bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in
Mortierella alpina
1S-4
Authors: Zhang Shuo , Sakuradani Eiji , Shimizu Sakayu
Published: January 2007

Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus
Mortierella alpina
1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ
7SR
, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from
Xenopus laevis
(African clawed frog). Heterologous expression of the Mo
Δ7SR
gene in yeast
Saccharomyces cerevisiae
revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of Mo
Δ7SR
gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in
M. alpina
1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.
Microbial Diversity within Early-Stage CulturedPanulirus ornatusPhyllosomas
Authors: Payne Matthew S. , Hall Mike R. , Sly Lindsay , Bourne David G.
Published: January 2007

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster,Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PIIP. ornatusphyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated byThiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also containedThiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria asThiothrixsp., being present on dead phyllosomas. FISH also identifiedLeucothrixsp. andVibriosp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE).Vibriosp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population ofVibriosp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolatedVibrio harveyistrain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenicVibriosp. to cause larval mortalities.
Redirection of Metabolism for Biological Hydrogen Production
Authors: Rey Federico E. , Heiniger Erin K. , Harwood Caroline S.
Published: January 2007

A major route for hydrogen production by purple photosynthetic bacteria is biological nitrogen fixation. Nitrogenases reduce atmospheric nitrogen to ammonia with the concomitant obligate production of molecular hydrogen. However, hydrogen production in the context of nitrogen fixation is a rather inefficient process because about 75% of the reductant consumed by the nitrogenase is used to generate ammonia. In this study we describe a selection strategy to isolate strains of purple photosynthetic bacteria in which hydrogen production is necessary for growth and independent of nitrogen fixation. We obtained four mutant strains of the photosynthetic bacterium
Rhodopseudomonas palustris
that produce hydrogen constitutively, even in the presence of ammonium, a condition where wild-type cells do not accumulate detectable amounts of hydrogen. Some of these strains produced up to five times more hydrogen than did wild-type cells growing under nitrogen-fixing conditions. Transcriptome analyses of the hydrogen-producing mutant strains revealed that in addition to the nitrogenase genes, 18 other genes are potentially required to produce hydrogen. The mutations that caused constitutive hydrogen production mapped to four different sites in the NifA transcriptional regulator in the four different strains. The strategy presented here can be applied to the large number of diverse species of anoxygenic photosynthetic bacteria that are known to exist in nature to identify strains for which there are fitness incentives to produce hydrogen.
Effects of Abiotic Factors on the Phylogenetic Diversity of Bacterial Communities in Acidic Thermal Springs
Authors: Mathur Jayanti , Bizzoco Richard W. , Ellis Dean G. , Lipson David A. , Poole Alexander W. , Levine Richard , Kelley Scott T.
Published: January 2007

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E
h
of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to
Hydrogenobaculum
spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of
Hydrogenobaculum
spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.
Cdc37p Is Required for Stress-Induced High-Osmolarity Glycerol and Protein Kinase C Mitogen-Activated Protein Kinase Pathway Functionality by Interaction with Hog1p and Slt2p (Mpk1p)
Authors: Hawle Patricija , Horst Danielle , Bebelman Jan Paul , Yang Xiao Xian , Siderius Marco , van der Vies Saskia M.
Published: January 2007

The yeast
Saccharomyces cerevisiae
utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a
cdc37
-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the
cdc37
-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.
Yersiniabactin Is a Virulence Factor for
Klebsiella pneumoniae
during Pulmonary Infection
Authors: Lawlor Matthew S. , O'Connor Christopher , Miller Virginia L.
Published: January 2007

Iron acquisition systems are essential for the in vivo growth of bacterial pathogens. Despite the epidemiological importance of
Klebsiella pneumoniae
, few experiments have examined the importance of siderophores in the pathogenesis of this species. A previously reported signature-tagged mutagenesis screen identified an attenuated strain that featured an insertional disruption in
ybtQ
, which encodes a transporter for the siderophore yersiniabactin. We used this finding as a starting point to evaluate the importance of siderophores in the physiology and pathogenesis of
K. pneumoniae
. Isogenic strains carrying in-frame deletions in genes required for the synthesis of either enterobactin or yersiniabactin were constructed, and the growth of these mutants was examined both in vitro and in vivo using an intranasal infection model. The results suggest divergent functions for each siderophore in different environments, with enterobactin being more important for growth in vitro under iron limitation than in vivo and the reverse being true for the yersiniabactin locus. These observations represent the first examination of isogenic mutants in iron acquisition systems for
K. pneumoniae
and may indicate that the acquisition of nonenterobactin siderophores is an important step in the evolution of virulent enterobacterial strains.
Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice againstCryptococcus neoformansInfection
Authors: Beenhouwer David O. , Yoo Esther M. , Lai Chun-Wei , Rocha Miguel A. , Morrison Sherie L.
Published: January 2007

The encapsulated yeastCryptococcus neoformansis a significant cause of meningitis and death in patients with AIDS. Some murine monoclonal antibodies (MAbs) against the glucuronoxylomannan (GXM) component of theC. neoformanscapsular polysaccharide can prolong the lives of infected mice, while others have no effect or can even shorten survival. To date, no one has systematically compared the efficacies of antibodies with the same variable regions and different human constant regions with their unique combination of effector functions in providing protection against murineC. neoformansinfection. In the present study, we examined the efficacies of anti-GXM MAbs of the four human immunoglobulin G (IgG) subclasses, which have identical variable regions but differ in their capacities to bind the three types of Fc receptors for IgG (FcγR), their abilities to activate complement, and their half-lives. IgG2 and IgG4 anti-GXM prolonged the lives of infected BALB/c mice, IgG3 anti-GXM did not affect animal survival, while mice treated with IgG1 anti-GXM died earlier than mice treated with phosphate-buffered saline or irrelevant isotype-matched MAbs. All MAbs decreased serum GXM in infected animals. Effector pathways traditionally believed to be important in defense against microbes, such as opsonophagocytosis and complement binding, negatively correlated with antibody efficacy. It is generally accepted that human IgG1 has the most favorable combination of effector functions for therapeutic use against infections. Therefore, our findings have significant implications for humanization of the mouse IgG1 currently in clinical trials for cryptococcal meningitis and for the design of antibody therapeutics to treat other infectious diseases as well.
Native Microbial Colonization of
Drosophila melanogaster
and Its Use as a Model of
Enterococcus faecalis
Pathogenesis
Authors: Cox Christopher R. , Gilmore Michael S.
Published: January 2007

Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the
Drosophila melanogaster
microflora and examined the occurrence of enterococci in the gastrointestinal consortium of
Drosophila
. In a survey of laboratory-reared
Drosophila
and wild-captured flies, we found that
Drosophila
was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including
Enterococcus faecalis
,
Enterococcus faecium
,
Enterococcus gallinaraum
, and
Enterococcus durans
, as well as a previously detected but uncultured
Enterococcus
species.
Drosophila
could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to
Drosophila
compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the
Drosophila
GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies.
Burkholderia cenocepacia
Requires a Periplasmic HtrA Protease for Growth under Thermal and Osmotic Stress and for Survival In Vivo
Authors: Flannagan Ronald S. , Aubert Daniel , Kooi Cora , Sokol Pamela A. , Valvano Miguel A.
Published: January 2007

Burkholderia cenocepacia
, a member of the
B. cepacia
complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the
B. cenocepacia
stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including
htrA
, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPΩTp. Genetic analyses and complementation studies revealed that HtrA
BCAL2829
was required for growth of
B. cenocepacia
upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44°C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA
BCAL2829
PDZ domains demonstrated that these areas are required for protein function. HtrA
BCAL2829
also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the
htrA
gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA
BCAL2829
is a virulence factor in
B. cenocepacia
.
Mutagenesis of
hetR
Reveals Amino Acids Necessary for HetR Function in the Heterocystous Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Risser Douglas D. , Callahan Sean M.
Published: January 2007

HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium
Anabaena
sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking
hetR
on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het
−
phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type
hetR
under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a
hetR
deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het
−
phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in
Anabaena
sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.
Inferring a Population Structure for
Staphylococcus epidermidis
from Multilocus Sequence Typing Data
Authors: Miragaia M. , Thomas J. C. , Couto I. , Enright M. C. , de Lencastre H.
Published: January 2007

Despite its importance as a human pathogen, information on population structure and global epidemiology of
Staphylococcus epidermidis
is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of
S. epidermidis
isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCC
mec
) carrying the genetic determinant of methicillin resistance. The 217
S. epidermidis
isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCC
mec
was acquired at least 56 times by
S. epidermidis
. Although geographic dissemination of
S. epidermidis
strains and the value of the index of association between the alleles, 0.2898 (
P
< 0.05), support the clonality of
S. epidermidis
species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that
S. epidermidis
has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCC
mec
.
Multiple Roles of Biosurfactants in Structural Biofilm Development by
Pseudomonas aeruginosa
Authors: Pamp Sünje Johanna , Tolker-Nielsen Tim
Published: January 2007

Recent studies have indicated that biosurfactants produced by
Pseudomonas aeruginosa
play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. Through the use of flow cell technology and enhanced confocal laser scanning microscopy, we have obtained results which suggest that the biosurfactants produced by
P. aeruginosa
play additional roles in structural biofilm development. We present genetic evidence that during biofilm development by
P. aeruginosa
, biosurfactants promote microcolony formation in the initial phase and facilitate migration-dependent structural development in the later phase.
P. aeruginosa rhlA
mutants, deficient in synthesis of biosurfactants, were not capable of forming microcolonies in the initial phase of biofilm formation. Experiments involving two-color-coded mixed-strain biofilms showed that
P. aeruginosa rhlA
mutants were defective in migration-dependent development of mushroom-shaped multicellular structures in the later phase of biofilm formation. Experiments involving three-color-coded mixed-strain
P. aeruginosa
biofilms demonstrated that the wild-type and
rhlA
and
pilA
mutant strains formed distinct subpopulations on top of each other dependent on their ability to migrate and produce biosurfactants.
A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of
Bacillus subtilis
, Is Involved in Spore Germination
Authors: Masayama Atsushi , Kuwana Ritsuko , Takamatsu Hiromu , Hemmi Hisashi , Yoshimura Tohru , Watabe Kazuhito , Moriyama Ryuichi
Published: January 2007

The predicted amino acid sequence of
Bacillus subtilis ycsK
exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that
ycsK
mRNA was first detected from 4 h after the onset of sporulation and that transcription of
ycsK
was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the
ycsK
gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of
ycsK
spores in a mixture of
l
-asparagine,
d
-glucose,
d
-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in
l
-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in
Escherichia coli
showed lipolytic activity. We therefore propose that
ycsK
should be renamed
lipC
. This is the first study of a bacterial spore germination-related lipase.
The
rbmBCDEF
Gene Cluster Modulates Development of Rugose Colony Morphology and Biofilm Formation in
Vibrio cholerae
Authors: Fong Jiunn C. N. , Yildiz Fitnat H.
Published: January 2007

Vibrio cholerae
, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the
vps
genes, which are organized into
vps-
I and
vps-
II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the
vps
intergenic region, termed
rbmB
to -
F
(
r
ugosity and
b
iofilm structure
m
odulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of
rbmB
, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the
rbmC bap1
double deletion mutant and
bap1
single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the
rbmC bap1
double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of
rbmDEF
mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that
vps
intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.
Interrelationships between Colonies, Biofilms, and Planktonic Cells of
Pseudomonas aeruginosa
Authors: Mikkelsen H. , Duck Z. , Lilley K. S. , Welch M.
Published: January 2007

Pseudomonas aeruginosa
is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.
Immunohistochemical study of nuclear factor-κB activity and interleukin-8 abundance in oesophageal adenocarcinoma; a useful strategy for monitoring these biomarkers
Authors: Jenkins G J S , Mikhail J , Alhamdani A , Brown T H , Caplin S , Manson J M , Bowden R , Toffazal N , Griffiths A P , Parry J M , Baxter J N
Published: January 2007

Aims:

To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-κB (NF-κB) activity in oesophageal adenocarcinoma and pre-malignant (Barrett’s) oesophageal tissues, relative to normal oesophageal mucosa. The pro-inflammatory cytokine interleukin-8 (IL-8), a transcriptional target of NF-κB, was also studied to better understand NF-κB functionality; its RNA and protein levels were assessed in oesophageal tissues.

Methods:

IHC was employed using an antibody against the nuclear localisation sequence (NLS) of the p65 subunit as well as an antibody against IL-8. To assess NF-κB function, changes in gene expression of NF-κB controlled genes (IL-8 and I-κB) were also assessed in the histological sequence using real-time PCR. More global expression changes were also studied using membrane arrays.

Results:

IHC was effective at monitoring overall NF-κB activity and IL-8 abundance. This method also allowed NF-κB activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-κB activity and IL-8 abundance across the histological series. Gene expression analysis also showed consistent up-regulation of IL-8, confirming the IHC data and showing enhanced transcriptional NF-κB activity. I-κB (another NF-κB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-κB gene expression may partly explain increased NF-κB activity.

Conclusion:

IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-κB activity in oesophageal tissues. As IHC is amenable to high-throughput screening (whereas traditional electrophoretic mobility shift assay methods are not), this may lead to the development of a better screening tool for early cancer risk.
Nonreceptor Protein-Tyrosine Phosphatases in Immune Cell Signaling
Authors: Pao Lily I. , Badour Karen , Siminovitch Katherine A. , Neel Benjamin G.
Published: January 2007

Tyrosyl phosphorylation plays a critical role in multiple signaling pathways regulating innate and acquired immunity. Although tyrosyl phosphorylation is a reversible process, we know much more about the functions of protein-tyrosine kinases (PTKs) than about protein-tyrosine phosphatases (PTPs). Genome sequencing efforts have revealed a large and diverse superfamily of PTPs, which can be subdivided into receptor-like (RPTPs) and nonreceptor (NRPTPs). The role of the RPTP CD45 in immune cell signaling is well known, but those of most other PTPs remain poorly understood. Here, we review the mechanism of action, regulation, and physiological functions of NRPTPs in immune cell signaling. Such an analysis indicates that PTPs are as important as PTKs in regulating the immune system.
Fc Receptor-Like Molecules
Authors: Davis Randall S.
Published: January 2007

Discovery of a large family of Fc receptor-like (FCRL) molecules, homologous to the well-known receptors for the Fc portion of immunoglobulin (FCR), has uncovered an impressive abundance of immunoglobulin superfamily (IgSF) genes in the human 1q21–23 chromosomal region and revealed significant diversity for these genes between humans and mice. The observation that FCRL representatives are members of an ancient multigene family that share a common ancestor with the classical FCR is underscored by their linked genomic locations, gene structure, shared extracellular domain composition, and utilization of common cytoplasmic tyrosine-based signaling elements. In contrast to the conventional FCR, however, FCRL molecules possess diverse extracellular frameworks, autonomous or dual signaling properties, and preferential B lineage expression. Most importantly, there is no strong evidence thus far to support a role for them as Ig-binding receptors. These characteristics, in addition to their identification in malignancies and autoimmune disorders, predict a fundamental role for these receptors as immunomodulatory agents in normal and subverted B lineage cells.
Structural Basis of Integrin Regulation and Signaling
Authors: Luo Bing-Hao , Carman Christopher V. , Springer Timothy A.
Published: January 2007

Integrins are cell adhesion molecules that mediate cell-cell, cell–extracellular matrix, and cell-pathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 Å couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
Mucosal Dendritic Cells
Authors: Iwasaki Akiko
Published: January 2007

The internal surfaces of the human body are covered by distinct types of epithelial cells and mucus-secreting cells. The mucosal surfaces serve many vital functions, such as respiration (nasal passage and lung), absorption (gastrointestinal tract), excretion (lung, urinary tract, large intestine), and reproduction (reproductive tract). In performing these functions, the host is inevitably exposed to environmental antigens, food particles, commensal flora, and pathogens. Mucosal surfaces contain specialized dendritic cells (DCs) capable of sensing these external stimuli and mounting appropriate local responses depending on the nature of the elements they encounter. In the absence of pathogens, mucosal DCs either ignore the antigen or induce regulatory responses. Upon recognition of microorganisms that invade the mucosal barrier, mucosal DCs mount robust protective immunity. This review highlights progress in our understanding of how mucosal DCs process external information and direct appropriate responses by mobilizing various cells of the innate and adaptive immune systems to achieve homeostasis and protection.
Impact of acute and enduring volume overload on mechanotransduction and cytoskeletal integrity of canine left ventricular myocardium
Authors: Donker Dirk W. , Maessen Jos G. , Verheyen Fons , Ramaekers Frans C. , Spätjens Roel L. H. M. G. , Kuijpers Helma , Ramakers Christian , Schiffers Paul M. H. , Vos Marc A. , Crijns Harry J. G. M. , Volders Paul G. A.
Published: January 2007

It is poorly understood how mechanical stimuli influence in vivo myocardial remodeling during chronic hemodynamic overload. Combined quantitation of ventricular mechanics and expression of key proteins involved in mechanotransduction can improve fundamental understanding. Adult anesthetized dogs ( n = 20) were studied at sinus rhythm (SR) and 0, 3, 10, and 35 days of complete atrioventricular block (AVB). Serial left ventricular (LV) myofiber mechanics were measured. Repeated LV biopsies were analyzed for mRNA and/or protein expression of β1D-integrin, melusin, Akt, GSK3β, muscle LIM protein (MLP), four-and-a-half LIM protein 2 (fhl2), desmin, and calpain. Upon AVB, increased ejection strain (0.29 ± 0.01 vs. 0.13 ± 0.02, SR) and end-diastolic stress (4.8 ± 1.1 vs. 2.7 ± 0.4 kPa) dominated mechanical changes. Brain natriuretic peptide plasma levels were correspondingly high (33 ± 4 vs. 19 ± 1 pg/ml, SR). β1D-Integrin protein expression increased chronically after AVB. Melusin was temporarily overexpressed (+33 ± 9%, 3 days AVB vs. SR), followed by elevated ratios of phosphorylated (P)-Akt to Akt and P-GSK3β to GSK3β (+26 ± 6% and +30 ± 8% at 10 days AVB vs. SR). These changes corresponded to peak hypertrophic growth at 3 to 10 days. MLP increased gradually to maxima at chronic AVB (+36 ± 7%). In contrast, fhl2 (−22 ± 3%, 3 days) and desmin (−30 ± 9%, 10 days AVB) transiently declined but recovered at chronic AVB. Calpain protein expression remained unaltered. In conclusion, volume overload after AVB causes a transient compromise of cytoskeletal integrity based, at least partly, on transcriptional downregulation. Subsequent cytoskeletal reorganization coincides with the upregulation of melusin, P-Akt, P-GSK3β, and MLP, indicating a strong drive to compensated hypertrophy.
W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition
Authors: Qu Yu-Jie , Bondarenko Vladimir E. , Xie Chang , Wang Shimin , Awayda Mouhamed S. , Strauss Harold C. , Morales Michael J.
Published: January 2007

Ca+-calmodulin (Ca2+-CaM)-dependent protein kinase II (Ca2+/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca2+-occupied CaM, and KN-93, an inhibitor of Ca2+/CaMKII, on the Kv4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC50of 92.4 μM. The block was voltage dependent, with an effective electrical distance of 0.18 ± 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of Kv4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 μM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated Kv4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 μM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca2+/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca2+-CaM, as well as a change in activity of Ca2+-CaM-dependent enzymes, including Ca2+/CaMKII.
Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature
Authors: Ishikawa Mami , Sekizuka Eiichi , Yamaguchi Noriyuki , Nakadate Hiromichi , Terao Satoshi , Granger D. Neil , Minamitani Haruyuki
Published: January 2007

Angiotensin II type 1 (AT1) receptor signaling has been implicated in cerebral microvascular alterations associated with ischemia, diabetes mellitus, hypercholesterolemia, and atherosclerosis. Platelets, which express AT1receptors, also appear to contribute to the thrombogenic and inflammatory responses that are elicited by these pathological conditions. This study assesses the role of AT1receptor activation on platelet-leukocyte-endothelial cell interactions elicited in cerebral microvasculature by ischemia and reperfusion. Intravital microscopy was used to monitor the adhesion of platelets and leukocytes that were labeled with different fluorochromes, whereas dihydrorhodamine-123 was used to quantify oxygen radical production in cerebral surface of mice that were either treated with the AT1receptor agonist Val-angiotensin II (ANG II) or subjected to bilateral common carotid artery occlusion (BCCAO) followed by reperfusion. ANG II elicited a dose- and time- dependent increase in platelet-leukocyte-endothelial cell interactions in cerebral venules that included rolling platelets, adherent platelets on the leukocytes and the endothelial cells, rolling leukocytes, and adherent leukocytes. All of these interactions were attenuated by treatment with either P-selectin or P-selectin glycoprotein ligand 1 (PSGL-1) antibody. The AT1receptor antagonist candesartan and losartan as well as diphenyleneiodonium, an inhibitor of flavoproteins including NAD(P)H oxidase, significantly reduced the platelet-leukocyte-endothelial cell interactions elicited by either ANG II administration or BCCAO/reperfusion. The increased oxygen radical generation elicited by BCCAO/reperfusion was also attenuated by candesartan. These findings are consistent with an AT1receptor signaling mechanism, which involves oxygen radical production and ultimately results in P-selectin- and PSGL-1-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation
Effects of hypercapnia and hypoxemia on respiratory sinus arrhythmia in conscious humans during spontaneous respiration
Authors: Tzeng Y. C. , Larsen P. D. , Galletly D. C.
Published: January 2007

Normally, at rest, the amplitude of respiratory sinus arrhythmia (RSA) appears to correlate with cardiac vagal tone. However, recent studies showed that, under stress, RSA dissociates from vagal tone, indicating that separate mechanisms might regulate phasic and tonic vagal activity. This dissociation has been linked to the hypothesis that RSA improves pulmonary gas exchange through preferential distribution of heartbeats in inspiration. We examined the effects of hypercapnia and mild hypoxemia on RSA-vagal dissociation in relation to heartbeat distribution throughout the respiratory cycle in 12 volunteers. We found that hypercapnia, but not hypoxemia, was associated with significant increases in heart rate (HR), tidal volume, and RSA amplitude. The RSA amplitude increase remained statistically significant after adjustment for respiratory rate, tidal volume, and HR. Moreover, the RSA amplitude increase was associated with a paradoxical rise in HR and decrease in low-frequency-to-high-frequency mean amplitude ratio derived from spectral analysis, which is consistent with RSA-vagal dissociation. Although hypercapnia was associated with a significant increase in the percentage of heartbeats during inspiration, this association was largely secondary to increases in the inspiratory period-to-respiratory period ratio, rather than RSA amplitude. Additional model analyses of RSA were consistent with the experimental data. Heartbeat distribution did not change during hypoxemia. These results support the concept of RSA-vagal dissociation during hypercapnia; however, the putative role of RSA in optimizing pulmonary perfusion matching requires further experimental validation.
E-wave deceleration time may not provide an accurate determination of LV chamber stiffness if LV relaxation/viscoelasticity is unknown
Authors: Shmuylovich Leonid , Kovács Sándor J.
Published: January 2007

Average left ventricular (LV) chamber stiffness (ΔPavg/ΔVavg) is an important diastolic function index. An E-wave-based determination of ΔPavg/ΔVavg (Little WC, Ohno M, Kitzman DW, Thomas JD, Cheng CP. Circulation 92: 1933–1939, 1995) predicted that deceleration time (DT) determines stiffness as follows: ΔPavg/ΔVavg = N(π/DT)2 (where N is constant), which implies that if the DTs of two LVs are indistinguishable, their stiffness is indistinguishable as well. We observed that LVs with indistinguishable DTs may have markedly different ΔPavg/ΔVavg values determined by simultaneous echocardiography-catheterization. To elucidate the mechanism by which LVs with indistinguishable DTs manifest distinguishable chamber stiffness, we use a validated, kinematic E-wave model (Kovács SJ, Barzilai B, Perez JE. Am J Physiol Heart Circ Physiol 252: H178–H187, 1987) with stiffness ( k) and relaxation/viscoelasticity ( c) parameters. Because the predicted linear relation between k and ΔPavg/ΔVavg has been validated, we reexpress the DT-stiffness (ΔPavg/ΔVavg) relation of Little et al. as follows: DT k ≈ [Formula: see text]. Using the kinematic model, we derive the general DT-chamber stiffness/viscoelasticity relation as follows: DT k, c = [Formula: see text](where c and k are determined directly from the E-wave), which reduces to DT k when c ≪ k. Validation involved analysis of 400 E-waves by determination of five-beat averaged k and c from 80 subjects undergoing simultaneous echocardiography-catheterization. Clinical E-wave DTs were compared with model-predicted DT k and DT k, c. Clinical DT was better predicted by stiffness and relaxation/viscoelasticity ( r2 = 0.84, DT vs. DT k, c) jointly rather than by stiffness alone ( r2 = 0.60, DT vs. DT k). Thus LVs can have indistinguishable DTs but significantly different ΔPavg/ΔVavg if chamber relaxation/viscoelasticity differs. We conclude that DT is a function of both chamber stiffness and chamber relaxation viscoelasticity. Quantitative diastolic function assessment warrants consideration of simultaneous stiffness and relaxation/viscoelastic effects.
Restoration of coronary endothelial function in obese Zucker rats by a low-carbohydrate diet
Authors: Focardi Marta , Dick Gregory M. , Picchi Andrea , Zhang Cuihua , Chilian William M.
Published: January 2007

A popular diet used for weight reduction is the low-carbohydrate diet, which has most calories derived from fat and protein, but effects of this dietary regimen on coronary vascular function have not been identified. We tested the hypothesis that obesity-induced impairment in coronary endothelial function is reversed by a low-carbohydrate diet. We used four groups of male Zucker rats: lean and obese on normal and low-carbohydrate diets. Rats were fed ad libitum for 3 wk; total caloric intake and weight gain were similar in both diets. To assess endothelial and vascular function, coronary arterioles were cannulated and pressurized for diameter measurements during administration of acetylcholine or sodium nitroprusside or during flow. When compared with lean rats, endothelium-dependent acetylcholine-induced vasodilation was impaired by ∼50% in obese rats (normal diet), but it was restored to normal by the low-carbohydrate diet. When the normal diet was fed, flow-induced dilation (FID) was impaired by >50% in obese compared with lean rats. Similar to acetylcholine, responses to FID were restored to normal by a low-carbohydrate diet. Nω-nitro-l-arginine methyl ester (10 μM), an inhibitor of nitric oxide (NO) synthase, inhibited acetylcholine- and flow-induced dilation in lean rats, but it had no effect on acetylcholine- or flow-induced vasodilation in obese rats on a low-carbohydrate diet. Tetraethylammonium, a nonspecific K+channel antagonist, blocked flow-dependent dilation in the obese rats, suggesting that the improvement in function was mediated by a hyperpolarizing factor independent of NO. In conclusion, obesity-induced impairment in endothelium-dependent vasodilation of coronary arterioles can be dramatically improved with a low-carbohydrate diet most likely through the production of a hyperpolarizing factor independent of NO.
Bleomycin treatment causes enhancement of virus replication in the lungs of SHIV-infected macaques
Authors: Dhillon Navneet Kaur , Pinson David , Dhillon Sukhbir , Tawfik Ossama , Danley Marsha , Davis Marilyn , Nemon Olga , Mayo Matthew , Kumar Anil , Tsai Yi-Jou , Kumar Amit , Buch Shilpa
Published: January 2007

Pneumonia is a major complication of human immunodeficiency virus (HIV) pathogenesis but it develops only after prolonged infection. We used the macaque model to explore a hypothesis that the disease is a two-stage process, the first stage being establishment of the viral infection in the lung and the second being amplification of virus replication by host factors induced by chemical agents or opportunistic pathogens in the lung. Bleomycin, a chemical known to induce diffuse alveolar damage and pulmonary fibrosis with accumulation of macrophages and a rich T helper type 2 (Th2) cytokine environment, was inoculated intratracheally into five of eight SHIV 89.6P-infected macaques and into one uninfected macaque. Three additional simian HIV (SHIV)-infected macaques without bleomycin treatment served as untreated virus controls. Although none of the animals became clinically ill, bleomycin induced classical host responses in the lungs of all the treated, virus-infected macaques. There was enhanced production of the chemokine, monocyte chemotactic protein-1 (MCP-1), that had previously been shown to cause enhanced replication of the virus. Four of the five treated animals developed more productive SHIV infection in the lungs compared with the infected untreated animals. Enhanced virus replication was found primarily in infiltrating macrophages. Enhanced replication of the virus in the lungs was associated with host factors induced by the drug and supported the hypothesis for a two-stage process of pulmonary pathogenesis.