bluuebunny/crossref_metadata_2025 huggingface dataset:
prtH2
, Not
prtH
, Is the Ubiquitous Cell Wall Proteinase Gene in
Lactobacillus
helveticus
Authors: Genay M. , Sadat L. , Gagnaire V. , Lortal S.
Published: March 2009

Lactobacillus
helveticus
strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of
L
.
helveticus
, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of
L
.
helveticus
isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the
prtH
and
prtH2
genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The
prtH2
gene was ubiquitous in the 29 strains of
L
.
helveticus
studied, whereas only 18 of them also exhibited the
prtH
gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by
L
.
helveticus
at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening.
Suppression of the Bacterial Spot PathogenXanthomonas euvesicatoriaon Tomato Leaves by an Attenuated Mutant ofXanthomonas perforans
Authors: Hert A. P. , Marutani M. , Momol M. T. , Roberts P. D. , Olson S. M. , Jones J. B.
Published: March 2009

A bacteriocin-producing strain of the bacterial spot of tomato plant pathogen,Xanthomonas perforans, with attenuated pathogenicity was deployed for biocontrol of a bacteriocin-sensitive strain of the genetically closely related bacterial spot of tomato plant pathogen,X.euvesicatoria. The attenuated mutant (91-118ΔopgHΔbcnB) ofX.perforanswas tested in leaf tissue and shown to significantly inhibit internal populations of the wild-typeX.euvesicatoriastrain although significantly less than the wild-type 91-118 strain, whereas in a phyllosphere inhibition assay, the mutant strain reduced epiphytic populations comparably to 91-118. Thus, the attenuated mutant limited the sensitive bacterium more efficiently on the leaf surface than inside the leaf. In field experiments, weekly application of 91-118ΔopgHΔbcnBsignificantly reducedX.euvesicatoriapopulations compared to the growers’ standard control (copper hydroxide and mancozeb applied weekly and acibenzolar-S-methyl applied every 2 weeks). The biological control agent, 91-118ΔopgHΔbcnB, applied every 2 weeks also significantly reducedX.euvesicatoriapopulations in one season but was not significantly different from the growers’ standard control. Potentially, attenuated pathogenic strains could be deployed as biological control agents in order to improve disease control of foliar plant pathogens.
Characterization of a New Plasmid-Like Prophage in a Pandemic
Vibrio parahaemolyticus
O3:K6 Strain
Authors: Lan Shih-Feng , Huang Chung-Ho , Chang Chuan-Hsiung , Liao Wei-Chao , Lin I-Hsuan , Jian Wan-Neng , Wu Yueh-Gin , Chen Shau-Yan , Wong Hin-chung
Published: March 2009

Vibrio parahaemolyticus
is a common food-borne pathogen that is normally associated with seafood. In 1996, a pandemic O3:K6 strain abruptly appeared and caused the first pandemic of this pathogen to spread throughout many Asian countries, America, Europe, and Africa. The role of temperate bacteriophages in the evolution of this pathogen is of great interest. In this work, a new temperate phage, VP882, from a pandemic O3:K6 strain of
V
.
parahaemolyticu
s was purified and characterized after mitomycin C induction. VP882 was a
Myoviridae
bacteriophage with a polyhedral head and a long rigid tail with a sheath-like structure. It infected and lysed high proportions of
V
.
parahaemolyticus
,
Vibrio vulnificus
, and
Vibrio cholerae
strains. The genome of phage VP882 was sequenced and was 38,197 bp long, and 71 putative open reading frames were identified, of which 27 were putative functional phage or bacterial genes. VP882 had a linear plasmid-like genome with a putative protelomerase gene and cohesive ends. The genome does not integrate into the host chromosome but was maintained as a plasmid in the lysogen. Analysis of the reaction sites of the protelomerases in different plasmid-like phages revealed that VP882 and ΦHAP-1 were highly similar, while N15, ΦKO2, and PY54 made up another closely related group. The presence of DNA adenine methylase and quorum-sensing transcriptional regulators in VP882 may play a specific role in this phage or regulate physiological or virulence-associated traits of the hosts. These genes may also be remnants from the bacterial chromosome following transduction.
Activation and Transfer of the Chromosomal Phage Resistance Mechanism AbiV in
Lactococcus lactis
Authors: Haaber Jakob , Moineau Sylvain , Hammer Karin
Published: March 2009

AbiV is a chromosomally encoded phage resistance mechanism that is silent in the wild-type phage-sensitive strain
Lactococcus lactis
subsp.
cremoris
MG1363. Spontaneous phage-resistant mutants of
L. lactis
MG1363 were analyzed by reverse transcriptase PCR and shown to express AbiV. This expression was related to a reorganization in the upstream region of
abiV
. Transfer of
abiV
between two lactococcal strains, most likely by conjugation, was also demonstrated. To our knowledge, this is the first report of natural transfer of a chromosomally encoded phage resistance mechanism.
Candicidin Biosynthesis Gene Cluster Is Widely Distributed among
Streptomyces
spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord, Norway
Authors: Jørgensen Hanne , Fjærvik Espen , Hakvåg Sigrid , Bruheim Per , Bredholt Harald , Klinkenberg Geir , Ellingsen Trond E. , Zotchev Sergey B.
Published: March 2009

A large number of
Streptomyces
bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (
can
). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with
can
-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained
can
genes. The majority of the isolates studied contained large plasmids, and two
can
-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the
can
genes and candicidin production, while mating of the “cured” strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete
can
gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.
Dual Role of the Oligopeptide Permease Opp3 during Growth of
Staphylococcus aureus
in Milk
Authors: Borezée-Durant Elise , Hiron Aurelia , Piard Jean-Christophe , Juillard Vincent
Published: March 2009

Staphylococcus aureus
RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases.
Fusobacterium nucleatum
ATCC 10953 Requires
Actinomyces naeslundii
ATCC 43146 for Growth on Saliva in a Three-Species Community That Includes
Streptococcus oralis
34
Authors: Periasamy Saravanan , Chalmers Natalia I. , Du-Thumm Laurence , Kolenbrander Paul E.
Published: March 2009

Formation of dental plaque is a developmental process involving initial and late colonizing species that form polymicrobial communities. Fusobacteria are the most numerous gram-negative bacteria in dental plaque, but they become prevalent after the initial commensal colonizers, such as streptococci and actinomyces, have established communities. The unusual ability of these bacteria to coaggregate with commensals, as well as pathogenic late colonizers, has been proposed to facilitate colonization by the latter organisms. We investigated the integration of
Fusobacterium nucleatum
into multispecies communities by employing two in vitro models with saliva as the sole nutritional source. In flow cell biofilms, numbers of cells were quantified using fluorescently conjugated antibodies against each species, and static biofilms were analyzed by quantitative real-time PCR (q-PCR) using species-specific primers. Unable to grow as single-species biofilms,
F. nucleatum
grew in two-species biofilms with
Actinomyces naeslundii
but not with
Streptococcus oralis
. However, enhanced growth of fusobacteria was observed in three-species biofilms, indicating that there was multispecies cooperation. Importantly, these community dynamics yielded an 18-fold increase in the
F. nucleatum
biomass between 4 h and 18 h in the flow cell inoculated with three species. q-PCR analysis of static biofilms revealed that maximum growth of the three species occurred at 24 h to 36 h. Lower numbers of cells were observed at 48 h, suggesting that saliva could not support higher cell densities as the sole nutrient. Integration of
F. nucleatum
into multispecies commensal communities was evident from the interdigitation of fusobacteria in coaggregates with
A. naeslundii
and
S. oralis
and from the improved growth of fusobacteria, which was dependent on the presence of
A. naeslundii
.
Microtubule-Nucleus Interactions in
Dictyostelium discoideum
Mediated by Central Motor Kinesins
Authors: Tikhonenko Irina , Nag Dilip K. , Robinson Douglas N. , Koonce Michael P.
Published: March 2009

Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division.
Dictyostelium discoideum
contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase.
kif9
null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in
kif9

−

cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.
Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast
Schizosaccharomyces pombe
Authors: Luo Jun , Matsuo Yasuhiro , Gulis Galina , Hinz Haylee , Patton-Vogt Jana , Marcus Stevan
Published: March 2009

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast
Schizosaccharomyces pombe
, we have characterized three predicted genes in this organism, designated
psd1
,
psd2
, and
psd3
, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms.
S. pombe
mutants carrying deletions in any one or two
psd
genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three
psd
genes (
psd1-3
Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the
psd1
to
psd3
gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to
psd1-3
Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for
S. pombe
cell growth.
psd1-3
Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in
S. pombe
.
psd1-3Δ
cells appear morphologically indistinguishable from wild-type
S. pombe
cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in
S. pombe
, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.
Altering Sphingolipid Metabolism in
Saccharomyces cerevisiae
Cells Lacking the Amphiphysin Ortholog Rvs161 Reinitiates Sugar Transporter Endocytosis
Authors: Morgan Jeanelle , McCourt Paula , Rankin Lauren , Swain Evelyn , Rice Lyndi M. , Nickels Joseph T.
Published: March 2009

Amphiphysins are proteins thought to be involved in synaptic vesicle endocytosis. Amphiphysins share a common BAR domain, which can sense and/or bend membranes, and this function is believed to be essential for endocytosis.
Saccharomyces cerevisiae
cells lacking the amphiphysin ortholog Rvs161 are inviable when starved for glucose. Altering sphingolipid levels in
rvs161
cells remediates this defect, but how lipid changes suppress remains to be elucidated. Here, we show that the sugar starvation-induced death of
rvs161
cells extends to other fermentable sugar carbon sources, and the loss of sphingolipid metabolism suppresses these defects. In all cases,
rvs161
cells respond to the starvation signal, elicit the appropriate transcriptional response, and properly localize the requisite sugar transporter(s). However, Rvs161 is required for transporter endocytosis.
rvs161
cells accumulate transporters at the plasma membrane under conditions normally resulting in their endocytosis and degradation. Transporter endocytosis requires the endocytosis (
endo
) domain of Rvs161. Altering sphingolipid metabolism by deleting the very-long-chain fatty acid elongase
SUR4
reinitiates transporter endocytosis in
rvs161
and
rvs161 endo
−
cells. The sphingolipid-dependent reinitiation of endocytosis requires the ubiquitin-regulating factors Doa1, Doa4, and Rsp5. In the case of Doa1, the phospholipase A
2
family ubiquitin binding motif is dispensable. Moreover, the conserved AAA-ATPase Cdc48 and its accessory proteins Shp1 and Ufd1 are required. Finally,
rvs161
cells accumulate monoubiquitin, and this defect is remediated by the loss of
SUR4
. These results show that defects in sphingolipid metabolism result in the reinitiation of ubiquitin-dependent sugar transporter endocytosis and suggest that this event is necessary for suppressing the nutrient starvation-induced death of
rvs161
cells.
Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe
Authors: Bagar Tanja , Altenbach Kirsten , Read Nick D. , Benčina Mojca
Published: March 2009

A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of
Aspergillus niger
. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in
Aspergillus.
Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 μm of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with
N
-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide
m
-chlorophenylhydrazone or cyanide
p
-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.
Nested Genes
CDA12
and
CDA13
Encode Proteins Associated with Membrane Trafficking in the Ciliate
Tetrahymena thermophila
Authors: Zweifel Erica , Smith Joshua , Romero Daniel , Giddings Thomas H. , Winey Mark , Honts Jerry , Dahlseid Jeff , Schneider Brent , Cole Eric S.
Published: March 2009

We describe a novel pair of nested genes,
CDA12
and
CDA13
, from
Tetrahymena thermophila
. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense
CDA12
RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense
CDA13
RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate
T. thermophila
. We also highlight an unusual aspect of genome organization in
Tetrahymena
, namely, the existence of nested, antisense genes.
A Comprehensive Survey of Single Nucleotide Polymorphisms (SNPs) across
Mycobacterium bovis
Strains and
M. bovis
BCG Vaccine Strains Refines the Genealogy and Defines a Minimal Set of SNPs That Separate Virulent
M. bovis
Strains and
M. bovis
BCG Strains
Authors: Garcia Pelayo M. Carmen , Uplekar Swapna , Keniry Andrew , Mendoza Lopez Pablo , Garnier Thierry , Nunez Garcia Javier , Boschiroli Laura , Zhou Xiangmei , Parkhill Julian , Smith Noel , Hewinson R. Glyn , Cole Stewart T. , Gordon Stephen V.
Published: March 2009

To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine
Mycobacterium bovis
BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of
Mycobacterium bovis
and
M. bovis
BCG. SNPs were assayed in
M. bovis
isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent
M. bovis
strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent
M. bovis
strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent
M. bovis
strains and the attenuated BCG strain that will underpin future functional analyses.
Whole-Genome Tiling Array Analysis of
Mycobacterium leprae
RNA Reveals High Expression of Pseudogenes and Noncoding Regions
Authors: Akama Takeshi , Suzuki Koichi , Tanigawa Kazunari , Kawashima Akira , Wu Huhehasi , Nakata Noboru , Osana Yasunori , Sakakibara Yasubumi , Ishii Norihisa
Published: March 2009

Whole-genome sequence analysis of
Mycobacterium leprae
has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some
M. leprae
pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the
M. leprae
genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with
M. leprae
RNA from the SHR/NCrj-
rnu
nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the
M. leprae
unique repetitive sequence RLEP and other sequences without any homology to known functional noncoding RNAs. Although the biological functions of RNA transcribed from
M. leprae
pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that
M. leprae
will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.
Identification and Characterization of Inorganic Pyrophosphatase and PAP Phosphatase from
Thermococcus onnurineus
NA1
Authors: Lee Hyun Sook , Kim Yun Jae , Lee Jung-Hyun , Kang Sung Gyun
Published: March 2009

Two hypothetical genes were functionally verified to be a pyrophosphatase and a PAP phosphatase in
Thermococcus onnurineus
NA1. This is the first report of the pyrophosphatases and the PAP phosphatases being organized in the gene clusters of the sulfate activation system only in
T. onnurineus
NA1 and “
Pyrococcus abyssi
.”
Competence for Genetic Transformation in
Streptococcus pneumoniae
: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW
Authors: Piotrowski Andrew , Luo Ping , Morrison Donald A.
Published: March 2009

Competence for genetic transformation in
Streptococcus pneumoniae
is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes,
comX
and
comW
, and 15 other “early” genes. ComX, a transient alternative sigma factor, drives transcription of “late” genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.
Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,
Mycoplasma gallisepticum
, Revealed by Negative-Staining Electron Microscopy
Authors: Nakane Daisuke , Miyata Makoto
Published: March 2009

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion.
Mycoplasma gallisepticum
, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of
M. gallisepticum
cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species,
Mycoplasma pneumoniae
. Next, to elucidate the cytoskeletal structure of
M. gallisepticum
, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of
M. gallisepticum
were compared with those of
M. pneumoniae
in detail, and the possible protein components of these structures were considered.
Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in
Enterococcus faecalis
Authors: Kline Kimberly A. , Kau Andrew L. , Chen Swaine L. , Lim Adeline , Pinkner Jerome S. , Rosch Jason , Nallapareddy Sreedhar R. , Murray Barbara E. , Henriques-Normark Birgitta , Beatty Wandy , Caparon Michael G. , Hultgren Scott J.
Published: March 2009

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in
Enterococcus faecalis
. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in
E. faecalis
were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.
Occupational exposure to potential endocrine disruptors: further development of a job exposure matrix
Authors: Brouwers M M , van Tongeren M , Hirst A A , Bretveld R W , Roeleveld N
Published: March 2009

Objectives:

The aim was to develop a new up-to-date and comprehensive job exposure matrix (JEM) for estimating exposure to potential endocrine disruptors in epidemiological research.

Methods:

Chemicals with endocrine disrupting properties were identified from the literature and classified into 10 chemical groups: polycyclic aromatic hydrocarbons (PAHs), polychlorinated organic compounds, pesticides, phthalates, organic solvents, bisphenol A, alkylphenolic compounds, brominated flame retardants, metals and a miscellaneous group. Most chemical groups were divided into three to six subgroups. Focusing on the years 1996–2006, three experts scored the probability of exposure to each chemical group and subgroup for 353 job titles as “unlikely” (0), “possible” (1) or “probable” (2). Job titles with positive exposure probability scores were provided with exposure scenarios that described the reasoning behind the scores.

Results:

Exposure to any chemical group was unlikely for 238 job titles (67%), whereas 102 (29%) job titles were classified as possibly (17%) or probably (12%) exposed to one or several endocrine disruptors. The remaining 13 job titles provided too little information to classify exposure. PAHs, pesticides, phthalates, organic solvents, alkylphenolic compounds and metals were often linked to a job title in the JEM. The remaining chemical groups were found to involve very few occupations.

Conclusions:

Despite some important limitations, this JEM could be a valuable tool for exposure assessment in studies on the health risks of endocrine disruptors, especially when task specific information is incorporated. The documented exposure scenarios are meant to facilitate further adjustments to the JEM to allow more widespread use.
The impact of growth history and flagellation on the adhesion of variousListeria monocytogenesstrains to polystyrene
Authors: Tresse Odile , Lebret Véronique , Garmyn Dominique , Dussurget Olivier
Published: March 2009

The contribution of growth history and flagella to adhesion of Listeria monocytogenes was analysed. An in-frame deletion on the flagellin encoding gene (flaA) was performed in L. monocytogenes EGD-e to compare its adhesion ability with the parental strain, after cultivation at various pH values and temperatures. The pH, as well as the temperature, affected the adhesion of L. monocytogenes EGD-e. In addition, the adhesion of L. monocytogenes EGD-e was reduced in energy-depressed cells. Conversely, the physicochemical bacterial surface characteristics affected by growth history did not influence the adhesion. Adhesion variations observed among environmental and clinical strains was attributed to the flagella. The naturally aflagellated strains resulted in an adhesion capacity similar to that observed for mutants and parental strains cultivated under flagellum expression repressing conditions. However, L. monocytogenes is able to adhere to inert surfaces through a residual adhesion process without flagella. All these observations emphasize the importance to consider the food environmental factors in the risk assessment of L. monocytogenes in food industry.
Wintering strategies by mountain goats in interior mountains
Authors: Poole Kim G. , Stuart-Smith Kari , Teske Irene E.
Published: March 2009

As with many ungulates inhabiting areas with potentially deep snow, winter is an important season for mountain goats ( Oreamnos americanus (de Blainville, 1816)) and is characterized by restricted movements and high juvenile mortality. We examined winter habitat selection and wintering strategies by mountain goats in two adjacent areas of southeastern British Columbia characterized by deep, moist snow and by shallow, dry snow. Fifteen GPS collars were placed on mountain goats in each area over two winters. Winter-range size did not differ between areas and comprised, on average, 2.2%–7.4% of male home range and 8.0%–14.1% of female home range. Topographic variables dominated habitat model selection. At the broad scale, mountain goats in both areas selected winter ranges closer to escape terrain on warmer aspects that contained lesser amounts of mature dense forest. At the fine scale, mountain goats in both areas selected rugged habitat at upper mid-elevations and on warmer aspects. Alpine areas were avoided in the deep snow area and selected in the shallow snow area. No selection for mature forests was observed in either area. Mountain goats, therefore, appeared to utilize open, high-elevation habitats in shallow snow zones, but they did not seek reduced snow levels in mature forest stands in deep snow areas.
ASYMPTOTIC DERIVATION OF THE SECTION-AVERAGED SHALLOW WATER EQUATIONS FOR NATURAL RIVER HYDRAULICS
Authors: DECOENE ASTRID , BONAVENTURA LUCA , MIGLIO EDIE , SALERI FAUSTO
Published: March 2009

The section-averaged shallow water model usually applied in river and open channel hydraulics is derived by an asymptotic analysis that accounts for terms up to second order in the vertical/longitudinal length ratio, starting from the three-dimensional Reynolds-averaged Navier–Stokes equations for incompressible free surface flows. The derivation is carried out under quite general assumptions on the geometry of the channel, thus allowing for the application of the resulting equations to natural rivers with arbitrarily shaped cross sections. As a result of the derivation, a generalized friction term is obtained, that does not rely on local uniformity assumptions and that can be computed directly from three-dimensional turbulence models, without need for local uniformity assumptions. The modified equations including the novel friction term are compared to the classical Saint Venant equations in the case of steady state open channel flows, where analytic solutions are available, showing that the solutions resulting from the modified equation set are much closer to the three-dimensional solutions than those of the classical equation set. Furthermore, it is shown that the proposed formulation yields results that are very similar to those obtained with empirical friction closures widely applied in computational hydraulics. The generalized friction term derived therefore justifies a posteriori these empirical closures, while allowing to avoid the assumptions on local flow uniformity on which these closures rely.
Contribution of nerve growth factor to augmented TRPV1 responses of muscle sensory neurons by femoral artery occlusion
Authors: Xing Jihong , Lu Jian , Li Jianhua
Published: March 2009

In rats, hindlimb muscle ischemia induced by femoral artery occlusion augments the sympathetic nervous response to stimulation of transient receptor potential vanilloid type 1 (TRPV1) by injection of capsaicin into the arterial blood supply of the hindlimb muscles. The enhanced sympathetic response is due to alterations in TRPV1 receptor expression and its responsiveness in sensory neurons. The underlying mechanism by which TRPV1 receptor responses are increased after muscle vascular insufficiency/ischemia is unclear. In this report we tested the hypothesis that muscle ischemia elevates nerve growth factor (NGF) levels in primary afferent neurons, thereby increasing TRPV1 responsiveness. Muscle vascular insufficiency induced by the femoral artery ligation significantly increased NGF in the dorsal root ganglion (DRG) compared with sham controls. Furthermore, when NGF was infused in the hindlimb muscles of healthy rats (72 h using an osmotic minipump), the magnitude of the DRG neuron response to capsaicin was augmented (5.4 ± 0.54 nA with NGF infusion vs. 3.0 ± 0.17 nA in control; P < 0.05). With the addition of NGF in the culture dish containing the DRG neurons, the magnitude of the DRG neuron response to capsaicin was greater (6.4 ± 0.27 nA; P < 0.05 vs. control) than that seen in control (2.9 ± 0.16 nA). Note that this NGF effect was seen in isolectin B4-negative DRG neurons, a group of thin fiber nerves that contain neuropeptides and depend on NGF for survival. These data suggest that NGF affects a selective subpopulation of the afferent neurons in mediating augmented TRPV1 responses after femoral artery occlusion.
Acidosis and ischemia increase cellular Ca2+transient alternans and repolarization alternans susceptibility in the intact rat heart
Authors: Kapur Sunil , Wasserstrom J. Andrew , Kelly James E. , Kadish Alan H. , Aistrup Gary L.
Published: March 2009

Cardiac cellular Ca2+transient (CaT) alternans and electrocardiographic T-wave alternans (TWA) often develop in myocardial ischemia, but the mechanisms for this relationship have not been elucidated. Acidosis is a major component of ischemia, but there is no direct evidence linking acidosis-induced cellular CaT alternans to ischemia-induced CaT alternans and TWA in whole heart. We used laser-scanning confocal microscopy to measure intracellular Ca2+(Cai2+) cycling in individual myocytes of fluo-4 AM-loaded rat hearts and simultaneously recorded pseudo-ECGs to investigate changes in CaTs and late-phase repolarization, respectively, during baseline and rapid pacing under control and either globally acidic or globally ischemic conditions. Acidosis (hypercapnia; pH 6.6) increased diastolic Cai2+levels, prolonged CaT duration, and shifted to slower heart rates both the development of pacing-induced acidosis-induced CaT alternans (both concordant and discordant) and of repolarization alternans (RPA, a measure of TWA in rat ECGs). The magnitudes of these shifts were equivalent for both CaT alternans and RPA, suggesting a close association between them. Nearly identical results were found in low-flow global ischemia. Additionally, ischemic preconditioning reduced the increased propensity for CaT alternans and RPA development and was mimicked by preconditioning by acidosis alone. Our results demonstrate that global acidosis or ischemia modifies Cai2+cycling in myocytes such that the diastolic Cai2+rises and the cellular CaT duration is prolonged, causing spatially concordant as well as spatially discordant cellular CaT alternans to develop at slower heart rates than in controls. Since RPA also developed at slower heart rates, our results suggest that acidosis is a major contributor to CaT alternans, which underlies the proarrhythmic state induced by myocardial ischemia and therefore may play a role in its modulation and prevention.
Antidiabetic drug pioglitazone protects the heart via activation of PPAR-γ receptors, PI3-kinase, Akt, and eNOS pathway in a rabbit model of myocardial infarction
Authors: Yasuda Shinji , Kobayashi Hiroyuki , Iwasa Masamitsu , Kawamura Itta , Sumi Shouhei , Narentuoya Bao , Yamaki Takahiko , Ushikoshi Hiroaki , Nishigaki Kazuhiko , Nagashima Kenshi , Takemura Genzou , Fujiwara Takako , Fujiwara Hisayoshi , Minatoguchi Shinya
Published: March 2009

The insulin-sensitizing drug pioglitazone has been reported to be protective against myocardial infarction. However, its precise mechanism is unclear. Rabbits underwent 30 min of coronary occlusion followed by 48 h of reperfusion. Rabbits were assigned randomly to nine groups ( n = 10 in each): the control group (fed a normal diet), pioglitazone group (fed diets containing 1 mg·kg−1·day−1 pioglitazone), pioglitazone + 5-hydroxydecanoic acid (HD) group [fed the pioglitazone diet + 5 mg/kg iv 5-HD, a mitochondrial ATP-sensitive K+ (KATP) channel blocker], pioglitazone + GW9662 group [fed the pioglitazone diet + 2 mg/kg iv GW9662, a peroxisome proliferator activated receptor (PPAR)-γ antagonist], GW9662 group (fed a normal diet + iv GW9662), pioglitazone + wortmannin group [fed the pioglitazone diet + 0.6 mg/kg iv wortmannin, a phosphatidylinositol (PI)3-kinase inhibitor], wortmannin group (fed a normal diet + iv wortmannin), pioglitazone + nitro-l-arginine methyl ester (l-NAME) group [fed the pioglitazone diet + 10 mg/kg iv l-NAME, a nitric oxide synthase (NOS) inhibitor], and l-NAME group (fed a normal diet + iv l-NAME). All groups were fed the diets for 7 days. The risk area and nonrisk area of the left ventricle (LV) were separated by Evans blue dye, and the infarct area was determined by triphenyltetrazolium chloride staining. The infarct size was calculated as a percentage of the LV risk area. Western blotting was performed to assess levels of Akt and phospho-Akt and phospho-endothelial NOS (eNOS) in the myocardium following reperfusion. The infarct size was significantly smaller in the pioglitazone group (21 ± 2%) than in the control group (43 ± 3%). This effect was abolished by GW9662 (42 ± 3%), wortmannin (40 ± 3%), or l-NAME (42 ± 7%) but not by 5-HD (24 ± 5%). Western blotting showed higher levels of phospho-Akt and phospho-eNOS in the pioglitazone group. Pioglitazone reduces the myocardial infarct size via activation of PPAR-γ, PI3-kinase, Akt, and eNOS pathways, but not via opening the mitochondrial KATP channel. Pioglitazone may be a novel strategy for the treatment of diabetes mellitus with coronary artery disease.
Prenatal cocaine exposure abolished ischemic preconditioning-induced protection in adult male rat hearts: role of PKCε
Authors: Meyer Kurt D. , Zhang Haitao , Zhang Lubo
Published: March 2009

Prenatal cocaine exposure in rats resulted in decreased PKCε protein expression in the heart of adult male but not female offspring. The present study determined its functional consequence of inhibiting cardioprotection mediated by ischemic preconditioning. Pregnant Sprague-Dawley rats were administered intraperitoneally saline or cocaine (30 mg·kg−1·day−1) from day 15 to day 21 of gestational age. Hearts were isolated from 3-mo-old offspring and were subjected to ischemia and reperfusion injury in a Langendorff preparation, with or without prior ischemic preconditioning. Preischemic values of left ventricular function were the same between the saline control and cocaine-treated animals. Ischemic preconditioning of two episodes of 5-min ischemia significantly decreased infarct size and enhanced postischemic functional recovery of the left ventricle in the saline control animals. This ischemic preconditioning was associated with increased phospho-PKCε, but not phospho-PKCδ, levels and was blocked by a PKCε translocation inhibitor peptide. Prenatal cocaine treatment abolished the ischemic preconditioning-mediated increase in phospho-PKCε and cardioprotection in the heart of male offspring. In contrast, the cardioprotective effect was fully maintained in female offspring that were exposed to cocaine before birth. The results suggest that prenatal cocaine exposure causes a sex-specific loss of cardioprotection by ischemic preconditioning in adult offspring, which is most likely due to fetal programming of PKCε gene repression, resulting in a downregulation of PKCε function in the heart of adult male offspring.
Angiotensin II upregulates hypothalamic AT1receptor expression in rats via the mitogen-activated protein kinase pathway
Authors: Wei Shun-Guang , Yu Yang , Zhang Zhi-Hua , Felder Robert B.
Published: March 2009

ANG II type 1 receptors (AT1R) mediate most of the central effects of ANG II on cardiovascular function, fluid homeostasis, and sympathetic drive. The mechanisms regulating AT1R expression in the brain are unknown. In some tissues, the AT1R can be upregulated by prolonged exposure to ANG II. We examined the hypothesis that ANG II upregulates the AT1R in the brain by stimulating the intracellular mitogen-activated protein kinase (MAPK) signaling pathway. Using molecular and immunochemical approaches, we examined expression of the AT1R and phosphorylated MAPK in the paraventricular nucleus of the hypothalamus (PVN) and the subfornical organ (SFO) of rats receiving a chronic (4-wk) subcutaneous infusion of ANG II (0.6 μg/h) or saline (vehicle control), with or without concomitant (4-wk) intracerebroventricular (ICV) infusions of MAPK inhibitors or the AT1R blocker losartan. Subcutaneous infusion of ANG II markedly increased phosphorylation of MAPK and expression of AT1R mRNA and protein and AT1R-like immunoreactivity in the PVN and SFO. ANG II-induced AT1R expression was blocked by ICV infusion of the p44/42 MAPK inhibitor PD-98059 (0.025 μg/h) and the JNK inhibitor SP-600125 (0.125 μg/h), but not by the p38 MAPK inhibitor SB-203580 (0.125 μg/h). Upregulation of the AT1R in the PVN and SFO by peripheral ANG II was abolished by ICV losartan (10 μg/h). The data indicate that blood-borne ANG II upregulates brain AT1R by activating intracellular p44/42 MAPK and JNK signaling pathways.
Sex-dependent impairment of cardiac action potential conduction in type 1 diabetic rats
Authors: Shimoni Yakhin , Emmett Teresa , Schmidt Robyn , Nygren Anders , Kargacin Gary
Published: March 2009

The incidence of diabetes mellitus is increasing. Cardiac dysfunction often develops, resulting in diverse arrhythmias. These arise from ion channel remodeling or from altered speed and pattern of impulse propagation. Few studies have investigated impulse propagation in the diabetic heart. We previously showed a reduced conduction reserve in the diabetic heart, with associated changes in intercellular gap junctions. The present study investigated whether these effects are sex specific. Hearts from control and streptozotocin-diabetic male and female rats were used. Optical mapping was performed with the voltage-sensitive dye di-4-ANEPPS, using Langendorff-perfused hearts. Isolated ventricular cells and tissue sections were used for immunofluorescent labeling of the gap junction protein connexin43 (Cx43). The gap junction uncoupler heptanol (0.75 mM) or elevated K+(9 mM, to reduce cell excitability) produced significantly greater slowing of propagation in diabetic males than females. In ovariectomized diabetic females, 9 mM K+slowed conduction significantly more than in nonovariectomized females. The subcellular redistribution (lateralization) of the gap junction protein Cx43 was smaller in diabetic females. Pretreatment of diabetic males with the angiotensin-converting enzyme inhibitor quinapril reduced Cx43 lateralization and the effects of 9 mM K+on propagation. In conclusion, the slowing of cardiac impulse propagation in type 1 diabetes is smaller in female rats, partly due to the presence of female sex hormones. This difference is (partly) mediated by sex differences in activation of the cardiac renin-angiotensin system.