bluuebunny/crossref_metadata_2025 huggingface dataset:
Biodegradation of 5-Nitroanthranilic Acid by
Bradyrhizobium
sp. Strain JS329
Authors: Qu Yi , Spain Jim C.
Published: January 2010

Biodegradation of synthetic compounds has been studied extensively, but the metabolic diversity required for catabolism of many natural compounds has not been addressed. 5-Nitroanthranilic acid (5NAA), produced in soil by
Streptomyces scabies
, is also the starting material for synthetic dyes and other nitroaromatic compounds.
Bradyrhizobium
JS329 was isolated from soil by selective enrichment with 5NAA. When grown on 5NAA, the isolate released stoichiometric amounts of nitrite and half of the stoichiometric amounts of ammonia. Enzyme assays indicate that the initial step in 5NAA degradation is an unusual hydrolytic deamination for formation of 5-nitrosalicylic acid (5NSA). Cloning and heterologous expression revealed the genes that encode 5NAA deaminase (
naaA
) and the 5NSA dioxygenase (
naaB
) that cleaves the aromatic ring of 5NSA without prior removal of the nitro group. The results provide the first clear evidence for the initial steps in biodegradation of amino-nitroaromatic compounds and reveal a novel deamination reaction for aromatic amines.
Selection from Industrial Lager Yeast Strains of Variants with Improved Fermentation Performance in Very-High-Gravity Worts
Authors: Huuskonen Anne , Markkula Tuomas , Vidgren Virve , Lima Luis , Mulder Linda , Geurts Wim , Walsh Michael , Londesborough John
Published: January 2010

There are economic and other advantages if the fermentable sugar concentration in industrial brewery fermentations can be increased from that of currently used high-gravity (ca. 14 to 17°P [degrees Plato]) worts into the very-high-gravity (VHG; 18 to 25°P) range. Many industrial strains of brewer's yeast perform poorly in VHG worts, exhibiting decreased growth, slow and incomplete fermentations, and low viability of the yeast cropped for recycling into subsequent fermentations. A new and efficient method for selecting variant cells with improved performance in VHG worts is described. In this new method, mutagenized industrial yeast was put through a VHG wort fermentation and then incubated anaerobically in the resulting beer while maintaining the α-glucoside concentration at about 10 to 20 g·liter
−1
by slowly feeding the yeast maltose or maltotriose until most of the cells had died. When survival rates fell to 1 to 10 cells per 10
6
original cells, a high proportion (up to 30%) of survivors fermented VHG worts 10 to 30% faster and more completely (residual sugars lower by 2 to 8 g·liter
−1
) than the parent strains, but the sedimentation behavior and profiles of yeast-derived flavor compounds of the survivors were similar to those of the parent strains.
Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing
Authors: Jaé Nicolas , Wang Pingping , Gu Tianpeng , Hühn Martin , Palfi Zsofia , Urlaub Henning , Bindereif Albrecht
Published: January 2010

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate byin vitroreconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.
Role for Parasite Genetic Diversity in Differential Host Responses toTrypanosoma bruceiInfection
Authors: Morrison Liam J. , McLellan Sarah , Sweeney Lindsay , Chan Chi N. , MacLeod Annette , Tait Andy , Turner C. Michael R.
Published: January 2010

The postgenomic era has revolutionized approaches to defining host-pathogen interactions and the investigation of the influence of genetic variation in either protagonist upon infection outcome. We analyzed pathology induced by infection with two genetically distinctTrypanosoma bruceistrains and found that pathogenesis is partly strain specific, involving distinct host mechanisms. Infections of BALB/c mice with one strain (927) resulted in more severe anemia and greater erythropoietin production compared to infections with the second strain (247), which, contrastingly, produced greater splenomegaly and reticulocytosis. Plasma interleukin-10 (IL-10) and gamma interferon levels were significantly higher in strain 927-infected mice, whereas IL-12 was higher in strain 247-infected mice. To define mechanisms underlying these differences, expression microarray analysis of host genes in the spleen at day 10 postinfection was undertaken. Rank product analysis (RPA) showed that 40% of the significantly differentially expressed genes were specific to infection with one or the other trypanosome strain. RPA and pathway analysis identified LXR/RXR signaling, IL-10 signaling, and alternative macrophage activation as the most significantly differentially activated host processes. These data suggest that innate immune response modulation is a key determinant in trypanosome infections, the pattern of which can vary, dependent upon the trypanosome strain. This strongly suggests that a parasite genetic component is responsible for causing disease in the host. Our understanding of trypanosome infections is largely based on studies involving single parasite strains, and our results suggest that an integrated host-parasite approach is required for future studies on trypanosome pathogenesis. Furthermore, it is necessary to incorporate parasite variation into both experimental systems and models of pathogenesis.
F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation
Authors: Arutyunov Denis , Arenson Barbara , Manchak Jan , Frost Laura S.
Published: January 2010

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.
The Receiver Domain of Hybrid Histidine Kinase VirA: an Enhancing Factor for
vir
Gene Expression in
Agrobacterium tumefaciens
Authors: Wise Arlene A. , Fang Fang , Lin Yi-Han , He Fanglian , Lynn David G. , Binns Andrew N.
Published: January 2010

The plant pathogen
Agrobacterium tumefaciens
expresses virulence (
vir
) genes in response to chemical signals found at the site of a plant wound. VirA, a hybrid histidine kinase, and its cognate response regulator, VirG, regulate
vir
gene expression. The receiver domain at the carboxyl end of VirA has been described as an inhibitory element because its removal increased
vir
gene expression relative to that of full-length VirA. However, experiments that characterized the receiver region as an inhibitory element were performed in the presence of constitutively expressed
virG
. We show here that VirA's receiver domain is an activating factor if
virG
is expressed from its native promoter on the Ti plasmid. When
virA
ΔR was expressed from a multicopy plasmid, both sugar and the phenolic inducer were essential for
vir
gene expression. Replacement of wild-type
virA
on pTi with
virA
ΔR precluded
vir
gene induction, and the cells did not accumulate VirG or induce transcription of a
virG
-
lacZ
fusion in response to acetosyringone. These phenotypes were corrected if the
virG
copy number was increased. In addition, we show that the VirA receiver domain can interact with the VirG DNA-binding domain.
Identification of Tail Genes in the Temperate Phage
16
-
3
of
Sinorhizobium meliloti
41
Authors: Deák Veronika , Lukács Rita , Buzás Zsuzsanna , Pálvölgyi Adrienn , Papp Péter P. , Orosz László , Putnoky Péter
Published: January 2010

Genes encoding the tail proteins of the temperate phage
16
-
3
of the symbiotic nitrogen-fixing bacterium
Sinorhizobium meliloti
41 have been identified. First, a new host range gene, designated
hII
, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage
16
-
3
possesses an icosahedral head and a long, noncontractile tail characteristic of the
Siphoviridae
. By using a lysogenic
S. meliloti
41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in
ORF017
,
ORF018a
,
ORF020
,
ORF021
, the previously described
h
gene, and
hII
resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the H
II
tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original
h
gene recognizes a proteinaceous receptor.
PcaO Positively Regulates
pcaHG
of the β-Ketoadipate Pathway in
Corynebacterium glutamicum
Authors: Zhao Ke-Xin , Huang Yan , Chen Xi , Wang Nan-Xi , Liu Shuang-Jiang
Published: January 2010

We identified a new regulator, PcaO, which is involved in regulation of the protocatechuate (PCA) branch of the β-ketoadipate pathway in
Corynebacterium glutamicum
. PcaO is an atypical
l
arge
A
TP-binding
L
uxR family (LAL)-type regulator and does not have a Walker A motif. A mutant of
C. glutamicum
in which
pcaO
was disrupted (RES167Δ
pcaO
) was unable to grow on PCA, and growth on PCA was restored by complementation with
pcaO
. Both an enzymatic assay of PCA 3,4-dioxygenase activity (encoded by
pcaHG
) and transcriptional analysis of
pcaHG
by reverse transcription-PCR revealed that PcaO positively regulated
pcaHG
. A promoter-LacZ transcriptional fusion assay suggested that PcaO interacted with the sequence upstream of
pcaHG
. Electrophoretic mobility shift assay (EMSA) analysis indicated that an imperfect palindromic sequence (
−78
AACCCCTGACCTTCGGGGTT
−59
) that was located upstream of the −35 region of the
pcaHG
promoter was essential for PcaO regulation. DNase I footprinting showed that this imperfect palindrome was protected from DNase I digestion. Site-directed mutation and EMSA tests revealed that this palindrome sequence was essential for PcaO binding to the DNA fragment.
In vitro
EMSA results showed that ATP weakened the binding between PcaO and its target sequence but ADP strengthened this binding, while the effect of protocatechuate on PcaO binding was dependent on the protocatechuate concentration.
Identification and Regulation of the
N
-Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium
Xanthomonas campestris
pv. campestris
Authors: Boulanger Alice , Déjean Guillaume , Lautier Martine , Glories Marie , Zischek Claudine , Arlat Matthieu , Lauber Emmanuelle
Published: January 2010

Xanthomonas campestris
pv. campestris, the causal agent of black rot disease of brassicas, is known for its ability to catabolize a wide range of plant compounds. This ability is correlated with the presence of specific
c
arbohydrate
u
tilization loci containing
T
onB-dependent transporters (CUT loci) devoted to scavenging specific carbohydrates. In this study, we demonstrate that there is an
X. campestris
pv. campestris CUT system involved in the import and catabolism of
N
-acetylglucosamine (GlcNAc). Expression of genes belonging to this GlcNAc CUT system is under the control of GlcNAc via the LacI family NagR and GntR family NagQ regulators. Analysis of the NagR and NagQ regulons confirmed that GlcNAc utilization involves NagA and NagB-II enzymes responsible for the conversion of GlcNAc-6-phosphate to fructose-6-phosphate. Mutants with mutations in the corresponding genes are sensitive to GlcNAc, as previously reported for
Escherichia coli
. This GlcNAc sensitivity and analysis of the NagQ and NagR regulons were used to dissect the
X. campestris
pv. campestris GlcNAc utilization pathway. This analysis revealed specific features, including the fact that uptake of GlcNAc through the inner membrane occurs via a major facilitator superfamily transporter and the fact that this amino sugar is phosphorylated by two proteins belonging to the glucokinase family, NagK-IIA and NagK-IIB. However, NagK-IIA seems to play a more important role in GlcNAc utilization than NagK-IIB under our experimental conditions. The
X. campestris
pv. campestris GlcNAc NagR regulon includes four genes encoding TonB-dependent active transporters (TBDTs). However, the results of transport experiments suggest that GlcNAc passively diffuses through the bacterial envelope, an observation that calls into question whether GlcNAc is a natural substrate for these TBDTs and consequently is the source of GlcNAc for this nonchitinolytic plant-associated bacterium.
The
Sinorhizobium meliloti
RNA Chaperone Hfq Mediates Symbiosis of
S. meliloti
and Alfalfa
Authors: Barra-Bily Lise , Pandey Shree P. , Trautwetter Annie , Blanco Carlos , Walker Graham C.
Published: January 2010

There exist commonalities between symbiotic
Sinorhizobium meliloti
and pathogenic
Brucella
bacteria in terms of extensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperone Hfq is essential for virulence for several bacterial groups, including
Brucella
; however, its role in
S. meliloti
has not been investigated. Our studies of an
S. meliloti
loss-of-function
hfq
mutant have revealed that Hfq plays a key role in the establishment of the symbiosis between
S. meliloti
and its host
Medicago sativa. S. meliloti
Hfq is involved in controlling the population density under a free-living state and affects the growth parameters and nodulation. An
hfq
mutant poorly colonizes the infection threads that are necessary for the bacteria to invade the developing nodule. An
hfq
mutant is severely impaired in its ability to invade plant cells within the nodule, which leads to the formation of small, ineffective nodules unable to fix nitrogen. In culture, the
hfq
mutant did not accumulate transcripts of
nifA
, which encodes a key regulator necessary for nitrogen fixation. Hfq may be involved in regulation of several proteins relevant to
hfq
mutant phenotypes. The crucial role of Hfq in symbiosis suggests that small regulatory RNAs are important for its interactions with its plant host.
Proteomic Alterations Explain Phenotypic Changes in
Sinorhizobium meliloti
Lacking the RNA Chaperone Hfq
Authors: Barra-Bily Lise , Fontenelle Catherine , Jan Gwenael , Flechard Maud , Trautwetter Annie , Pandey Shree P. , Walker Graham C. , Blanco Carlos
Published: January 2010

The ubiquitous bacterial RNA-binding protein Hfq is involved in stress resistance and pathogenicity. In
Sinorhizobium meliloti
, Hfq is essential for the establishment of symbiosis with
Medicago sativa
and for nitrogen fixation. A proteomic analysis identifies 55 proteins with significantly affected expression in the
hfq
mutant; most of them are involved in cell metabolism or stress resistance. Important determinants of oxidative stress resistance, such as CysK, Gsh, Bfr, SodC, KatB, KatC, and a putative peroxiredoxine (SMc00072), are downregulated in the
hfq
mutant. The
hfq
mutant is affected for H
2
O
2
, menadione, and heat stress resistance. Part of these defects could result from the reductions of
rpoE1
,
rpoE2
,
rpoE3
, and
rpoE4
expression levels in the
hfq
mutant. Some proteins required for efficient symbiosis are reduced in the
hfq
mutant, contributing to the drastic defect in nodulation observed in this mutant.
Systematic Domain Deletion Analysis of the Major Sporulation Kinase in
Bacillus subtilis
Authors: Eswaramoorthy Prahathees , Fujita Masaya
Published: January 2010

To characterize the role of the three PAS domains in KinA, the major sporulation kinase in
Bacillus subtilis
, we constructed a series of systematic PAS domain deletion mutants and analyzed their activities using an IPTG (isopropyl-β-
d
-thiogalactopyranoside)-inducible artificial sporulation induction system, which we have developed recently. The results showed that any one of the three PAS domains is sufficient to maintain the kinase activity and trigger sporulation, if not fully then at least partially, when the protein levels increase beyond a certain level.
RamA, a Member of the AraC/XylS Family, Influences Both Virulence and Efflux inSalmonella entericaSerovar Typhimurium
Authors: Bailey Andrew M. , Ivens Al , Kingsley Rob , Cottell Jennifer L. , Wain John , Piddock Laura J. V.
Published: January 2010

The transcriptomes ofSalmonella entericaserovar Typhimurium SL1344 lacking a functionalramAorramRor with plasmid-mediated high-level overexpression oframAwere compared to those of the wild-type parental strain. Inactivation oframAled to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption oframAled to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressedramAled to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, includingacrAB,acrEF, andtolC. Decreased expression of 34Salmonellapathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization ofCaenorhabditis eleganswere also seen. Disruption oframRled to the increased expression oframA,acrAB, andtolC, but not to the same level as whenramAwas overexpressed on a plasmid. Inactivation oframRhad a more limited effect on pathogenicity gene expression.In silicoanalysis of a suggested RamA-binding consensus sequence identified target genes, includingramR,acrA,tolC,sipABC, andssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.
The influence of Rhizobium leguminosarum biovar. trifolii on the growth and neighbour relationships of Trifolium repens and three grasses
Authors: Thompson J. D. , Turkington R. , Holl F. B.
Published: January 2010

Trifolium repens was sampled from 10 areas dominated by each of Dactylis glomerata, Holcus lanatus, and Lolium perenne in each of three different-aged pastures. Cell extracts of nodule isolates of Rhizobium leguminosarum biovar. trifolii were compared using polyacrylamide gel electrophoresis. Each pasture contained a range of Rhizobium strains as defined by the protein profiles. No single strain was common to Trifolium sampled from the same grass species neighbourhood. The diversity of Rhizobium strains encountered was negatively correlated (r = −0.67; n = 9) with pasture age; old pastures showed a decrease in strain diversity. In addition, in the oldest pasture (45-year-old), tillers of the associated grasses were also sampled. In a glasshouse, ramets from each Trifolium clone and each of the three grass species were grown in all possible combinations with each of the three Rhizobium strains, plus Trifolium monocultures and an uninoculated control. All Trifolium clones gave increased yield in mixture with Lolium compared with yields with Dactylis and Holcus. Trifolium from the Holcus patches, in most cases, had higher yield than Trifolium from other grass patches. Rhizobium inoculation did not consistently increase Trifolium yield; it actually caused a decrease in yield when in mixture with Lolium. A significant Rhizobium × Trifolium interaction indicated a variable performance of each Trifolium genotype with different Rhizobium strains. The identity of the Trifolium clone had a significant effect on the yield of Holcus and Lolium, but not Dactylis. Total mixture yields were highest when Trifolium from a Lolium patch was part of the mixture. These results are discussed in the context of the hypothesis that Trifolium–grass coexistence is significantly influenced by Rhizobium strain differences.
A method for assessing resistance to the snow molds Typhula incarnata and Microdochium nivale in winter wheat incubated at the optimum growth temperature ranges of the fungi
Authors: Nakajima T. , Abe J.
Published: January 2010

Studies were carried out to determine whether incubation of wheat plants at the temperatures optimum for growth of fungi could reduce the time normally required for determination of resistance to Typhula incarnata and Microdochium nivale in wheat, comparing with conventional under-snow incubation methods in field plots. Typhula incarnata produced greater damage to the winter wheat plants at temperatures of 5 and 10 °C than M. nivale. At 15 and 18 °C, there existed little difference in virulence and we could complete incubation in a period of weeks. Incubation of the plants for varying periods of time was of use for quantitative determination of the degree of resistance expressed as LI50 (the number of incubation days when 50% of the plants are killed) values. The relative order of resistance among cultivars of wheat was consistent with that obtained from an under-snow incubation method. The new technique permitted wheat breeders to screen genotypes of wheat for resistance to snow mold pathogens in a shorter period, with less expensive facilities than the conventional "snow mold chamber method."
La végétation des contreforts des Laurentides : une analyse des gradients écologiques et du niveau successionnel des communautés
Authors: Gauthier Sylvie , Gagnon Daniel
Published: January 2010

The forest vegetation of the foothills of the Laurentians was studied to determine which ecological factors are most strongly correlated with vegetation variation. Vegetation and ecological factors were sampled within 78 plots distributed systematically on 19 transects. Ordination and classification analyses show that soil moisture and richness, two factors strongly associated to topographical variations, are the ecological factors that best explain vegetation distribution. Eleven community types were identified. Oligotrophic–xeric community types, characterized by Pinus strobus and Quercus rubra, are generally found in sites at high elevation, where soils are thin, well drained and nutrient poor. The Acer saccharum community types, of the mesotrophic–mesic group, are situated at the base of slopes with deep, rich soils. Fagus grandifolia, Acer rubrum, and Tsuga canadensis characterize a third group of oligotrophic–mesic community types, found on sites with relatively poorly drained and nutrient-poor soils, in topographic positions intermediate to the first two groups. A community type successionnal status index, based on the homogeneity and similarity of the tree and sapling strata, shows that natural and anthropogenic disturbances are also responsible for part of the vegetation variation. Hemlock and beech forests are the most successionnally advanced community types of the oligotrophic–mesic group. On richer soils fed by seepage, the maple–hickory community type is also relatively stable. These results contribute to the ongoing discussion about the eventual outcome of succession on mesic sites in northeastern North America.
Androecial development and the problem of monophyly of Loasaceae
Authors: Hufford Larry
Published: January 2010

Patterns of androecial diversity, especially developmental attributes, led some investigators to question the monophyly of Loasaceae. Polystaminate members of the family may possess either centripetal or centrifugal androecial development; however, these characterizations are shown to be too simple to explain the complex developmental patterns. Loasoideae with putatively centrifugal androecial development also have centripetally initiated stamens. Antesepalous regions among Loasoideae have stamens in positions that are similar to those of the earliest formed stamens of Mentzelia and are thus hypothesized to be homologous. Important transitional character states among the Klaprothieae show that complex patterns among some Loasoideae are not as divergent from those of Mentzelia as previously suggested. Haplostemony of Gronovioideae and diplostemony of Schismocarpus also show homologies with the developmental patterns present in Mentzelia and Eucnide.
The Role of Knowledge Management in Innovation
Authors: Dasgupta Meeta , Sahay A. , Gupta R. K.
Published: January 2010

Research on innovation practices reinforces the notion that majority of innovation projects fail systematically due to being managed as raw technology projects. Open forms of innovation where internal and external ideas are leveraged across the organisation are gaining importance over the internal and centralised approach to R&D. India, during the last 15 years, has been spiralling ahead towards ultimate economic success based on its 'knowledge-based talent pool'. Innovation has become a top strategic focus for most Indian companies. Seeing the growing importance of innovation and knowledge management, a need was felt to understand how organisations manage their knowledge capital so as to promote innovation.

This paper aims to provide insights into how an organisation manages knowledge through culture, structure, technology and leadership, so as to promote acquisition and application of knowledge which leads to innovation. The study brings out the importance of tacit knowledge embedded in the firm's culture, structure and leadership as a complement to the explicit knowledge embedded in the firm's technology and documents.
Effective Information Flow in the Internal Supply Chain: Results from a Snowball Method to Map Information Flows
Authors: Chibba Aron , Rundquist Jonas
Published: January 2010

Information sharing is an important factor for effectiveness within the internal supply chain. In this paper we use a methodology for mapping information flows in an internal supply chain, and case studies of two Swedish multinational organisations. Eight retrospective cases were used to map, describe and analyse the information flow that supports the physical material flow from the receipt of an order to point of delivery. Every involved person was interviewed on at least one occasion each. The interviews were conducted to map and describe the information and physical material flow. The aim was to identify factors that could improve and rationalise information flows and generate a better flow within the organisation.

The study shows the importance of an integrated information system, but also clearly indicates the importance of a collaborative culture and an awareness of the human–technology interface. The study also shows that three factors of interface distortions are most frequent in the cases: (1) changes registered in the database trigger no action among the staff, (2) new knowledge to staff is stored only orally and not in the database, and (3) interface between the paper system and the database, and between the old and the new information storage culture.
Genomewide RNA expression profiling in lung identifies distinct signatures in idiopathic pulmonary arterial hypertension and secondary pulmonary hypertension
Authors: Rajkumar Revathi , Konishi Kazuhisa , Richards Thomas J. , Ishizawar David C. , Wiechert Andrew C. , Kaminski Naftali , Ahmad Ferhaan
Published: January 2010

Idiopathic pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by pulmonary arteriolar remodeling. This investigation aimed to identify genes involved specifically in the pathogenesis of PAH and not other forms of pulmonary hypertension (PH). Using genomewide microarray analysis, we generated the largest data set to date of RNA expression profiles from lung tissue specimens from 1) 18 PAH subjects and 2) 8 subjects with PH secondary to idiopathic pulmonary fibrosis (IPF) and 3) 13 normal subjects. A molecular signature of 4,734 genes discriminated among these three cohorts. We identified significant novel biological changes that were likely to contribute to the pathogenesis of PAH, including regulation of actin-based motility, protein ubiquitination, and cAMP, transforming growth factor-β, MAPK, estrogen receptor, nitric oxide, and PDGF signaling. Bone morphogenic protein receptor type II expression was downregulated, even in subjects without a mutation in this gene. Women with PAH had higher expression levels of estrogen receptor 1 than normal women. Real-time quantitative PCR confirmed differential expression of the following genes in PAH relative to both normal controls and PH secondary to IPF: a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9, cell adhesion molecule with homology to L1CAM, cytochrome b558and β-polypeptide, coagulation factor II receptor-like 3, A-myb myeloblastosis viral oncogene homolog 1, nuclear receptor coactivator 2, purinergic receptor P2Y, platelet factor 4, phospholamban, and tropomodulin 3. This study shows that PAH and PH secondary to IPF are characterized by distinct gene expression signatures, implying distinct pathophysiological mechanisms.
Tenascin-C may aggravate left ventricular remodeling and function after myocardial infarction in mice
Authors: Nishioka Tomohiro , Onishi Katsuya , Shimojo Naoshi , Nagano Yuka , Matsusaka Hidenori , Ikeuchi Masaki , Ide Tomomi , Tsutsui Hiroyuki , Hiroe Michiaki , Yoshida Toshimichi , Imanaka-Yoshida Kyoko
Published: January 2010

Tenascin-C (TN-C) is an extracellular matrix glycoprotein with high bioactivity. It is expressed at low levels in normal adult heart, but upregulated under pathological conditions, such as myocardial infarction (MI). Recently, we (Ref. 34 ) reported that MI patients with high serum levels of TN-C have a greater incidence of maladaptive cardiac remodeling and a worse prognosis. We hypothesized that TN-C may aggravate left ventricular remodeling. To examine the effects of TN-C, MI was induced by ligating coronary arteries of TN-C knockout (KO) mice under anesthesia and comparing them with sibling wild-type (WT) mice. In WT+MI mice, TN-C expression was upregulated at day 1, peaked at day 5, downregulated and disappeared by day 28, and the molecule was localized in the border zone between intact myocardium and infarct lesions. The morphometrically determined infarct size and survival rate on day 28 were comparable between the WT+MI and KO+MI groups. Echocardiography and hemodynamic analyses demonstrated left ventricular end-diastolic diameter, myocardial stiffness, and left ventricular end-diastolic pressure to be significantly increased in both WT+MI and KO+MI mice compared with sham-operated mice. However, end-diastolic pressure and dimension and myocardial stiffness of KO+MI were lower than those of the WT+MI mice. Histological examination revealed normal tissue healing, but interstitial fibrosis in the residual myocardium in peri-infarcted areas was significantly less pronounced in KO+MI mice than in WT+MI mice. TN-C may thus accelerate adverse ventricular remodeling, cardiac failure, and fibrosis in the residual myocardium after MI.
Pivotal role of JNK-dependent FOXO1 activation in downregulation of kallistatin expression by oxidative stress
Authors: Shen Bo , Chao Lee , Chao Julie
Published: January 2010

Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor forkhead box O 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahl salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, H2O2dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO1 expression with small-interfering RNA. H2O2rapidly induced FOXO1 nuclear translocation, but the effect was blocked by c-Jun NH2-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed H2O2's effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells.
Cardiovascular and metabolic effects of 48-h glucagon-like peptide-1 infusion in compensated chronic patients with heart failure
Authors: Halbirk Mads , Nørrelund Helene , Møller Niels , Holst Jens Juul , Schmitz Ole , Nielsen Roni , Nielsen-Kudsk Jens Erik , Nielsen Søren Steen , Nielsen Torsten Toftegaard , Eiskjær Hans , Bøtker Hans Erik , Wiggers Henrik
Published: January 2010

The incretin hormone glucagon-like peptide-1 (GLP-1) and its analogs are currently emerging as antidiabetic medications. GLP-1 improves left ventricular ejection fraction (LVEF) in dogs with heart failure (HF) and in patients with acute myocardial infarction. We studied metabolic and cardiovascular effects of 48-h GLP-1 infusions in patients with congestive HF. In a randomized, double-blind crossover design, 20 patients without diabetes and with HF with ischemic heart disease, EF of 30 ± 2%, New York Heart Association II and III ( n = 14 and 6) received 48-h GLP-1 (0.7 pmol·kg−1·min−1) and placebo infusion. At 0 and 48 h, LVEF, diastolic function, tissue Doppler regional myocardial function, exercise testing, noninvasive cardiac output, and brain natriuretic peptide (BNP) were measured. Blood pressure, heart rate, and metabolic parameters were recorded. Fifteen patients completed the protocol. GLP-1 increased insulin (90 ± 17 pmol/l vs. 69 ± 12 pmol/l; P = 0.025) and lowered glucose levels (5.2 ± 0.1 mmol/l vs. 5.6 ± 0.1 mmol/l; P < 0.01). Heart rate (67 ± 2 beats/min vs. 65 ± 2 beats/min; P = 0.016) and diastolic blood pressure (71 ± 2 mmHg vs. 68 ± 2 mmHg; P = 0.008) increased during GLP-1 treatment. Cardiac index (1.5 ± 0.1 l·min−1·m−2 vs. 1.7 ± 0.2 l·min−1·m−2; P = 0.54) and LVEF (30 ± 2% vs. 30 ± 2%; P = 0.93), tissue Doppler indexes, body weight, and BNP remained unchanged. Hypoglycemic events related to GLP-1 treatment were observed in eight patients. GLP-1 infusion increased circulating insulin levels and reduced plasma glucose concentration but had no major cardiovascular effects in patients without diabetes but with compensated HF. The impact of minor increases in heart rate and diastolic blood pressure during GLP-1 infusion requires further studies. Hypoglycemia was frequent and calls for caution in patients without diabetes but with HF.
Activation of L-type Ca2+channels by protein kinase C is reduced in smooth muscle-specific Na+/Ca2+exchanger knockout mice
Authors: Ren Chongyu , Zhang Jin , Philipson Kenneth D. , Kotlikoff Michael I. , Blaustein Mordecai P. , Matteson Donald R.
Published: January 2010

L-type voltage-gated Ca2+channels (LVGCs) are functionally downregulated in arterial smooth muscle (SM) cells (ASMCs) of mice with SM-specific knockout of Na+/Ca2+exchanger type-1 (NCX1SM−/−) ( 32 ). Here, using activators and inhibitors of protein kinase C (PKC), we explore the regulation of these channels by a PKC-dependent mechanism. In both wild-type (WT) and NCX1SM−/−myocytes, the PKC activator phorbol 12,13-dibutyrate (PDBu) increases LVGC conductance, decreases channel closing rate, and shifts the voltage dependence of channel opening to more negative potentials. Three different PKC inhibitors, bisindolylmaleimide, Ro-31-8220, and PKC 19-31, all decrease LVGC currents in WT myocytes and prevent the PDBu-induced increase in LVGC current. Dialysis of WT ASMCs with activated PKC increases LVGC current and decreases channel closing rate. These results demonstrate that PKC activates LVGCs in ASMCs. The phosphatase inhibitor calyculin A increases LVGC conductance by over 50%, indicating that the level of LVGC activation is a balance between phosphatase and PKC activities. PDBu causes a larger increase in LVGC conductance and a larger shift in voltage dependence in NCX1SM−/−myocytes than in WT myocytes. The inhibition of PKC with PKC 19-31 decreased LVGC conductance by 65% in WT myocytes but by only 37% in NCX1SM−/−myocytes. These results suggest that LVGCs are in a state of low PKC-induced phosphorylation in NCX1SM−/−myocytes. We conclude that in NCX1SM−/−myocytes, reduced Ca2+entry via NCX1 lowers cytosolic [Ca2+], thereby reducing PKC activation that lowers LVGC activation.
Anti-inflammatory effect of MUC1 during respiratory syncytial virus infection of lung epithelial cells in vitro
Authors: Li Yusheng , Dinwiddie Darrell L. , Harrod Kevin S. , Jiang Yong , Kim K. Chul
Published: January 2010

MUC1 is a transmembrane glycoprotein expressed on the apical surface of airway epithelial cells and plays an anti-inflammatory role during airway bacterial infection. In this study, we determined whether the anti-inflammatory effect of MUC1 is also operative during the respiratory syncytial virus (RSV) infection. The lung epithelial cell line A549 was treated with RSV, and the production of TNFα and the levels of MUC1 protein were monitored temporally during the course of infection by ELISA and Western blot analysis. Small inhibitory RNA (siRNA) transfection was utilized to assess the role of MUC1 in regulating RSV-mediated inflammatory responses by lung epithelial cells. Our results revealed that: 1) following RSV infection, an increase in MUC1 level was preceded by an increase in TNFα production and completely inhibited by soluble TNF receptor (TNFR); and 2) knockdown of MUC1 using MUC1 siRNA resulted in a greater increase in TNFα level following RSV infection compared with control siRNA treatment. We conclude that the RSV-induced increase in the TNFα levels upregulates MUC1 through its interaction with TNFR, which in turn suppresses further increase in TNFα by RSV, thus forming a negative feedback loop in the control of RSV-induced inflammation. This is the first demonstration showing that MUC1 can suppress the virus-induced inflammatory responses.
Adenovirus-mediated gene transfer of hIGF-IB in mouse lungs induced prolonged inflammation but no fibroproliferation
Authors: Léger Caroline , Ni Ai , Andonegui Graciela , Wong Josée , Mowat Connie , Winston Brent W.
Published: January 2010

Pulmonary fibrosis (PF), the end stage of a variety of fibroproliferative lung diseases, is characterized by excessive lung mesenchymal cell activation and extracellular matrix deposition. Most PF is induced after repetitive or chronic lung inflammation; however, a significant portion of PF occurs without apparent inflammation. The mechanisms of fibroproliferation are poorly understood. Studies have shown that cytokines regulating inflammation and tissue repair processes play essential roles in the development of PF. Insulin-like growth factor I (IGF-I) has been shown to stimulate lung mesenchymal cell proliferation and extracellular matrix synthesis in vitro and is significantly elevated in patients with PF. In this study, we investigated whether human IGF-IB (hIGF-IB) expression in the lungs induces PF in a C57BL/6 mouse model. Mice were subjected to adenoviral gene transfer, and the effects of hIGF-IB expression on the lungs were examined 3, 7, 14, 21, and 42 days after gene delivery. hIGF-IB expression induced significant and prolonged inflammatory cell infiltration into the lungs, with an early neutrophil infiltration followed by a late macrophage infiltration. No significant fibroblast or matrix accumulation could be detected in the lungs of these mice. No significant collagen accumulation could be detected in vivo, despite in vitro evidence that hIGF-IB induces collagen mRNA expression in fibroblasts. Therefore, IGF-IB alone is not sufficient to induce fibrosis, and it is possible that a coactivator is required to induce significant fibroproliferation in vivo.
Airway smooth muscle remodels pericellular collagen fibrils: implications for proliferation
Authors: Schuliga Michael , Ong Siau Chi , Soon Lilian , Zal Fatemeh , Harris Trudi , Stewart Alastair G.
Published: January 2010

Airway wall remodeling comprises a broad range of structural changes including increases in the volume of airway smooth muscle (ASM) and fibrillar collagen. The impact of fibrillar collagen remodeling on ASM proliferation was investigated. Human ASM cultured on type I fibrillar collagen remodeled the collagen substrate by both degradation (collagenolysis) and formation of networks comprised of thicker reticular collagen fibrils (fibrillogenesis). In cultures maintained on fibrillar collagen, the levels of matrix metalloproteases (MMPs) -1 and -14 mRNA and active MMP-2 were higher than in cultures maintained on nonfibrillar type I collagen (gelatin) or plastic. Although there was no apparent increase in cytotoxicity or apoptosis, the number of ASM was lower on fibrillar collagen than on gelatin or plastic for control conditions. Furthermore, maintenance on fibrillar collagen attenuated basic fibroblast growth factor-stimulated increases in cell number and the percentage of cells entering S-phase. In cultures maintained on fibrillar collagen, the MMP inhibitor ilomastat (2.5 μM) 1) attenuated collagenolysis, 2) enhanced fibrillogenesis, and 3) inhibited proliferation. In contrast, knockdown of the β1-integrin gene in ASM maintained on fibrillar collagen led to an increase in proliferation and reduced MMP-1 and -14 expression. Thus, ASM remodel the pericellular environment by degrading collagen fibrils and spinning them into larger collagen assemblies. Moreover, the collagen fibrils limit proliferation and activate autocrine MMPs in a β-integrin-dependent manner, suggesting a potential negative feedback on modeling executed through fibrillar collagen activation of β1-integrins.
Visual Flicker in the Gamma-Band Range Does Not Draw Attention
Authors: van Diepen Rosanne M. , Born Sabine , Souto David , Gauch Angélique , Kerzel Dirk
Published: January 2010

External transients, such as a flash or a startling sound, are believed to capture attention. Bauer, Cheadle, Parton, Müller, and Usher reported that attention can also be captured by a stimulus that flickers subliminally at 50 Hz, presumably by entrainment of neurons to the flicker frequency. In their reaction time (RT) task, participants had to locate a subtle change in the spatial frequency content of one of three Gabors (the target). Prior to target onset, presumably subliminal 50-Hz flicker in one of the Gabors served as a spatial cue. Bauer et al. found faster RTs when the cued location was congruent with the target location than when the cue was incongruent with the target location. In their experiments, the cue stopped to flicker at 50 Hz at target onset and was replaced by a stimulus flickering at 100 Hz (i.e., the screen refresh rate). In the present study, we show that the transition from 50 to 100 Hz results in a flash-like impression that can be localized above chance. We suggest that the illusory transition flash interfered with the localization of the subtle target, which contributed to the congruency effect. In support of this view, participants selected the flickering object more often than the non-flickering object when they failed to respond to the target. Further, no cueing effects were observed when the cue continued to flicker until the end of the trial or when the target was a salient change in polarity. In our view, the cueing effect occurs because observers confuse the illusory transition flash with the target when the two are similar. When truly subliminal flicker is used (70-Hz flicker), very small cueing effects persist in the absence of an illusory transition flash but may be accounted for by small effects on reaction time unrelated to attention.
Characterization of Major Surface Protease Homologues ofTrypanosoma congolense
Authors: Marcoux Veronica , Wei Guojian , Tabel Henry , Bull Harold J.
Published: January 2010

Trypanosomes encode a family of proteins known as Major Surface Metalloproteases (MSPs). We have identified six putative MSPs encoded within the partially sequencedT. congolensegenome. Phylogenic analysis indicates thatT. congolenseMSPs belong to five subfamilies that are conserved among African trypanosome species. Molecular modeling, based on the known structure ofLeishmania MajorGP63, reveals subfamily-specific structural variations around the putative active site despite conservation of overall structure, suggesting that each MSP subfamily has evolved to recognize distinct substrates. We have cloned and purified a protein encoding the amino-terminal domain of theT. congolensehomologue TcoMSP-D (most closely related toLeishmaniaGP63). We detect TcoMSP-D in the serum ofT. congolense-infected mice. Mice immunized with the amino-terminal domain of TcoMSP-D generate a persisting IgG1 antibody response. Surprisingly, a low-dose challenge of immunized mice withT. congolensesignificantly increases susceptibility to infection, indicating that immunity to TcoMSP-D is a factor affecting virulence.
Anaemia Management in Non-Dialysis Chronic Kidney Disease: Flexibility of Target to Target Stability?
Authors: De Nicola Luca , Minutolo Roberto , Conte Giuseppe
Published: January 2010

International clinical practice guidelines have recently recognized for the first time the concrete difficulty of keeping chronic kidney disease (CKD) patients within the narrow haemoglobin (Hb) target recommended of 11–12 g/dl (110–120 g/l) because of the variability of individual patient Hb levels. This emerging clinical problem has been the topic of several editorials that, however, exclusively focused on haemodialysis patients. Recently, 3 retrospective studies have been published on Hb variability in non-dialysis CKD. The studies which overall included more than 6,000 patients showed that Hb variability in non-dialysis CKD is greatly prevalent and is associated with a worse cardiorenal outcome. This minireview summarizes the results and limits of these studies and discusses the potential implications for clinical practice.