bluuebunny/crossref_metadata_2025 huggingface dataset:
Moraxella catarrhalisStrain O35E Expresses Two Filamentous Hemagglutinin-Like Proteins That Mediate Adherence to Human Epithelial Cells
Authors: Balder Rachel , Hassel Jonathan , Lipski Serena , Lafontaine Eric R.
Published: March 2007

Two-partner secretion (TPS) systems are a family of proteins being rapidly identified and characterized in a growing number of gram-negative bacteria. TPS systems mediate the secretion of proteins, many involved in virulence traits such as hemolysis, adherence to epithelial cells, inhibition of bacterial growth, and immunomodulation of the host. A TPS system typically consists of a transporter located in the bacterial outer membrane (OM) which is responsible for the recognition and secretion of at least one large exoprotein. Two of the better-characterized TPS systems specify theBordetella pertussisFHA andHaemophilus influenzaeHMW1/HMW2 proteins. We identified three gene products ofMoraxella catarrhalisstrain O35E that resemble TPS proteins and designated them MhaC (transporter), MhaB1 (exoprotein), and MhaB2 (exoprotein). Western blot analysis using anti-MhaC, or antibodies reacting to both MhaB1 and MhaB2 (MhaB-reactive), revealed that these antigens are expressed in the OM of 63% of isolates tested. Mutations in themhaCgene specifying the putative transporter of theM. catarrhaliswild-type strains O35E, O12E, and McGHS1 resulted in the absence of MhaB1/MhaB2 in the OM of mutants. These results are therefore consistent with the Mha proteins functioning as a TPS system. Furthermore, we discovered that thesemhaCmutants exhibit markedly decreased binding to human epithelial cells relevant to pathogenesis byM. catarrhalis(Chang, HEp2, A549, and/or 16HBE14o−). Expression of O12E MhaC and MhaB1 in a nonadherent strain ofEscherichia coliwas found to increase the adherence of recombinant bacteria to HEp2 monolayers by sevenfold, thereby demonstrating that thisM. catarrhalisTPS system directly mediates binding to human epithelial cells. The construction of isogenic mutants in themhaB1andmhaB2genes of strain O35E also suggests that the MhaB proteins play distinct roles inM. catarrhalisadherence.
The Relative Susceptibility of Mouse Strains to PulmonaryCryptococcus neoformansInfection Is Associated with Pleiotropic Differences in the Immune Response
Authors: Zaragoza Oscar , Alvarez Mauricio , Telzak Andrew , Rivera Johanna , Casadevall Arturo
Published: March 2007

CBA/J mice were highly susceptible to intratracheal (i.t.)Cryptococcus neoformansinfection relative to BALB/c mice, while both strains were equally susceptible to intravenous (i.v.) infection. Increased susceptibility in i.t. infection was associated with higher brain CFU, lower serum immunoglobulin M (IgM) and IgG responses to glucuronoxylomannan (GXM), lack of IgE regulation during infection, and alveolar macrophage permissiveness to intracellular replication in vitro. In contrast, for BALB/c mice, relative resistance was associated with increased interleukin-12 (IL-12) and decreased IL-10 pulmonary levels. In CBA/J mice, relative susceptibility was associated with a decreased proportion of CD4+and CD8+T cells and an increase in macrophage percentage in pulmonary infiltrates. In contrast, no significant differences in these cytokines or cell recruitment were observed in the i.v. model, consistent with no differences in the survival rate. Passive antibody (Ab) protection experiments revealed a prozone effect in the BALB/c mice with i.v. infection, such that Ab efficacy decreased at higher doses. In the i.t. model using CBA/J mice, low Ab doses were disease enhancing and protection was observed only at high doses. Our results show (i) that differences in mouse strain susceptibility are a function of the infection model, (ii) that susceptibility to pulmonary infection was associated with macrophage permissiveness for intracellular replication, and (iii) that the efficacy of passive Ab in pulmonary infection is a function of dose and mouse strain. The results highlight significant differences in the pathogenesis of cryptococcal infection among inbred mice and associate their relative susceptibility with differences in numerous components of the innate and adaptive immune responses.
Transcutaneous Immunization withClostridium difficileToxoid A Induces Systemic and Mucosal Immune Responses and Toxin A-Neutralizing Antibodies in Mice
Authors: Ghose Chandrabali , Kalsy Anuj , Sheikh Alaullah , Rollenhagen Julianne , John Manohar , Young John , Rollins Sean M. , Qadri Firdausi , Calderwood Stephen B. , Kelly Ciaran P. , Ryan Edward T.
Published: March 2007

Clostridium difficileis the leading cause of nosocomial infectious diarrhea.C. difficileproduces two toxins (A and B), and systemic and mucosal anti-toxin A antibodies prevent or limitC. difficile-associated diarrhea. To evaluate whether transcutaneous immunization with formalin-treatedC. difficiletoxin A (CDA) induces systemic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able to neutralizeC. difficiletoxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous immunization with CDA toxoid may be a feasible immunization strategy againstC. difficile, an important cause of morbidity and mortality against which current preventative strategies are failing.
Infection of Mice with Lyme Disease Spirochetes Constitutively Producing Outer Surface Proteins A and B
Authors: Strother Keith O. , Hodzic Emir , Barthold Stephen W. , de Silva Aravinda M.
Published: March 2007

Outer surface protein A (OspA) of the Lyme disease spirochete is primarily produced in the tick vector. OspA, which is a receptor for attaching spirochetes to the tick gut, is down regulated as the spirochetes leave the tick and enter the mammalian host. Although OspA is not a major antigen produced in the mammal, the protein appears to be produced under some conditions and production has been linked to more severe disease. A Lyme disease vaccine based on recombinant OspA has been approved for human use. However, the vaccine is no longer available, in part because of fears that OspA causes arthritis in people. To further understand the consequences of OspA production in the host, we created a
Borrelia burgdorferi
mutant that was unable to down regulate OspA. C3H/HeN mice infected with this mutant developed a specific anti-OspA immune response, and the spirochetes were unable to persist in these mice. In contrast, immunodeficient SCID mice were persistently infected with the mutant. We conclude that spirochetes producing OspA and B from the
flaB
promoter in immunocompetent mice stimulate an immune response that clear the bacteria without any signs of disease development in the mice.
The
pyrH
Gene of
Vibrio vulnificus
Is an Essential In Vivo Survival Factor
Authors: Lee Shee Eun , Kim Soo Young , Kim Choon Mee , Kim Mi-Kwang , Kim Young Ran , Jeong Kwangjoon , Ryu Hwa-Ja , Lee Youn Suhk , Chung Sun Sik , Choy Hyon E. , Rhee Joon Haeng
Published: March 2007

We have suggested an important role of the
pyrH
gene during the infectious process of
Vibrio vulnificus.
Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes,
pyrH
encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the
pyrH
gene was lethal to
V. vulnificus
, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the
pyrH
gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in
trans
complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that
pyrH
is essential for the in vivo survival and growth of
V. vulnificus
and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.
Flagella Promote
Escherichia coli
K1 Association with and Invasion of Human Brain Microvascular Endothelial Cells
Authors: Parthasarathy G. , Yao Y. , Kim K. S.
Published: March 2007

Escherichia coli
containing the K1 capsule is the leading cause of gram-negative meningitis, but the pathogenesis of this disease is not completely understood. Recent microarray experiments in which we compared the gene expression profile of
E. coli
K1 associated with human brain microvascular endothelial cells (HBMEC) to the gene expression profile of
E. coli
K1 not associated with HBMEC revealed that there was a threefold increase in the expression of the
fliI
gene, encoding an ATP synthase involved in flagellar synthesis and motility, in HBMEC-associated
E. coli
. In this study, we examined the role of flagella in
E. coli
K1 association with and invasion of HBMEC by constructing isogenic Δ
flhDC
, Δ
fliI
, Δ
fliC
, and Δ
cheW
mutants that represented each class of flagellar genes. Mutations that affected the flagellum structure and flagellum formation (Δ
flhDC
, Δ
fliI
, and Δ
fliC
) resulted in significant defects in motility, as well as in HBMEC association and invasion, compared to the characteristics of the wild-type strain when preparations were examined with or without centrifugation. Transcomplementation with the corresponding genes restored the levels of these mutants to the levels of the parent strain. These findings suggest that the HBMEC association and invasion defects of the mutants are most likely related to flagella and less likely due to their motility defects. This conclusion was supported by our demonstration that the
cheW
mutant was not motile but was able to associate with and invade HBMEC. In addition, purified recombinant flagellin reduced the association of the wild-type strain with HBMEC by ∼40%, while it had no effect on the
fliC
mutant's association with HBMEC. Together, these findings indicate that flagella promote
E. coli
K1 binding to HBMEC.
Response of
Leptospira interrogans
to Physiologic Osmolarity: Relevance in Signaling the Environment-to-Host Transition
Authors: Matsunaga James , Lo Miranda , Bulach Dieter M. , Zuerner Richard L. , Adler Ben , Haake David A.
Published: March 2007

Transmission of pathogenic
Leptospira
between mammalian hosts usually involves dissemination via soil or water contaminated by the urine of carrier animals. The ability of
Leptospira
to adapt to the diverse conditions found inside and outside the host is reflected in its relatively large genome size and high percentage of signal transduction genes. An exception is
Leptospira borgpetersenii
serovar Hardjo, which is transmitted by direct contact and appears to have lost genes necessary for survival outside the mammalian host. Invasion of host tissues by
Leptospira interrogans
involves a transition from a low osmolar environment outside the host to a higher physiologic osmolar environment within the host. Expression of the lipoprotein LigA and LigB adhesins is strongly induced by an upshift in osmolarity to the level found in mammalian host tissues. These data suggest that
Leptospira
utilizes changes in osmolarity to regulate virulence characteristics. To better understand how
L. interrogans
serovar Copenhageni adapts to osmolar conditions that correspond with invasion of a mammalian host, we quantified alterations in transcript levels using whole-genome microarrays. Overnight exposure in leptospiral culture medium supplemented with sodium chloride to physiologic osmolarity significantly altered the transcript levels of 6% of
L. interrogans
genes. Repressed genes were significantly more likely to be absent or pseudogenes in
L. borgpetersenii
, suggesting that osmolarity is relevant in studying the adaptation of
L. interrogans
to host conditions. Genes induced by physiologic osmolarity encoded a higher than expected number of proteins involved in signal transduction. Further, genes predicted to encode lipoproteins and those coregulated by temperature were overrepresented among both salt-induced and salt-repressed genes. In contrast, leptospiral homologues of hyperosmotic or general stress genes were not induced at physiologic osmolarity. These findings suggest that physiologic osmolarity is an important signal for regulation of gene expression by pathogenic leptospires during transition from ambient conditions to the host tissue environment.
Anti-Lewis X Antibody PromotesHelicobacter pyloriAdhesion to Gastric Epithelial Cells
Authors: Sheu Shew-Meei , Sheu Bor-Shyang , Yang Hsiao-Bai , Lei Huan-Yao , Wu Jiunn-Jong
Published: March 2007

Lewis X (Lex) antigen is expressed on the human gastric mucosa and the O-specific chain of lipopolysaccharides ofHelicobacter pylori. This antigen can induce autoantibodies, which may be involved in bacterial colonization and thus deserve further investigation. Flow cytometry was used to examine the effects of anti-Le monoclonal antibodies (MAbs) onH. pyloriadhesion. AbabA2mutant was also constructed to evaluate the effect of an anti-LexMAb on adhesion. The bacterial agglutination and in situ adhesion assays were used to confirm the anti-LexMAb effect onH. pyloriadhesion. This study revealed that an anti-LexMAb, but not an anti-LebMAb or an anti-LeyMAb, could enhance the adhesion ofH. pyloristrains that expressed high levels of Lexantigen to AGS cells. The enhancement was not found on anH. pyloristrain with a low level of Lexantigen. Anti-LexMAb could increase the adhesion of both the wild-type strain and its isogenicbabA2mutant to AGS cells. When AGS cells were pretreated with anti-LexMAb, the adhesion of thebabA2mutant also increased. Only anti-LexMAb could promote bacterial agglutination, and the in situ adhesion assay further confirmed that adding anti-LexMAb resulted in denser bacterial adhesion on the gastric epithelia collected from clinical patients. These results suggest anti-LexMAb could specifically enhance the adhesion abilities ofH. pyloristrains through a mechanism by which anti-LexMAb promotes bacterial aggregation and mediates bivalent interaction (antigen-antibody-antigen) between bacteria and host cells.
High-Throughput mRNA Profiling Characterizes the Expression of Inflammatory Molecules in Sepsis Caused by
Burkholderia pseudomallei
Authors: Wiersinga W. Joost , Dessing Mark C. , Kager Piet A. , Cheng Allen C. , Limmathurotsakul Direk , Day Nicholas P. , Dondorp Arjen M. , van der Poll Tom , Peacock Sharon J.
Published: March 2007

Sepsis is characterized by an uncontrolled inflammatory response to invading microorganisms. We describe the inflammatory mRNA profiles in whole-blood leukocytes, monocytes, and granulocytes using a multigene system for 35 inflammatory markers that included pro- and anti-inflammatory cytokines, chemokines, and signal transduction molecules in a case-control study with 34 patients with sepsis caused by the gram-negative bacterium
Burkholderia pseudomallei
(the pathogen causing melioidosis) and 32 healthy volunteers. Relative to healthy controls, patients with sepsis showed increased transcription of a whole array of inflammatory genes in peripheral blood leukocytes, granulocytes, and monocytes. Specific monocyte and granulocyte mRNA profiles were identified. Strong correlations were found between inflammatory mRNA expression levels in monocytes and clinical outcome. These data underline the notion that circulating leukocytes are an important source for inflammatory mediators in patients with gram-negative sepsis. Gene profiling such as was done here provides an excellent tool to obtain insight into the extent of inflammation activation in patients with severe infection.
Protein F1 andStreptococcus pyogenesResistance to Phagocytosis
Authors: Hyland Kendra A. , Wang Beinan , Cleary P. Patrick
Published: March 2007

Streptococcus pyogenesis a major cause of pharyngitis in humans and encodes several fibronectin-binding proteins. M protein and protein F1 (PrtF1/SfbI) are differentially regulated by CO2and O2, respectively, and both mediate the invasion of epithelial cells. This study examined whether PrtF1/SfbI shares other properties with M protein. Expression of the PrtF1/SfbI protein by an M-negative mutant conferred resistance to phagocytosis and partial inhibition of C3 deposition on theS. pyogenessurface.
Environmental Strains of
Mycobacterium avium
Interfere with Immune Responses Associated with
Mycobacterium bovis
BCG Vaccination
Authors: Young Sarah L. , Slobbe Lynn , Wilson Rachel , Buddle Bryce M. , de Lisle Geofferey W. , Buchan Glenn S.
Published: March 2007

Prior exposure of a vaccinee to certain species of environmental mycobacteria can prime the immune system against common mycobacterial antigens, which can in turn reduce the subsequent efficacy of live attenuated mycobacterial vaccines (such as
Mycobacterium bovis
BCG), in both human and livestock vaccination programs. In this study, two strains of
Mycobacterium avium
, both isolated from New Zealand livestock, were investigated to determine their growth characteristics and effects on the immune system in murine models. Markedly different effects on the immune system were observed; an IS
901
-negative strain (WAg 207) induced significant up-regulation of cell surface activation markers (major histocompatibility complex II, CD80, and CD86) on in vitro-derived dendritic cells and induced the release of proinflammatory monokines (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha) in dendritic cell-macrophage cocultures following direct in vitro contact of cells with bacteria. In contrast, an IS
901
-positive strain (WAg 206) had none of these effects. When mice were exposed to
M. avium
via oral infection prior to BCG parenteral immunization, both strains were shown to be capable of decreasing subsequent antigen-stimulated gamma interferon secretion by splenic lymphocytes, although this effect was more significant for strain WAg 206. Both strains also induced a mycobacterial antigen-specific serological response in
M. avium
-sensitized and BCG-immunized mice; this response was greater in WAg 206-sensitized mice, and there was a predominance of immunoglobulin G1 antibody. The down-regulation of IFN-γ responses and the up-regulation of antibody responses are characteristic of a switch to a type 2 immune response. The different results may be linked to the inherent growth characteristics of the two strains, since WAg 206 was shown to grow slowly in murine macrophages in vitro and to cause a persistent systemic infection following infection in vivo, while WAg 207 grew fast and did not persist in mice. The implications of these findings for BCG vaccination protocols are discussed.
Trivalent Vaccine against Botulinum Toxin Serotypes A, B, and E That Can Be Administered by the Mucosal Route
Authors: Ravichandran Easwaran , Al-Saleem Fetweh H. , Ancharski Denise M. , Elias Mohammad D. , Singh Ajay K. , Shamim Mohammad , Gong Yujing , Simpson Lance L.
Published: March 2007

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.
Biological and Immunological Characteristics of Lipooligosaccharide-Based Conjugate Vaccines for Serotype C
Moraxella catarrhalis
Authors: Yu Shengqing , Gu Xin-Xing
Published: March 2007

Moraxella catarrhalis
is an important bacterial cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS), a major surface antigen of this bacterium, is a potential vaccine component against the organism. There are three major LOS serotypes (serotypes A, B, and C) in clinical isolates of
M. catarrhalis
. Our previous studies demonstrated that serotype A and B LOS-based conjugates were immunogenic in animals and elicited bactericidal antibodies. In this study, LOS from serotype C strain 26404 was isolated, detoxified, and conjugated to tetanus toxoid (TT) or the cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT, dLOS-CRM-1, and dLOS-CRM-2 vaccine candidates. The molar ratios (dLOS/protein) of the resulting conjugates were 47:1, 19:1, and 32:1, respectively, while the weight ratios were 0.94, 0.84 and 1.44, respectively. All conjugates were highly immunogenic in both mouse and rabbit models. Three subcutaneous injections of each conjugate formulated with the Ribi adjuvant elicited >700-fold increases in serum anti-LOS immunoglobulin G levels in mice (5 μg of dLOS) and >2,000-fold increases in rabbits (50 μg of dLOS). The resulting mouse and rabbit antisera showed complement-mediated bactericidal activity against the homologous strain. In addition, a representative rabbit antiserum showed bactericidal activity against 14 of 18 testable strains, and this bactericidal activity could be 100% inhibited by the serotype C or A LOS but only 30% inhibited by the serotype B LOS. These results indicate that the serotype C LOS-based conjugates can be used as vaccine components for further investigation in humans.
Inhibitory Effect of Enterohepatic
Helicobacter hepaticus
on Innate Immune Responses of Mouse Intestinal Epithelial Cells
Authors: Sterzenbach Torsten , Lee Sae Kyung , Brenneke Birgit , von Goetz Franz , Schauer David B. , Fox James G. , Suerbaum Sebastian , Josenhans Christine
Published: March 2007

Enterohepatic
Helicobacter
species infect the intestinal tracts and biliary trees of various mammals, including mice and humans, and are associated with chronic inflammatory diseases of the intestine, gallstone formation, and malignant transformation. The recent analysis of the whole genome sequence of the mouse enterohepatic species
Helicobacter hepaticus
allowed us to perform a functional analysis of bacterial factors that may play a role in these diseases. We tested the hypothesis that
H. hepaticus
suppresses or evades innate immune responses of mouse intestinal epithelial cells, which allows this pathogen to induce or contribute to chronic inflammatory disease. We demonstrated in the present study that the innate immune responses of intestinal epithelial cells to lipopolysaccharide (LPS) via Toll-like receptor 4 (TLR4) and to flagellin-mediated activation via TLR5 are reduced by
H. hepaticus
infection through soluble bacterial factors. In particular,
H. hepaticus
lysate and the soluble component LPS antagonized TLR4- and TLR5-mediated immune responses of intestinal epithelial cells.
H. hepaticus
lysate and LPS inhibited development of endotoxin tolerance to
Escherichia coli
LPS. Suppression of innate immune responses by
H. hepaticus
LPS thus may affect intestinal responses to the resident microbial flora, epithelial homeostasis, and intestinal inflammatory conditions.
Legionella pneumophila
Infection Up-Regulates Dendritic Cell Toll-Like Receptor 2 (TLR2)/TLR4 Expression and Key Maturation Markers
Authors: Rogers James , Hakki Amal , Perkins Izabella , Newton Catherine , Widen Ray , Burdash Nicholas , Klein Thomas , Friedman Herman
Published: March 2007

Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of
Legionella pneumophila
infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live
L. pneumophila
resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.
Glycine Residues in the Hydrophobic Core of the GspB Signal Sequence Route Export toward the Accessory Sec Pathway
Authors: Bensing Barbara A. , Siboo Ian R. , Sullam Paul M.
Published: March 2007

The
Streptococcus gordonii
cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of
S. gordonii
and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.
Initial Characterization of the Photosynthetic Apparatus of “
Candidatus
Chlorothrix halophila,” a Filamentous, Anoxygenic Photoautotroph
Authors: van de Meene Allison M. L. , Olson Tien Le , Collins Aaron M. , Blankenship Robert E.
Published: March 2007

“
Candidatus
Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol.
181:
17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the
Chloroflexi
and
Chlorobi
phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll
c
(BChl
c
) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl
c
is present in the chlorosomes of “
Ca
. Chlorothrix halophila,” that BChl
a
is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ∼6-kDa band, which was found to be related to the BChl
c
binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “
Ca
. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of
Chlorobium tepidum
and
Chloroflexus aurantiacus
.
Pigment Analysis of “
Candidatus
Chlorothrix halophila,” a Green Filamentous Anoxygenic Phototrophic Bacterium
Authors: Olson Tien Le , van de Meene Allison M. L. , Francis J. Nicholas , Pierson Beverly K. , Blankenship Robert E.
Published: March 2007

The pigment composition of “
Candidatus
Chlorothrix halophila,” a filamentous anoxygenic phototrophic bacterium found in Baja California Sur, Mexico, was determined. Previous work showed that bacteriochlorophyll
c
(BChl
c
) was the major pigment in “
Ca
. Chlorothrix halophila,” but it was not clear if this bacterium also contains BChl
a
(J. A. Klappenbach and B. K. Pierson, Arch. Microbiol.
181:
17-25, 2004). Here we show that in addition to BChl
c
, a small amount of a pigment that is spectrally indistinguishable from BChl
a
is present in cell extracts of “
Ca
. Chlorothrix halophila.” Nevertheless, the BChl
a
-like pigment from “
Ca
. Chlorothrix halophila” has a different molecular weight and a different high-performance liquid chromatography elution time than BChl
a
from other photosynthetic bacteria. Based on mass spectrometry and other spectroscopic analysis, we determined that the BChl
a
-like pigment in “
Ca
. Chlorothrix halophila” contains a tetrahydrogeranylgeraniol tail rather than the phytol tail that is present in BChl
a
. The carotenoids and major BChl
c
homologs in “
Ca
. Chlorothrix halophila” were also identified. BChls
c
were found to be farnesol esterified and geranylgeraniol esterified.
Heterologous Expression and Identification of the Genes Involved in Anaerobic Degradation of 1,3-Dihydroxybenzene (Resorcinol) in
Azoarcus anaerobius
Authors: Darley Paula I. , Hellstern Jutta A. , Medina-Bellver Javier I. , Marqués Silvia , Schink Bernhard , Philipp Bodo
Published: March 2007

Azoarcus anaerobius
, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the
A. anaerobius
genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species
Thauera aromatica
, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R
+
, carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of
T. aromatica
transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R
+
carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of
T. aromatica
transconjugants carrying transposon insertions in different genes of cosmid R
+
, eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.
Telmisartan prevents the glitazone-induced weight gain without interfering with its insulin-sensitizing properties
Authors: Zanchi Anne , Dulloo Abdul G. , Perregaux Christine , Montani Jean-Pierre , Burnier Michel
Published: March 2007

Glitazones are peroxisome proliferator-activated receptor (PPAR)-γ agonists with powerful insulin-sensitizing properties. They promote the development of metabolically active adipocytes that can lead to a substantial gain in fat mass. Telmisartan is an ANG II type 1 receptor antagonist with partial PPAR-γ agonistic properties. Recently, telmisartan has been reported to prevent weight gain and improve insulin sensitivity in diet-induced obese rodents. The goal of this study was to examine the influence of telmisartan on pioglitazone-induced weight gain and insulin-sensitizing properties in the following two models of insulin resistance: a nongenetic model (high-fat-fed Sprague Dawley rats) and the genetically obese fa/ fa Zucker rat. After a 4-wk treatment, the pioglitazone-induced increase in fat mass was modest in the Sprague Dawley rats and severe in the Zucker rats. In both models, these effects were substantially decreased by concomitant treatment with telmisartan. The effects of telmisartan on body weight and fat mass in the Zucker rats were abolished by pair feeding, suggesting that it is the result of a decrease in food intake. Telmisartan did not interfere with the insulin-sensitizing properties of pioglitazone. This study demonstrates that telmisartan attenuates the glitazone-induced increase in fat mass without interfering with its insulin-sensitizing properties.
Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism
Authors: Morrison Christopher D. , Xi Xiaochun , White Christy L. , Ye Jianping , Martin Roy J.
Published: March 2007

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake ( P < 0.05) and hypothalamic Agouti-related protein ( Agrp) gene expression ( P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 μg) suppressed 24-h food intake ( P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1–7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1–7 cells ( P < 0.01), and this effect was attenuated by replacement of the amino acid leucine ( P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1–7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids ( P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.
Growth analysis of the mouse adrenal gland from weaning to adulthood: time- and gender-dependent alterations of cell size and number in the cortical compartment
Authors: Bielohuby Max , Herbach Nadja , Wanke Rüdiger , Maser-Gluth Christiane , Beuschlein Felix , Wolf Eckhard , Hoeflich Andreas
Published: March 2007

The adrenal gland is of critical importance for a plethora of biological processes. We performed the first systematic analysis of adrenal gland growth using unbiased stereological methods in male and female mice from weaning to adulthood ( weeks 3, 5, 7, 9, and 11) at the organ, compartment, and cellular levels. Adrenal weights increased from week 3 to week 7 in male and female mice, remained at this level in females, but decreased by 25% between week 7 and week 9 in males. Female adrenal glands displayed a higher weight at any stage investigated. The volume of the zona fasciculata was consistently higher in female vs. male mice. In both genders, the number of zona fasciculata cells reached a maximum at the age of 7 wk and decreased significantly until week 9. Serum corticosterone concentrations decreased from 3 to 11 wk of age both in male and female mice. However, the estimated total amounts of corticosterone in the circulation were similar in 3- and 11-wk-old mice. Furthermore, total circulating corticosterone was higher in females than in males at an age of 5 and 11 wk. In the zona glomerulosa and in the X-zone, time- and gender-dependent growth effects were observed. In conclusion, our results demonstrate that growth and function of the adrenal glands are markedly influenced by gender and age. These factors require careful consideration in studies aiming at the functional dissection of genetic and environmental factors affecting adrenal growth and function.
Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta
Authors: Atkins Kevin B. , Prezkop Antoine , Park James L. , Saha Jharna , Duquaine Damon , Charron Maureen J. , Olson Ann Louise , Brosius Frank C.
Published: March 2007

We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.
Microparticles from preeclamptic women induce vascular hyporeactivity in vessels from pregnant mice through an overproduction of NO
Authors: Tesse Angela , Meziani Ferhat , David Eric , Carusio Nunzia , Kremer Helene , Schneider Francis , Andriantsitohaina Ramaroson
Published: March 2007

Preeclampsia is associated with an increase of circulating levels of microparticles (MPs), but their role in vascular dysfunction during the course of preeclampsia is not understood. Inasmuch as preeclampsia is a gestational disease, we tested the effect of MPs from preeclamptic women (PrMPs) and MPs from normal pregnant women (CMPs) on vessels from pregnant mice. We exposed aortic rings from pregnant mice to circulating levels of PrMPs or CMPs for 24 h and evaluated their response to serotonin (5-HT). PrMPs, but not CMPs, were able to induce hyporeactivity in response to 5-HT in aortas from pregnant mice. The nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine strongly enhanced the response to 5-HT in PrMP-treated vessels but had no significant effect on CMP-treated vessels. The 5-HT-induced contraction in PrMP-treated vessels was completely abolished by the selective cyclooxygenase-2 (COX-2) inhibitor NS-398 but was only reduced in CMP-treated vessels, suggesting an increased participation of COX-2 vasoconstrictor products in the effect of PrMPs. Consistent with this hypothesis, PrMPs enhanced levels of 8-isoprostane and PGE2in vessels, despite reduction of thromboxane B2. These results strengthen the main concept that MPs in preeclampsia could act as vectors to stimulate intracellular cascades in vascular cells, leading to an enhanced NO production to counteract increased COX-2 vasoconstrictor metabolites by taking into account pregnancy.
Mediating δ-opioid-initiated heart protection via the β2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell
Authors: Huang Ming-He , Wang Hui-Qun , Roeske William R. , Birnbaum Yochai , Wu Yewen , Yang Ning-Ping , Lin Yu , Ye Yumei , McAdoo David J. , Hughes Michael G. , Lick Scott D. , Boor Paul J. , Lui Charles Y. , Uretsky Barry F.
Published: March 2007

Stimulation of cardiac β2-adrenergic receptor (β2-AR) or δ-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that δ-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca2+concentration ([Ca2+]i) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective δ-opioid agonist d-[Pen2,5]enkephalin (DPDPE) to ICA cells increased [Ca2+]itransients in a concentration-dependent manner. Such an effect was abolished by the Ca2+channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in δ-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 ± 15% or myocyte death by 26 ± 4%, respectively. β2-AR blockade markedly attenuated δ-opioid-initiated infarct size-limiting effect and abolished δ-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, δ-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that δ-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/β2-AR signaling pathway as a result of ICA cell activation.
Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury
Authors: Grad Etty , Golomb Mordechai , Mor-Yosef Irit , Koroukhov Nikolay , Lotan Chaim , Edelman Elazer R. , Danenberg Haim D.
Published: March 2007

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 ± 9, 18.6 ± 6.9, and 58.4 ± 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice ( n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.
Molecular effects of loss of BMPR2 signaling in smooth muscle in a transgenic mouse model of PAH
Authors: Tada Yuji , Majka Susan , Carr Michelle , Harral Julie , Crona Daniel , Kuriyama Takayuki , West James
Published: March 2007

Idiopathic pulmonary arterial hypertension (IPAH) in human patients is associated with mutations in type 2 receptor for the bone morphogenic protein pathway (BMPR2). Mice expressing an inducible dominant negative form of BMPR2 in smooth muscle develop elevated right ventricular pressures when the transgene is activated. We hypothesized that transcriptional changes in these mice may allow insight into the early molecular events leading to IPAH. Microarray analysis was used to examine the transcriptional changes induced in whole lung by loss of normal smooth muscle cell (SMC) BMPR2 signaling in adult male or female mice (12 wk at time of death) expressing the transgene for either 1 or 8 wk. Our key results include a decrease in markers of smooth muscle differentiation, an increase in cytokines and markers of immune response, particularly in female mice, and a decrease in angiogenesis-related genes. These broad patterns of gene expression appear as early as 1 wk and are well established by 8 wk. Results were confirmed by quantitative RT-PCR to RNA from individual mice. Primary pulmonary artery SMC cultures transfected with small interfering RNA to BMPR2 also show loss of SMC markers myosin heavy chain 11 and calponin by quantitative RT-PCR and Western blot. These studies show classes of genes differentially regulated in response to loss of BMPR2 in SMC in vivo with clear relevance to the IPAH disease process, suggesting that the relevance of BMPR2 dysregulation may extend beyond proliferation.