Published: March 2007

YABBY and WUSCHEL-LIKE HOMEOBOX (WOX) genes have been shown to play important roles in lateral organ formation and meristem function. Here, we report the characterization of functional relationship between rice (Oryza sativa) YAB3 and WOX3 in rice leaf development. Rice YAB3 is closely related to maize (Zea mays) ZmYAB14 and Arabidopsis (Arabidopsis thaliana) FILAMENTOUS FLOWER (FIL), whereas rice WOX3 is highly conserved with maize narrow sheath1 (NS1) and NS2 and Arabidopsis PRESSED FLOWER (PRS). In situ hybridization experiments revealed that the expression of both genes was excluded from the shoot apical meristem, but the transcripts were detected in leaf primordia, young leaves, and reproductive organs without any polar distribution. The function of the two genes was studied by both overexpression and RNA interference (RNAi) in transgenic rice. YAB3 RNAi induced twisted and knotted leaves lacking specialized structures such as ligule and auricles, while no phenotypic change was observed in YAB3 overexpression plants, suggesting that rice YAB3 may be required for leaf cell growth and differentiation. Overexpression of WOX3 repressed YAB3 and showed a YAB3 RNAi phenotype. The expression of class I KNOTTED-LIKE HOMEOBOX (KNOX) genes was ectopically induced in leaves of YAB3 RNAi or WOX3 overexpression plants. Data from inducible WOX3 expression and DNA-protein interaction assays suggested that WOX3 acted as a transcriptional repressor of YAB3. These data reveal a regulatory network involving YAB3, WOX3, and KNOX genes required for rice leaf development.
Sampling the Arabidopsis Transcriptome with Massively Parallel Pyrosequencing
Authors: Weber Andreas P.M. , Weber Katrin L. , Carr Kevin , Wilkerson Curtis , Ohlrogge John B.
Published: March 2007

Massively parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts, we analyzed mRNA from Arabidopsis (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 cDNA models indicated: (1) massively parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10%, but increased the overall sequence coverage by 50%. (2) Mapping of the ESTs to their predicted full-length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium, and long transcripts were equally represented. (3) Over 16,000 of the ESTs that mapped to the genome were not represented in the existing dbEST database. In some cases, the ESTs provide the first experimental evidence for transcripts derived from predicted genes, and, for at least 60 locations in the genome, pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together, the results indicate massively parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of nonmodel plants with less complete genomic information.
The Thylakoid Lumen Protease Deg1 Is Involved in the Repair of Photosystem II from Photoinhibition in Arabidopsis
Authors: Kapri-Pardes Einat , Naveh Leah , Adam Zach
Published: March 2007

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.
A Core Subunit of the RNA-Processing/Degrading Exosome Specifically Influences Cuticular Wax Biosynthesis inArabidopsis
Authors: Hooker Tanya S. , Lam Patricia , Zheng Huanquan , Kunst Ljerka
Published: March 2007

The cuticle is an extracellular matrix composed of cutin polyester and waxes that covers aerial organs of land plants and protects them from environmental stresses. The Arabidopsis thaliana cer7 mutant exhibits reduced cuticular wax accumulation and contains considerably lower transcript levels of ECERIFERUM3/WAX2/YORE-YORE (CER3/WAX2/YRE), a key wax biosynthetic gene. We show here that CER7 protein is a putative 3′-5′ exoribonuclease homologous to yeast Ribonuclease PH45 (RRP45p), a core subunit of the RNA processing and degrading exosome that controls the expression of CER3/WAX2/YRE. We propose that CER7 acts by degrading a specific mRNA species encoding a negative regulator of CER3/WAX2/YRE transcription. A second RRP45p homolog found in Arabidopsis, designated At RRP45a, is partially functionally redundant with CER7, and complete loss of RRP45 function in Arabidopsis is lethal. To our knowledge, CER7 is currently the only example of a core exosomal subunit specifically influencing a cellular process.
Neurophysiological characterization of mammalian osmosensitive neurones
Authors: Bourque Charles W. , Ciura Sorana , Trudel Eric , Stachniak Tevye J. E. , Sharif‐Naeini Reza
Published: March 2007

In mammals, the osmolality of the extracellular fluid is maintained near a predetermined set‐point through a negative feedback regulation of thirst, diuresis, salt appetite and natriuresis. This homeostatic control is believed to be mediated by osmosensory neurones which synaptically regulate the electrical activity of command neurones that mediate each of these osmoregulatory effector responses. Our present understanding of the molecular, cellular and network basis that underlies the central control of osmoregulation is largely derived from studies on primary osmosensory neurones in the organum vasculosum lamina terminalis (OVLT) and effector neurones in the supraoptic nucleus (SON), which release hormones that regulate diuresis and natriuresis. Primary osmosensory neurones in the OVLT exhibit changes in action potential firing rate that vary in proportion with ECF osmolality. This effect results from the intrinsic depolarizing receptor potential which these cells generate via a molecular transduction complex that may comprise various members of the transient receptor potential vanilloid (TRPV) family of cation channel proteins, notably TRPV1 and TRPV4. Osmotically evoked changes in the firing rate of OVLT neurones then regulate the electrical activity of downstream neurones in the SON through graded changes in glutamate release.
Role of TRPV ion channels in sensory transduction of osmotic stimuli in mammals
Authors: Liedtke Wolfgang
Published: March 2007

In signal transduction of metazoan cells, ion channels of the family of transient receptor potential (TRP) have been identified to respond to diverse external and internal stimuli, amongst them osmotic stimuli. This report highlights findings pertaining to the TRPV subfamily, focusing on mammalian members. Of the six mammalian TRPV channels, TRPV1, 2 and 4 were demonstrated to function in transduction of osmotic stimuli. TRPV channels have been found to function in cellular as well as systemic osmotic homeostasis. In a striking example of evolutionary conservation of function, mammalian TRPV4 has been found to rescue osmosensory deficits of the TRPV mutant strain osm‐9 in Caenorhabditis elegans, despite not more than 26% orthology of the respective proteins.
Effect of the Mycorrhizosphere on the Genotypic and Metabolic Diversity of the Bacterial Communities Involved in Mineral Weathering in a Forest Soil
Authors: Uroz S. , Calvaruso C. , Turpault M. P. , Pierrat J. C. , Mustin C. , Frey-Klett P.
Published: March 2007

To date, several bacterial species have been described as mineral-weathering agents which improve plant nutrition and growth. However, the possible relationships between mineral-weathering potential, taxonomic identity, and metabolic ability have not been investigated thus far. In this study, we characterized a collection of 61 bacterial strains isolated from
Scleroderma citrinum
mycorrhizae, the mycorrhizosphere, and the adjacent bulk soil in an oak forest. The ability of bacteria to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from this mineral. We showed that weathering bacteria occurred more frequently in the vicinity of
S. citrinum
than in the bulk soil. Moreover, the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera
Burkholderia
,
Collimonas
,
Pseudomonas
, and
Sphingomonas
, and their carbon metabolism was characterized by the BIOLOG method. The most efficient isolates belonged to the genera
Burkholderia
and
Collimonas
. Multivariate analysis resulted in identification of three metabolic groups, one of which contained mainly bacterial isolates associated with
S. citrinum
and exhibiting high mineral-weathering potential. Therefore, our results support the hypothesis that by its carbon metabolism this fungus selects in the bulk soil reservoir a bacterial community with high weathering potential, and they also address the question of functional complementation between mycorrhizal fungi and bacteria in the ectomycorrhizal complex for the promotion of tree nutrition.
Characterization of
Fusarium oxysporum
β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan
Authors: Sakamoto Tatsuji , Taniguchi Yuya , Suzuki Shiho , Ihara Hideshi , Kawasaki Haruhiko
Published: March 2007

A type II arabinogalactan-degrading enzyme (FoGal1) was purified from
Fusarium oxysporum
12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.
VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn
1546
Typing and Location, and Virulence Determinants
Authors: Biavasco F. , Foglia G. , Paoletti C. , Zandri G. , Magi G. , Guaglianone E. , Sundsfjord A. , Pruzzo C. , Donelli G. , Facinelli B.
Published: March 2007

VanA-type human (
n
= 69), animal (
n
= 49), and food (
n
= 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (
n
= 4) and M49 (
n
= 13), representing
Enterococcus faecium
isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17.
purK
allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn
1546
type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal
E. faecium
isolates bore large (>150 kb)
vanA
plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn
1546
in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork
E. faecium
isolates. There were no significant associations (
P
> 0.5) between Tn
1546
type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (
P
< 0.02) among clinical strains. Multiple determinants were found in clinical and meat
Enterococcus faecalis
isolates. The presence of indistinguishable
vanA
elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific
purK
housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.
The
dasABC
Gene Cluster, Adjacent to
dasR
, Encodes a Novel ABC Transporter for the Uptake of
N
,
N
′-Diacetylchitobiose in
Streptomyces coelicolor
A3(2)
Authors: Saito Akihiro , Shinya Tomonori , Miyamoto Katsushiro , Yokoyama Tomofumi , Kaku Hanae , Minami Eiichi , Shibuya Naoto , Tsujibo Hiroshi , Nagata Yoshiho , Ando Akikazu , Fujii Takeshi , Miyashita Kiyotaka
Published: March 2007

N
,
N
′-Diacetylchitobiose [(GlcNAc)
2
] induces the transcription of chitinase (
chi
) genes in
Streptomyces coelicolor
A3(2). Physiological studies showed that (GlcNAc)
2
addition triggered
chi
expression and increased the rate of (GlcNAc)
2
concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)
2
, suggesting that (GlcNAc)
2
induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of
chi
transcription. SCO5232, named
dasA
, showed transcriptional induction by (GlcNAc)
2
and
N
,
N
′,
N
‴-triacetylchitotriose [(GlcNAc)
3
]. Surface plasmon resonance analysis showed that recombinant DasA protein exhibited the highest affinity for (GlcNAc)
2
(equilibrium dissociation constant [

K
D

] = 3.22 × 10
−8
). In the
dasA
-null mutant, the rate of decline of the (GlcNAc)
2
concentration in the culture supernatant was about 25% of that in strain M145. The in vitro and in vivo data clearly demonstrated that
dasA
is involved in (GlcNAc)
2
uptake. Upstream and downstream of
dasA
, the transcriptional regulator gene (
dasR
) and two putative integral membrane protein genes (
dasBC
) are located in the opposite and same orientations, respectively. The expression of
dasR
and
dasB
, which seemed independent of
dasA
transcription, was also induced by (GlcNAc)
2
and (GlcNAc)
3
.
Adherence of Enterohemorrhagic
Escherichia coli
O157, O26, and O111 Strains to Bovine Intestinal Explants Ex Vivo
Authors: Girard Francis , Dziva Francis , van Diemen Pauline , Phillips Alan D. , Stevens Mark P. , Frankel Gad
Published: March 2007

We used bovine intestinal organ culture to study infection by enterohemorrhagic
Escherichia coli
serogroups O157, O26, and O111. We show colonization and attaching and effacing lesion formation on explants derived from the ileum, colon, and rectum. Intimin and Tir were detected at the sites of adherent bacteria; Tir was essential for colonization.
Second Acyl Homoserine Lactone Production System in the Extreme AcidophileAcidithiobacillus ferrooxidans
Authors: Rivas Mariella , Seeger Michael , Jedlicki Eugenia , Holmes David S.
Published: March 2007

The acidophilic proteobacteriumAcidithiobacillus ferrooxidansis involved in the industrial biorecovery of copper. It is found in acidic environments in biofilms and is important in the biogeochemical cycling of metals and nutrients. Its genome contains a cluster of four genes,glyQ,glysS,gph, andact, that are predicted to encode the α and β subunits of glycine tRNA synthetase, a phosphatase, and an acyltransferase, respectively (GenBank accession no. DQ149607).act, cloned and expressed inEscherichia coli, produces acyl homoserine lactones (AHLs) principally of chain length C14according to gas chromatography and mass spectrometry measurements. The AHLs have biological activity as shown by in vivo studies using the reporter strainSinorhizobium melilotiRm41 SinI−. Reverse transcription-PCR (RT-PCR) experiments indicate that the four genes are expressed as a single transcript, demonstrating that they constitute an operon. According to semiquantitative RT-PCR results,actis expressed more highly whenA. ferrooxidansis grown in medium containing iron than when it is grown in medium containing sulfur. Since AHLs are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression, this result suggests thatA. ferrooxidanshas two quorum-sensing systems, one based on Act, as described herein, and the other based on a Lux-like quorum-sensing system, reported previously. The latter system was shown to be upregulated inA. ferrooxidansgrown in sulfur medium, suggesting that the two quorum-sensing systems respond to different environmental signals that may be related to their abilities to colonize and use different solid sulfur- and iron-containing minerals.
Microbial Reductive Dechlorination of Aroclor 1260 in Baltimore Harbor Sediment Microcosms Is Catalyzed by Three Phylotypes within the Phylum
Chloroflexi
Authors: Fagervold Sonja K. , May Harold D. , Sowers Kevin R.
Published: March 2007

The specific dechlorination pathways for Aroclor 1260 were determined in Baltimore Harbor sediment microcosms developed with the 11 most predominant congeners from this commercial mixture and their resulting dechlorination intermediates. Most of the polychlorinated biphenyl (PCB) congeners were dechlorinated in the
meta
position, and the major products were tetrachlorobiphenyls with unflanked chlorines. Using PCR primers specific for the 16S rRNA genes of known PCB-dehalogenating bacteria, we detected three phylotypes within the microbial community that had the capability to dechlorinate PCB congeners present in Aroclor 1260 and identified their selective activities. Phylotype DEH10, which has a high level of sequence identity to
Dehalococcoides
spp., removed the double-flanked chlorine in 234-substituted congeners and exhibited a preference for
para
-flanked
meta
-chlorines when no double-flanked chlorines were available. Phylotype SF1 had similarity to the
o
-17/DF-1 group of PCB-dechlorinating bacteria. Phylotype SF1 dechlorinated all of the 2345-substituted congeners, mostly in the double-flanked
meta
position and 2356-, 236-, and 235-substituted congeners in the
ortho
-flanked
meta
position, with a few exceptions. A phylotype with 100% sequence identity to PCB-dechlorinating bacterium
o
-17 was responsible for an
ortho
and a double-flanked
meta
dechlorination reaction. Most of the dechlorination pathways supported the growth of all three phylotypes based on competitive PCR enumeration assays, which indicates that PCB-impacted environments have the potential to sustain populations of these PCB-dechlorinating microorganisms. The results demonstrate that the variation in dechlorination patterns of congener mixtures typically observed at different PCB impacted sites can potentially be mediated by the synergistic activities of relatively few dechlorinating species.
Adopting Selected Hydrogen Bonding and Ionic Interactions fromAspergillus fumigatusPhytase Structure Improves the Thermostability ofAspergillus nigerPhyA Phytase
Authors: Zhang Wanming , Mullaney Edward J. , Lei Xin Gen
Published: March 2007

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry,Aspergillus nigerPhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostableAspergillus fumigatusphytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-S42 region and ionic interactions between R168 and D161 and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into seven mutants of Afp. All seven mutants displayed decreases in thermostability, with the highest loss (25% [P< 0.05]) in the triple mutant (E35A R168A R248A). Subsequently, a set of corresponding substitutions were introduced into nine mutants of PhyA to strengthen the hydrogen bonding and ionic interactions. While four mutants showed improved thermostability, the best response came from the quadruple mutant (A58E P65S Q191R T271R), which retained 20% greater (P< 0.05) activity after being heated at 80°C for 10 min and had a 7°C higher melting temperature than that of wild-type PhyA. This study demonstrates the functional importance of the hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units to improve the thermostability of a homologous PhyA phytase.
The
Escherichia coli
Regulator of Sigma 70 Protein, Rsd, Can Up-Regulate Some Stress-Dependent Promoters by Sequestering Sigma 70
Authors: Mitchell Jennie E. , Oshima Taku , Piper Sarah E. , Webster Christine L. , Westblade Lars F. , Karimova Gouzel , Ladant Daniel , Kolb Annie , Hobman Jon L. , Busby Stephen J. W. , Lee David J.
Published: March 2007

The
Escherichia coli
Rsd protein forms complexes with the RNA polymerase σ
70
factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative σ
38
factor, and this was confirmed by experiments with
lac
fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-
lac
fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with σ
70
. Our experiments support a model in which the role of Rsd is primarily to sequester σ
70
, thereby increasing the levels of RNA polymerase containing the alternative σ
38
factor.
XphA/XqhA, a Novel GspCD Subunit for Type II Secretion in
Pseudomonas aeruginosa
Authors: Michel Gérard P. F. , Durand Eric , Filloux Alain
Published: March 2007

The opportunistic human pathogen bacterium
Pseudomonas aeruginosa
secretes various exoproteins in its surrounding environment. Protein secretion involves different secretory systems, including the type II secretion system, or T2SS, that is one of the most efficient secretory pathways of
P. aeruginosa
. There are two T2SS in this bacterium, the quorum-sensing-regulated Xcp system and the Hxc system, which is only present under phosphate-limiting conditions. Like T2SS of other bacteria, the Xcp T2SS is species specific, and this specificity mainly involves two proteins, XcpP (GspC family) and the secretin XcpQ (GspD family), which are the gatekeepers of the system. Interestingly, an orphan secretin, XqhA, was previously reported as being able to functionally replace the XcpQ secretin. In this study, we identified another gene, which we named
xphA
(
x
cp
P h
omologue
A
), which is located next to
xqhA
. We showed that deletion of the
xphA
gene in an
xcpP
mutant caused the disappearance of the residual secretion observed in this mutant strain, indicating that the protein XphA plays a role in the secretion process. Our results also revealed that complementation of an
xcpP/xcpQ
mutant can be obtained with the gene couple
xphA/xqhA
. The XphA and XqhA proteins (the P
A
Q
A
subunit) could thus form, together with XcpR-Z, a functional hybrid T2SS. A two-dimensional polyacrylamide gel electrophoresis analysis showed that except for the aminopeptidase PaAP, for which secretion is not restored by the P
A
Q
A
subunit in the
xcpP/xcpQ
deletion mutant, each major Xcp-dependent exoprotein is secreted by the new hybrid machinery. Our work supports the idea that components of the GspC/GspD families, such as XphA/XqhA or XcpP/XcpQ, are assembled as a specific tandem within the T2SS. Each of these pairs may thus confer a different level of secretion specificity, as is the case with respect to PaAP. Finally, using a chromosomal
xphA
-
lacZ
fusion, we showed that the
xphA-xqhA
genes are transcribed from an early stage of bacterial growth. We thus suggest that the P
A
Q
A
subunit might be involved in the secretion process at a different growth stage than XcpP/XcpQ.
MarT Activates Expression of the MisL Autotransporter Protein of
Salmonella enterica
Serotype Typhimurium
Authors: Tükel Çagla , Akçelik Mustafa , de Jong Maarten F. , Şimşek Ömer , Tsolis Renée M. , Bäumler Andreas J.
Published: March 2007

MisL is a
Salmonella enterica
serotype Typhimurium fibronectin binding protein whose expression is induced during infection of mice. T-POP transposon mutagenesis identified
marT
as a positive regulatory element controlling expression of a
misL
::
lacZYA
transcriptional fusion. Gel shift analysis identified MarT as a transcriptional activator of the
misL
promoter.
Identification of Critical Residues in the Propeptide of LasA Protease of
Pseudomonas aeruginosa
Involved in the Formation of a Stable Mature Protease
Authors: Grande Kerian K. , Gustin Jean K. , Kessler Efrat , Ohman Dennis E.
Published: March 2007

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by
Pseudomonas aeruginosa
. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing
lasA
as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.
Biosynthesis of Isoprenoid Wax Ester inMarinobacter hydrocarbonoclasticusDSM 8798: Identification and Characterization of Isoprenoid Coenzyme A Synthetase and Wax Ester Synthases
Authors: Holtzapple Erik , Schmidt-Dannert Claudia
Published: March 2007

Marinobacter hydrocarbonoclasticusDSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described fromAcinetobactersp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain,M. aquaeoleiVT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizingMarinobacterstrain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of theMarinobacterwax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.
VEGF-C is associated with lymphatic status and invasion in oral cancer
Authors: Siriwardena B S M S , Kudo Y , Ogawa I , Udagama M N G P K , Tilakaratne W M , Takata T
Published: March 2007

Background:

Nodal metastasis is a major prognostic indicator for oral squamous cell carcinoma (OSCC) progression. Recently, it has been revealed that lymphangiogenic growth factor VEGF-C and its receptor Flt-4 play an important role for invasion and metastasis in cancer cells.

Aim:

To examine VEGF-C expression and its correlation with lymphatic status, including the number of lymph vessels and lymphatic invasion, tumour invasion and metastasis in OSCC.

Methods:

Intratumoural and peritumoural lymphatic vessels were examined using D2-40 in 54 OSCC cases and correlated with VEGF-C expression and clinicopathological findings. The histological pattern of invasion and pathological findings were compared.

Results:

High expression of VEGF-C was frequently observed in OSCC and was associated with increased number of lymph vessels and lymphatic invasion. VEGF-C was well correlated with invasion pattern and metastasis.

Conclusions:

Results suggest that VEGF-C may play an important role for lymphangiogenesis and invasion in the metastatic process and can be a strong predicting factor for metastasis of OSCC.
Effects of posture on peripheral vascular responses to lower body positive pressure
Authors: Nishiyasu Takeshi , Hayashida Shigeko , Kitano Asami , Nagashima Kei , Ichinose Masashi
Published: March 2007

We tested the hypothesis that peripheral vascular responses (in the lower and upper limbs) to application of lower body positive pressure (LBPP) are dependent on the posture of the subjects. We measured heart rate, stroke volume, mean arterial pressure, leg and forearm blood flow (using the Doppler ultrasound technique), and leg (LVC) and forearm (FVC) vascular conductance in 11 subjects (9 men, 2 women) without and with LBPP (25 and 50 mmHg) in supine and upright postures. Mean arterial pressure increased in proportion to increases in LBPP and was greater in supine than in upright subjects. Heart rate was unchanged when LBPP was applied to supine subjects but was reduced in upright ones. Leg blood flow and LVC were both reduced by LBPP in supine subjects [LVC: 4.8 (SD 4.0), 3.6 (SD 3.5), and 1.4 (SD 1.8) ml·min−1·mmHg−1 before LBPP and during 25 and 50 mmHg LBPP, respectively; P < 0.05] but were increased in upright ones [LVC: 2.0 (SD 1.2), 3.4 (SD 3.4), and 3.0 (SD 2.0) ml·min−1·mmHg−1, respectively; P < 0.05]. Forearm blood flow and FVC both declined when LBPP was applied to supine subjects [FVC: 1.3 (SD 0.6), 1.0 (SD 0.4), and 0.9 (SD 0.6) ml· min−1·mmHg−1, respectively; P < 0.05] but remained unchanged in upright ones [FVC: 0.7 (SD 0.4), 0.7 (SD 0.4), and 0.6 (SD 0.5) ml·min−1·mmHg−1, respectively]. Together, these findings indicate that the leg vascular response to application of LBPP is posture dependent and that the response differs in the lower and upper limbs when subjects assume an upright posture.
Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane
Authors: Garg Vivek , Hu Keli
Published: March 2007

The ATP-sensitive K+ (KATP) channels in both sarcolemmal (sarcKATP) and mitochondrial inner membrane (mitoKATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcKATP contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoKATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing KATP into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing KATP channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing KATP channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional KATP channels in mitochondria was significantly increased by PMA, as shown by KATP-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing KATP channels was blocked by a selective PKCε inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the KATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCε. PKC isoform-regulated mitochondrial import of KATP channels may have significant implication in cardioprotection of IPC.