bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
Authors: Zhou Ping , Altman Eleonora , Perry Malcolm B. , Li Jianjun
Published: April 2010

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from
Escherichia coli
O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 μM, 100 μM, and 10 μM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including
Helicobacter pylori
,
Salmonella enterica
serovar Riogrande, and
Francisella novicida
.
Translocation of NopP by
Sinorhizobium fredii
USDA257 into
Vigna unguiculata
Root Nodules
Authors: Schechter Lisa M. , Guenther Jeanette , Olcay Elizabeth A. , Jang Sungchan , Krishnan Hari B.
Published: April 2010

Sinorhizobium fredii
is a nitrogen-fixing legume symbiont that stimulates the formation of root nodules.
S. fredii
nodulation of roots is influenced by Nop proteins, which are secreted through a type III secretion system (T3SS). We demonstrate that
S. fredii
injects NopP into
Vigna unguiculata
nodules in a T3SS-dependent manner.
Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica
Authors: Yousuf Mohammad Abu , Mi-ichi Fumika , Nakada-Tsukui Kumiko , Nozaki Tomoyoshi
Published: April 2010

Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite
Entamoeba histolytica
based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-
E. histolytica
PNT (
Eh
PNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in
E. histolytica
.
Cryptococcal Interactions with the Host Immune System
Authors: Voelz Kerstin , May Robin C.
Published: April 2010

Opportunistic pathogens have become of increasing medical importance over the last decade due to the AIDS pandemic. Not only is cryptococcosis the fourth-most-common fatal infectious disease in sub-Saharan Africa, but alsoCryptococcusis an emerging pathogen of immunocompetent individuals. The interaction betweenCryptococcusand the host's immune system is a major determinant for the outcome of disease. Despite initial infection in early childhood withCryptococcus neoformansand frequent exposure toC. neoformanswithin the environment, immunocompetent individuals are generally able to contain the fungus or maintain the yeast in a latent state. However, immune deficiencies lead to disseminating infections that are uniformly fatal without rapid clinical intervention. This review will discuss the innate and adaptive immune responses toCryptococcusand cryptococcal strategies to evade the host's defense mechanisms. It will also address the importance of these strategies in pathogenesis and the potential of immunotherapy in cryptococcosis treatment.
Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae
Authors: Nishizawa Masafumi , Tanigawa Mirai , Hayashi Michio , Maeda Tatsuya , Yazaki Yoshiaki , Saeki Yasushi , Toh-e Akio
Published: April 2010

The budding yeast
Saccharomyces cerevisiae
alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither
RIM101
transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that
PTK2
activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the
PHO
system when yeast cells cope with various environmental stresses potentially threatening their survival.
Human β-Defensin 3 Inhibits Cell Wall Biosynthesis in Staphylococci
Authors: Sass Vera , Schneider Tanja , Wilmes Miriam , Körner Christian , Tossi Alessandro , Novikova Natalia , Shamova Olga , Sahl Hans-Georg
Published: April 2010

Human β-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistantStaphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. Inin vitroassays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.
Functional Significance of the E Loop, a Novel Motif Conserved in the Lantibiotic Immunity ATP-Binding Cassette Transport Systems
Authors: Okuda Ken-ichi , Yanagihara Sae , Sugayama Tomomichi , Zendo Takeshi , Nakayama Jiro , Sonomoto Kenji
Published: April 2010

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.
RmpA Regulation of Capsular Polysaccharide Biosynthesis inKlebsiella pneumoniaeCG43
Authors: Cheng H. Y. , Chen Y. S. , Wu C. Y. , Chang H. Y. , Lai Y. C. , Peng H. L.
Published: April 2010

Sequence analysis of the large virulence plasmid pLVPK inKlebsiella pneumoniaeCG43 revealed the presence of another mucoid factor encoding genermpAbesidesrmpA2. Promoter activity measurement indicated that the deletion ofrmpAreduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carryingrmpArestored CPS production in thermpAorrmpA2mutant but not in thercsBmutant. Transformation of thermpAdeletion mutant with anrcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration ofin vivointeraction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region ofrmpA. The promoter activity analysis indicated that the deletion offurincreased thermpApromoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, thefurdeletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis inK. pneumoniaeCG43 via an RcsB-dependent manner. The expression ofrmpAis regulated by the availability of iron and is negatively controlled by Fur.
PdhS, an Old-Pole-Localized Histidine Kinase, Recruits the Fumarase FumC in
Brucella abortus
Authors: Mignolet Johann , Van der Henst Charles , Nicolas Cécile , Deghelt Michaël , Dotreppe Delphine , Letesson Jean-Jacques , De Bolle Xavier
Published: April 2010

The bacterial pathogen
Brucella abortus
was recently demonstrated to recruit the essential cytoplasmic histidine kinase PdhS to its old pole. Here, we report identification of the fumarase FumC as a specific partner for the N-terminal “sensing” domain of PdhS, using an ORFeome-based yeast two-hybrid screen. We observed that FumC and PdhS colocalize at the old pole of
B. abortus
, while the other fumarase FumA is not polarly localized. FumC is not required for PdhS localization, and polar FumC localization is not FumA dependent. FumC homologs are not polarly localized in
Sinorhizobium meliloti
and
Caulobacter crescentus
, suggesting that polar recruitment of FumC by PdhS is evolutionarily recent.
Self-Assembly of the Bacterial Cytoskeleton-Associated RNA Helicase B Protein into Polymeric Filamentous Structures
Authors: Taghbalout Aziz , Yang Qingfen
Published: April 2010

TheEscherichia coliRNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structuresin vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.
SpoIID-Mediated Peptidoglycan Degradation Is Required throughout Engulfment during
Bacillus subtilis
Sporulation
Authors: Gutierrez Jennifer , Smith Rachelle , Pogliano Kit
Published: April 2010

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in
Bacillus subtilis
. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the
in vivo
and
in vitro
activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in
B. subtilis
(R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.
Complete Genome Sequence of “
Candidatus
Puniceispirillum marinum” IMCC1322, a Representative of the SAR116 Clade in the
Alphaproteobacteria
Authors: Oh Hyun-Myung , Kwon Kae Kyoung , Kang Ilnam , Kang Sung Gyun , Lee Jung-Hyun , Kim Sang-Jin , Cho Jang-Cheon
Published: April 2010

The complete genome sequence of “
Candidatus
Puniceispirillum marinum” IMCC1322, the first cultured representative of the SAR116 clade in the
Alphaproteobacteria
, is reported here. The genome contains genes for proteorhodopsin, aerobic-type carbon monoxide dehydrogenase, dimethylsulfoniopropionate demethylase, and C
1
compound metabolism. The genome information proposes the SAR116 group to be metabolic generalists in ocean nutrient cycling.
Role of the FeoB Protein and Siderophore in Promoting Virulence ofXanthomonas oryzaepv. oryzae on Rice
Authors: Pandey Alok , Sonti Ramesh V.
Published: April 2010

Xanthomonas oryzaepv. oryzae causes bacterial blight, a serious disease of rice. Our analysis revealed that theX. oryzaepv. oryzae genome encodes genes responsible for iron uptake through FeoB (homolog of the major bacterial ferrous iron transporter) and a siderophore. A mutation in theX. oryzaepv. oryzaefeoBgene causes severe virulence deficiency, growth deficiency in iron-limiting medium, and constitutive production of a siderophore. We identified an iron regulatedxssgene cluster, in whichxssABCDE(Xanthomonassiderophoresynthesis) andxsuA(Xanthomonassiderophoreutilization) genes encode proteins involved in biosynthesis and utilization ofX. oryzaepv. oryzae siderophore. Mutations in thexssA,xssB, andxssEgenes cause siderophore deficiency and growth restriction under iron-limiting conditions but are virulence proficient. AnxsuAmutant displayed impairment in utilization of native siderophore, suggesting that XsuA acts as a specific receptor for a ferric-siderophore complex. Histochemical and fluorimetric assays withgusAfusions indicate that, duringin plantagrowth, thefeoBgene is expressed and that thexssoperon is not expressed. This study represents the first report describing a role forfeoBin virulence of any plant-pathogenic bacterium and the first functional characterization of a siderophore-biosynthetic gene cluster in any xanthomonad.
Dispersal of Arceuthobiumpusillum seeds
Authors: Baker F. A. , French D. W.
Published: April 2010

Seed dispersal of Arceuthobiumpusillum Peck on Piceamariana (Mill.) B.S.P. was studied at two locations in Minnesota from 1974–1980. Seed dispersal began as early as August 23 and was observed as late as September 27. Most seeds were dispersed in the mornings between 0400 and 0900. The mean annual distance of dispersal ranged from 1.0 to 2.3 m. The density of seeds trapped on the plots ranged from 4.2 to 16.7 seeds/m2. At both locations, the number of seeds trapped outside the stand decreased logarithmically with distance from the source trees. Within stands, seeds were more frequently trapped 1–4 m from source trees than under them. Most seed dispersal occurred 1-3 days after rain. Seeds were dispersed normally on 23 of 30 days when minimum temperatures were less than 0 °C. Fruit abortion was noted on four occasions. Insects were not found to carry dwarf mistletoe seeds.
Effects of the herbicides glyphosate, 2,4,5-trichlorophenoxyacetic acid, and 2,4-dichlorophenoxyacetic acid on forest litter decomposition
Authors: Fletcher K. , Freedman B.
Published: April 2010

Laboratory studies with two leaf litter and one forest floor substrate showed that the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a 50:50 mixture of these, and glyphosate all had toxic thresholds at which they reduced decomposition. However, in all cases, the thresholds were > 50 times higher than residue concentrations that occur in the field after silvicultural herbicide treatments. In a field study at one site, no measurable 1-year postspray effects on litter decomposition were found among treatment plots sprayed at 0.0, 3.4 or 6.7 kg 2,4,5-T/ha.
The influence of vesicular–arbuscular mycorrhizae on biomass production in willow
Authors: Backhaus Georg F. , Häggblom Per , Nilsson Lars Owe
Published: April 2010

Cuttings of Salixdasyclados and Salixdaphnoides were grown in clay, peat, or sand under greenhouse conditions and inoculated with endomycorrhizal roots. Uninoculated cuttings in the same substrates served as controls. All plants were fertilized with a commercial fertilizer solution. Shoot dry mass and colonization by vesicular–arbuscular mycorrhizal fungi were measured during the experiment. At the end of the experiment growth of inoculated plants was significantly enhanced in peat (p < 0.001) and sand (p < 0.01), while in clay, no effect of inoculation on growth was found. In peat and sand increased growth of plants could be correlated with frequency of vesicular–arbuscular mycorrhizal colonization. No significant differences in concentrations of P or N in leaves and stems were noted between mycorrhizal and nonmycorrhizal plants at the end of the experiment. It is concluded that vesicular–arbuscular mycorrhizae stimulate growth of willow under greenhouse conditions despite application of easily available nutrients.
Variable-form stem profile models for loblolly pine
Authors: Newberry James D. , Burkhart Harold E.
Published: April 2010

Stem profile models for loblolly pine (Pinustaeda L.) that allow for both taper and form changes were constructed and evaluated. In 1956, H. R. Gray defined form to be the basic shape of the tree (e.g., cone or paraboloid) and taper to be the rate of narrowing in diameter given a tree form. D. W. Ormerod's stem profile model was selected as the basic model since its parameters were readily interpretable in terms of Gray's taper and form definitions. Two-stage modeling procedures were used to relate individual tree taper and form parameters to tree and stand characteristics. Two second-stage parameter estimation alternatives were evaluated. Parameter estimates for both techniques, ordinary least-squares and random function analysis, were similar. Characteristics used to predict stem form were total tree height, crown ratio, height to the live crown, site index, and tree age. The taper parameter was related to diameter at breast height, crown ratio, and site index. Error evaluations suggest that a 10–20% gain can be made in predicting stem diameters using the variable-taper and variable-form stem profile models.
Destruction of shoots, flowers, and fruit of dwarf mistletoe by grasshoppers in California
Authors: Scharpf Robert F. , Koerber Thomas W.
Published: April 2010

Grasshoppers (Melanoplusdevastator) destroyed most shoots, flowers, and fruit of dwarf mistletoe (Arceuthobiumcampylopodum) in a young plantation of Jeffrey pines (PinusJeffreyi) in California. Damage resulted from the buildup of large populations of grasshoppers in herbaceous, understory vegetation in the spring of 1984. In the summer, when the preferred food sources became depleted or dried, the grasshoppers moved into the pines and fed upon the aerial portions of dwarf mistletoe plants. Little evidence of feeding was seen on the pines, but very young pine plantations are known to be seriously damaged by grasshoppers.
A causal relationship between shear stress and atherosclerotic lesions in apolipoprotein E knockout mice assessed by ultrasound biomicroscopy
Authors: Ding Shi Fang , Ni Mei , Liu Xiao Ling , Qi Li Hang , Zhang Mei , Liu Chun Xi , Wang Ying , Lv Hui Xia , Zhang Yun
Published: April 2010

The present study was undertaken to examine the hemodynamic state using the latest ultrasound biomicroscopy (UBM) technique and to investigate the effect of local shear stress on the development of atherosclerosis in the constrictive collar-treated carotid arteries of apolipoprotein E-deficient (apoE−/−) mice. Fifty-six male apoE−/− mice fed a high-lipid diet were divided into an interventional group ( n = 48) and the control group ( n = 8). Constrictive and nonconstrictive collars were placed around the carotid artery of the mice in the interventional group and the control group, respectively. The carotid lumen diameters and flow velocities were measured by UBM, and shear stress in the lesion region was calculated. Histopathology and electron microscopy were performed to observe the morphological changes in the carotid artery. In the region proximal to the constrictive collar, shear stress was significantly reduced 2 days after collar placement and remained low over time compared with the baseline level. In contrast, within the constrictive collar region, shear stress was increased significantly. Although endothelial permeability was enhanced in both regions, monocyte chemotaxis protein-1 (MCP-1) expression, macrophage infiltration, and atherosclerotic lesions were more prominent in the region proximal to the constrictive collar. Moreover, increased MCP-1 expression was observed as early as 2 days after constrictive collar placement, which preceded the morphological changes of the vessel wall. In conclusion, UBM offers a noninvasive and reliable technique for measuring shear stress in apoE−/− mice. Persistent low shear stress promotes endothelial permeability and enhances MCP-1 expression and macrophage recruitment, which were essential in the pathogenesis of atherosclerosis in apoE−/− mice.
VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model
Authors: Wu Florence T. H. , Stefanini Marianne O. , Gabhann Feilim Mac , Kontos Christopher D. , Annex Brian H. , Popel Aleksander S.
Published: April 2010

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, the growth of new capillaries from existing microvasculature. In peripheral arterial disease (PAD), lower extremity muscle ischemia develops downstream of atherosclerotic obstruction. A working hypothesis proposed that the maladaptive overexpression of soluble VEGF receptor 1 (sVEGFR1) in ischemic muscle tissues, and its subsequent antagonism of VEGF bioactivity, may contribute to the deficient angiogenic response in PAD, as well as the limited success of therapeutic angiogenesis strategies where exogenous VEGF genes/proteins are delivered. The objectives of this study were to develop a computational framework for simulating the systemic distributions of VEGF and sVEGFR1 (e.g., intramuscular vs. circulating, free vs. complexed) as observed in human PAD patients and to serve as a platform for the systematic optimization of diagnostic tools and therapeutic strategies. A three-compartment model was constructed, dividing the human body into the ischemic calf muscle, blood, and the rest of the body, connected through macromolecular biotransport processes. Detailed molecular interactions between VEGF, sVEGFR1, endothelial surface receptors (VEGFR1, VEGFR2, NRP1), and interstitial matrix sites were modeled. Our simulation results did not support a simultaneous decrease in plasma sVEGFR1 during PAD-associated elevations in plasma VEGF reported in literature. Furthermore, despite the overexpression in sVEGFR1, our PAD control demonstrated increased proangiogenic signaling complex formation, relative to our previous healthy control, due to sizeable upregulations in VEGFR2 and VEGF expression, thus leaving open the possibility that impaired angiogenesis in PAD may be rooted in signaling pathway disruptions downstream of ligand-receptor binding.
Genetic ablation of calcium-independent phospholipase A2β causes hypercontractility and markedly attenuates endothelium-dependent relaxation to acetylcholine
Authors: Dietrich Hans H. , Abendschein Dana R. , Moon Sung Ho , Nayeb-Hashemi Neema , Mancuso David J. , Jenkins Christopher M. , Kaltenbronn Kevin M. , Blumer Kendall J. , Turk John , Gross Richard W.
Published: April 2010

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A2β (iPLA2β) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA2β+/+) mice and those lacking the β-isoform (iPLA2β−/−) both ex vivo and in vivo. Vessels isolated from iPLA2β−/−mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA2β+/+mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA2β+/+vessels. In contrast, vessels from iPLA2β−/−constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA2β−/−vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA2β with ( S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA2β is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA2β causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.
Circulating endothelial progenitor cells are not affected by acute systemic inflammation
Authors: Padfield Gareth J. , Tura Olga , Haeck Marlieke L. A. , Short Abigail , Freyer Elizabeth , Barclay G. Robin , Newby David E. , Mills Nicholas L.
Published: April 2010

Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34+VEGF receptor (VEGF)R-2+CD133+, CD14+VEGFR-2+Tie2+, CD45−CD34+, as a surrogate for late outgrowth EPCs, and CD34+CXCR-4+], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 ± 0.6 vs. 5.1 ± 0.2 × 109cells/l, P < 0.0001), serum IL-6 [0.95 (0–1.7) vs. 0 (0–0) ng/l, P = 0.016], and VEGF-A [60 (45–94) vs. 43 (21–64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4–3.6) vs. 0.4 (0.2–0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 ± 7.6% increase in CD14+cells at 6 h ( P < 0.001) and a 22.4 ± 6.9% increase in CD34+cells at 7 days ( P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period ( P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury.
Activation becomes highly organized during long-duration ventricular fibrillation in canine hearts
Authors: Li Li , Jin Qi , Dosdall Derek J. , Huang Jian , Pogwizd Steven M. , Ideker Raymond E.
Published: April 2010

Little is known about the three-dimensional (3-D) intramural activation sequences during long-duration ventricular fibrillation (VF), including the role of the subendocardium and its Purkinje fibers (PFs) in long-duration VF maintenance. Our aim was to explore the mechanism of long-duration VF maintenance with 3-D electrical mapping. We recorded 10 min of electrically induced VF in the left ventricular anterior free wall of six 10-kg, open-chest dogs using a 3-D transmural unipolar electrode matrix (9 × 9 × 6, 2-mm spacing) that allowed us to map intramural activation sequences. At 2.5 ± 1.8 min of VF, although the body surface ECG continued to exhibit a disorganized VF pattern, intramurally a more organized, synchronous activation pattern was first observed [locally synchronized VF (LSVF)]. This pattern occurred one or more times in all dogs and was present 33.4 ± 31.4% of the time during 5–10 min of VF. As opposed to the preceding changing complex activation sequences of VF, during LSVF, wavefronts were large and highly repeatable near the endocardium, first exciting the endocardium almost simultaneously and then rapidly spreading toward the epicardium with different levels of conduction block en route. During LSVF, PF activations always preceded working myocardium activations near the endocardium. In conclusion, long-duration VF in dogs frequently becomes highly organized in the subendocardium, with activation fronts arising in this region and passing intramurally toward the epicardium, even though the surface ECG continues to exhibit a disorganized pattern. PFs appear to play an important role during this stage of VF.
Pathway to lamellar bodies for surfactant protein A
Authors: Fisher Aron B. , Dodia Chandra , Ruckert Peter , Tao Jian-Qin , Bates Sandra R.
Published: April 2010

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled35S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [35S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50–60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [35S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [35S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.
Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice
Authors: Métrailler-Ruchonnet Isabelle , Pagano Alessandra , Carnesecchi Stéphanie , Khatib Karim , Herrera Pedro , Donati Yves , Bron Camille , Barazzone Constance
Published: April 2010

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.
Chronic lung disease in preterm lambs: effect of daily vitamin A treatment on alveolarization
Authors: Albertine Kurt H. , Dahl Mar Janna , Gonzales Linda W. , Wang Zheng-ming , Metcalfe Drew , Hyde Dallas M. , Plopper Charles G. , Starcher Barry C. , Carlton David P. , Bland Richard D.
Published: April 2010

Neonatal chronic lung disease is characterized by failed formation of alveoli and capillaries, and excessive deposition of matrix elastin, which are linked to lengthy mechanical ventilation (MV) with O2-rich gas. Vitamin A supplementation has improved respiratory outcome of premature infants, but there is little information about the structural and molecular manifestations in the lung that occur with vitamin A treatment. We hypothesized that vitamin A supplementation during prolonged MV, without confounding by antenatal steroid treatment, would improve alveolar secondary septation, decrease thickness of the mesenchymal tissue cores between distal air space walls, and increase alveolar capillary growth. We further hypothesized that these structural advancements would be associated with modulated expression of tropoelastin and deposition of matrix elastin, phosphorylated Smad2 (pSmad2), cleaved caspase 3, proliferating cell nuclear antigen (PCNA), VEGF, VEGF-R2, and midkine in the parenchyma of the immature lung. Eight preterm lambs (125 days' gestation, term ∼150 days) were managed by MV for 3 wk: four were treated with daily intramuscular Aquasol A (vitamin A), 5,000 IU/kg, starting at birth; four received vehicle alone. Postmortem lung assays included quantitative RT-PCR and in situ hybridization, immunoblot and immunohistochemistry, and morphometry and stereology. Daily vitamin A supplementation increased alveolar secondary septation, decreased thickness of the mesenchymal tissue cores between the distal air space walls, and increased alveolar capillary growth. Associated molecular changes were less tropoelastin mRNA expression, matrix elastin deposition, pSmad2, and PCNA protein localization in the mesenchymal tissue core of the distal air space walls. On the other hand, mRNA expression and protein abundance of VEGF, VEGF-R2, midkine, and cleaved caspase 3 were increased. We conclude that vitamin A treatment partially improves lung development in chronically ventilated preterm neonates by modulating expression of tropoelastin, deposition of elastin, and expression of vascular growth factors.
Serotonin (5-HT) receptor 5A sequence variants affect human plasma triglyceride levels
Authors: Zhang Y. , Smith E. M. , Baye T. M. , Eckert J. V. , Abraham L. J. , Moses E. K. , Kissebah A. H. , Martin L. J. , Olivier M.
Published: April 2010

Neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) work closely with leptin and insulin to fine-tune the metabolic and neuroendocrine responses to dietary intake. Losing the sensitivity to excess food intake can lead to obesity, diabetes, and a multitude of behavioral disorders. It is largely unclear how different serotonin receptor subtypes respond to and integrate metabolic signals and which genetic variations in these receptor genes lead to individual differences in susceptibility to metabolic disorders. In an obese cohort of families of Northern European descent ( n = 2,209), the serotonin type 5A receptor gene, HTR5A, was identified as a prominent factor affecting plasma levels of triglycerides (TG), supported by our data from both genome-wide linkage and targeted association analyses using 28 publicly available and 12 newly discovered single nucleotide polymorphisms (SNPs), of which 3 were strongly associated with plasma TG levels ( P < 0.00125). Bayesian quantitative trait nucleotide (BQTN) analysis identified a putative causal promoter SNP (rs3734967) with substantial posterior probability ( P = 0.59). Functional analysis of rs3734967 by electrophoretic mobility shift assay (EMSA) showed distinct binding patterns of the two alleles of this SNP with nuclear proteins from glioma cell lines. In conclusion, sequence variants in HTR5A are strongly associated with high plasma levels of TG in a Northern European population, suggesting a novel role of the serotonin receptor system in humans. This suggests a potential brain-specific regulation of plasma TG levels, possibly by alteration of the expression of HTR5A.
Sclerosing Epithelioid Fibrosarcoma of the Bone: A Case Report of High Resistance to Chemotherapy and a Survey of the Literature
Authors: Grunewald Thomas G. P. , von Luettichau Irene , Weirich Gregor , Wawer Angela , Behrends Uta , Prodinger Peter M. , Jundt Gernot , Bielack Stefan S. , Gradinger Reiner , Burdach Stefan
Published: April 2010

Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue sarcoma mostly occurring in extraosseous sites. SEF represents a clinically challenging entity especially because no standardized treatment regimens are available. Intraosseous localization is an additional challenge with respect to the therapeutical approach. We report on a 16-year-old patient with SEF of the right proximal tibia. The patient underwent standardized neoadjuvant chemotherapy analogous to the EURAMOS-1 protocol for the treatment of osteosarcoma followed by tumor resection and endoprosthetic reconstruction. Histopathological analysis of the resected tumor showed>90% vital tumor cells suggesting no response to chemotherapy. Therefore, therapy was reassigned to the CWS 2002 High-Risk protocol for the treatment of soft tissue sarcoma. To date (22 months after diagnosis), there is no evidence of relapse or metastasis. Our data suggest that SEF may be resistant to a chemotherapy regimen containing Cisplatin, Doxorubicin, and Methotrexate, which should be considered in planning treatment for patients with SEF.
Effect of Stretching Force on the Cells of Epithelial Rests of Malassez In Vitro
Authors: Koshihara Teruyoshi , Matsuzaka Kenichi , Sato Toru , Inoue Takashi
Published: April 2010

Background and Objective. The aim of this study was to investigate the behavior of cells from epithelial rest of Malassez (ERM) against stretching force.Material and Methods. ERM-cultured cells were stretched for 1 hour, at the cycle of 18% elongation for 1 second followed by 1-second relaxation. The cells without addition of stretching force were used as controls. The cells were observed by immunohistochmical staining using actin 0, 12, 24, 36, 48, and 72 hours. Furthermore, expressions of HSP70-, VEGF-, and OPN-mRNAs of cells were also evaluated using quantitative RT-PCR.Results. Actin filaments were randomly orientated in the cytoplasm in the control group, whereas in the stretching group, actin filaments were orientated comparatively parallel to the stretching direction. Expression of HSP70-mRNA in the stretching group was significantly higher than that of control group at 12, 24, 36 hours (P<.05). Expression of VEGF-mRNA in the stretching group was significantly higher than that of control group at 24, 36, 48, and 72 hours (P<.05). Expression of OPN-mRNA in the stretching group was significantly higher than that of control group at 12 and 24 hours (P<.05).Conclusion. ERM cells response against the stretching force by expressing HSP70, VEGF, and OPN.
Complete Convergence for Weighted Sums of ρ∗‐Mixing Random Variables
Authors: Sung Soo Hak
Published: April 2010

We obtain the complete convergence for weighted sums of ρ∗‐mixing random variables. Our result extends the result of Peligrad and Gut (1999) on unweighted average to a weighted average under a mild condition of weights. Our result also generalizes and sharpens the result of An and Yuan (2008).
Neonatal Macrosomia Is an Independent Risk Factor for Adult Metabolic Syndrome
Authors: Hermann Gregory M. , Dallas Lindsay M. , Haskell Sarah E. , Roghair Robert D.
Published: April 2010

Background: Weight in infancy correlates with risk of type 2 diabetes, hypertension, and obesity in adulthood. Clinical observations have been confounded by obesity-prone genotypes and obesity-linked lifestyles. Objectives: To define the effects of isolated neonatal macrosomia in isogenic animals, we compared macrosomic and control C57Bl6 mice co-fostered by healthy dams receiving standard laboratory feed. Methods: Naturally occurring neonatal macrosomia was identified by a gender-specific weanling weight above the 90th percentile for the colony. Macrosomic and control mice were phenotyped in adulthood by exercise wheel, tail cuff and intraperitoneal insulin or glucose challenge. Results: Compared to control males, adult males with a history of neonatal macrosomia had significantly increased body weight, reduced voluntary activity, insulin resistance, fasting hyperinsulinemia, and impaired glucose tolerance. In contrast, adult females with neonatal macrosomia had no significant alteration in body weight or endocrine phenotypes, but did have higher blood pressures and lower heart rates than control females. After these baseline studies, all mice were switched to a hypercaloric, high fat diet (5 kcal/g, 45% of energy as fat). Twenty weeks later, male mice had impaired glucose tolerance and insulin resistance, independent of their weanling weight classification. While on high fat feeds, macrosomic males maintained a significantly higher body weight than control males. Conclusions: We conclude that (1) in our murine model, neonatal macrosomia is an independent risk factor of adult metabolic syndrome, and (2) neonatal macrosomia accentuates the sexually dimorphic predisposition of C57Bl6 male mice towards glucose intolerance and C57Bl6 female mice towards hypertension.
Towards a Noninvasive Approach to Malaria Diagnosis: Detection of Parasite DNA in Body Secretions and Surface Mucosa
Authors: A-Elgayoum Salwa M.E. , El-Rayah El-Amin , Giha Hayder A.
Published: April 2010

Invasive procedures for diagnostic or therapeutic purposes bear a relative risk of transmission of serious blood-borne infectious disease. In this study, a noninvasive approach to malaria diagnosis using polymerase chain reaction (PCR) for the detection of parasite DNA in saliva, buccal mucosa and urine (alternative samples) was examined. Saliva, buccal mucosa and urine samples were collected simultaneously with blood samples from 93 patients with microscopically confirmed Plasmodium falciparum infection. Species-specific primers detected the parasite DNA only in blood samples. However, when the PCR analysis was repeated using MSP1 and MSP2 primers in a subgroup of 21 complete sets of samples, the parasite DNA was detected in all except 3 samples, which were found to be negative with the MSP2 primers. Parasite density, body temperature or patient age did not influence the PCR results. In conclusion, P. falciparum parasite DNA was detected equally in saliva, buccal mucosa and urine of malaria patients, regardless of their ages, body temperatures or parasite density. Surprisingly, the parasite DNA was not amplified by species-specific primers in the alternative samples whereas it was in the blood samples.