bluuebunny/crossref_metadata_2025 huggingface dataset:
Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins
Authors: Park Sang-Youl , Fung Pauline , Nishimura Noriyuki , Jensen Davin R. , Fujii Hiroaki , Zhao Yang , Lumba Shelley , Santiago Julia , Rodrigues Americo , Chow Tsz-fung F. , Alfred Simon E. , Bonetta Dario , Finkelstein Ruth , Provart Nicholas J. , Desveaux Darrell , Rodriguez Pedro L. , McCourt Peter , Zhu Jian-Kang , Schroeder Julian I. , Volkman Brian F. , Cutler Sean R.
Published: May 2009

ABA Receptor Rumbled?

The plant hormone abscisic acid (ABA) is critical for normal development and for mediating plant responses to stressful environmental conditions. Now, two papers present analyses of candidate ABA receptors (see the news story by

Pennisi

).

Ma
et al.

(p. 1064; published online 30 April) and

Park
et al.

(p. 1068, published online 30 April) used independent strategies to search for proteins that physically interact with ABI family phosphatase components of the ABA response signaling pathway. Both groups identified different members of the same family of proteins, which appear to interact with ABI proteins to form a heterocomplex that can act as the ABA receptor. The variety of both families suggests that the ABA receptor may not be one entity, but rather a class of closely related complexes, which may explain previous difficulties in establishing its identity.
Transcriptional Regulation of β-Defensin Gene Expression in Tracheal Epithelial Cells
Authors: Diamond Gill , Kaiser Vicki , Rhodes Janice , Russell John P. , Bevins Charles L.
Published: May 2009

Innate immunity provides an ever-present or rapidly inducible initial defense against microbial infection. Among the effector molecules of this defense in many species are broad-spectrum antimicrobial peptides. Tracheal antimicrobial peptide (TAP) was the first discovered member of the β-defensin family of mammalian antimicrobial peptides. TAP is expressed in the ciliated epithelium of the bovine trachea, and its mRNA levels are dramatically increased upon stimulation with bacteria or bacterial lipopolysaccharide (LPS). We report here that this induction by LPS is regulated at the level of transcription. Furthermore, the transfection of reporter gene constructs into tracheal epithelial cells indicates that DNA sequences in the 5′ flanking region of the TAP gene, within 324 nucleotides of the transcription start site, are responsible in part for mediating gene induction. This region includes consensus binding sites for NF-κB and nuclear factor interleukin-6 (NF IL-6) transcription factors. Gel mobility shift assays indicate that LPS induces NF-κB binding activity in the nuclei of these cells, while NF IL-6 binding activity is constitutively present. The gene encoding human β-defensin 2, a human homologue of TAP with similar inducible expression patterns in the airway, was cloned and found to have conserved NF-κB and NF IL-6 consensus binding sites in its 5′ flanking region. Previous studies of antimicrobial peptides from insects indicated that their induction by infectious microbes and microbial products also occurs via activation of NF-κB-like and NF IL-6-like transcription factors. Together, these observations indicate that a strategy for the induction of peptide-based antimicrobial innate immunity is conserved among evolutionarily diverse organisms.
Host and Bacterial Factors Involved in the Innate Ability of Mouse Macrophages To Eliminate Internalized UnopsonizedEscherichia coli
Authors: Hamrick Terri S. , Havell Edward A. , Horton John R. , Orndorff Paul E.
Published: May 2009

In an effort to better understand genetic and cellular factors that influence innate immunity, we examined host and bacterial factors involved in the nonopsonic phagocytosis and killing ofEscherichia coliK-12 by mouse macrophages. Unelicited (resident) peritoneal macrophages from five different mouse strains, BALB/c, C57BL/6, CD-1, C3H/HeJ, and C3H/HeN, were employed. Additional macrophage populations were obtained from CD-1 mice (bone marrow-derived macrophages). Also, for BALB/c and C57BL/6 mice, peritoneal macrophages elicited with either thioglycolate or proteose peptone, bone marrow-derived macrophages, and macrophage-like cell lines derived from the two strains were employed. TwoE. coliK-12 strains that differed specifically in their abilities to produce type 1 pili containing the adhesive protein FimH were examined. The parameters used to assess macrophage bacteriocidal activity were (i) the killing of internalized (gentamicin-protected)E. coliduring the approximately 4-h assay and (ii) the initial rate at which internalizedE. coliwere eliminated. Data on these parameters allowed the following conclusions: (i) unelicited or proteose peptone-elicited peritoneal macrophages were significantly better at eliminating internalized bacteria than thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages, or macrophage cell lines; (ii) the host genetic background had no significant effect upon the ability of unelicited peritoneal macrophages to killE. coli(even though the mouse strains differ widely in their in vivo susceptibilities to bacterial infection); and (iii) the FimH phenotype had no significant effect uponE. colisurvival once the bacterium was inside a macrophage. Additionally, there was no correlation between the bacteriocidal effectiveness of a macrophage population and the number of bacteria bound per macrophage. However, macrophage populations that were the least bacteriocidal tended to bind higher ratios of FimH+to FimH−E. coli. The effect of gamma interferon, fetal calf serum, and the recombination proficiency ofE. coliwere examined as factors predicted to influence intracellular bacterial killing. These had no effect upon the rate ofE. colielimination by unelicited peritoneal macrophages.
Glycosylation of Homologous Immunodominant Proteins of
Ehrlichia chaffeensis
and
Ehrlichia canis
Authors: McBride Jere W. , Yu Xue-jie , Walker David H.
Published: May 2009

The glycoprotein genes of
Ehrlichia chaffeensis
(1,644 bp) and
Ehrlichia canis
(2,064 bp) encode proteins of 548 to 688 amino acids with predicted molecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene of
E. chaffeensis
contains four identical 240-bp tandem repeat units, and the 140-kDa protein gene of
E. canis
has 14 nearly identical, tandemly arranged 108-bp repeat units. Conserved serine-rich motifs identified in the repeat units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-associated adhesin protein Fap1 of
Streptococcus parasanguis
. Nearly the entire (99%)
E. chaffeensis
P120 gene (1,616 bp), the 14-repeat region (78%) of the
E. canis
P140 gene (1,620 bp), and a 2-repeat region from the
E. chaffeensis
P120 gene (520 bp) were expressed in
Escherichia coli
. The recombinant proteins exhibited molecular masses ranging from 1.6 to 2 times larger than those predicted by the amino acid sequences. Antibodies against the recombinant proteins reacted with
E. chaffeensis
P120 and
E. canis
P140, respectively. Carbohydrate was detected on the
E. chaffeensis
and
E. canis
recombinant proteins, including the two-repeat polypeptide region of
E. chaffeensis
P120. A carbohydrate compositional analysis identified glucose, galactose, and xylose on the recombinant proteins. The presence of only one site for N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of
N
-glycosidase F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydrate to protein suggest that the glycans may be O linked.
Analysis of Immune Responses against T- and B-Cell Epitopes fromPlasmodium falciparumLiver-Stage Antigen 1 in Rodent Malaria Models and Malaria-Exposed Human Subjects in India
Authors: Joshi Sunil K. , Bharadwaj Ashima , Chatterjee Shyama , Chauhan V. S.
Published: May 2009

Liver-stage antigen 1 (LSA-1) is a potential vaccine candidate against preerythrocytic stages of malaria. We report here the immunogenicity of linear synthetic constructs delineated as TH-cell determinants from the nonrepeat regions ofPlasmodium falciparumLSA-1 in murine models and human subjects from areas where malaria is endemic in Rajasthan State, India. Seven peptide constructs (LS1.1 to LS1.7) corresponding to predicted T-cell sites from both the N- and C-terminal regions and peptide LS1R from a repeat region of PfLSA-1 were synthesized to analyze the cellular immune responses. These linear peptides were also tested for humoral responses in order to determine if there were any overlapping B-cell epitopes in the predicted T-cell sites. Most peptides induced cellular responses in peptide-immunized BALB/c and C57BL/6 mice as measured by proliferation and cytokine analysis. Cross-reactive T-cell recognition ofP. falciparum-based peptides inPlasmodium berghei-immune animals was evaluated, but only one peptide, LS1.2 (amino acids 1742 to 1760) triggered T-cell proliferation and interleukin-2 and gamma interferon secretion inP. berghei-immune splenocytes of BALB/c and C57BL/6 mice as well as inThamnomys gazellae(natural host ofP. bergheiANKA). In an enzyme-linked immunosorbent assay with the peptides, only one peptide, LS1.1, was recognized by anti-P. bergheiliver-stage serum. Three peptides (LS1.1, LS1.2, and LS1.3) of the eight peptides tested in this study were recognized by a relatively large percentage ofP. falciparum-exposed human subjects; the reactivities ranged from ∼45% for LS1.3 to ∼60% for LS1.1 and LS1.2. Interestingly, all of the eight putative T-cell determinants were also recognized by the sera collected from malaria patients, although the response was variable in nature. These TH- and B-cell epitopes may be of potential value for preerythrocytic antigen-based malaria subunit vaccine formulations.
Involvement of CD4+Th1 Cells in Systemic Immunity Protective against Primary and Secondary Challenges withTrypanosoma cruzi
Authors: Hoft Daniel F. , Schnapp Anita R. , Eickhoff Christopher S. , Roodman Stanford T.
Published: May 2009

In general, gamma interferon (IFN-γ)-producing CD4+Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-γ responses and resistance to the intracellular protozoanTrypanosoma cruzi. To investigate a potential causal relationship between Th1 responses andT. cruziresistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulentT. cruzichallenges. We generatedT. cruzi-specific CD4+Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication inT. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection againstT. cruzito SCID mice. In addition, despite the fact that the adoptive transfer of CD4+T lymphocytes was shown to be necessary for the development of immunity protective against primaryT. cruziinfection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4+T lymphocytes. These results indicate that, although CD4+Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells inT. cruzisystemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.
In Vivo Characterization of the Murine Intranasal Model for Assessing the Immunogenicity of AttenuatedSalmonella entericaSerovar Typhi Strains as Live Mucosal Vaccines and as Live Vectors
Authors: Pickett Thames E. , Pasetti Marcela F. , Galen James E. , Sztein Marcelo B. , Levine Myron M.
Published: May 2009

AttenuatedSalmonella entericaserovar Typhi live vector vaccine strains are highly immunogenic in mice following intranasal but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrAfollowing intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyer's patches 2 min after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 109CFU (from a single 30- or 10-μl dose to four 2.5-μl doses given over the course of 1 h), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (P< 0.05) without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrAexpressing tetanus toxin fragment C under the control of the redox-responsivenir15promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutivelpppromoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated serovar Typhi vaccine organisms elicit serum IgG responses.
Murine Immune Response toNeisseria meningitidisGroup C Capsular Polysaccharide: Analysis of Monoclonal Antibodies Generated in Response to a Thymus-Independent Antigen and a Thymus-Dependent Toxoid Conjugate Vaccine
Authors: García-Ojeda Pablo A. , Monser Martha E. , Rubinstein Leonard J. , Jennings Harold J. , Stein Kathryn E.
Published: May 2009

Antibody (Ab) responses to polysaccharides (PSs) such asNeisseria meningitidisgroup C PS (MCPS) are characterized as being thymus independent (TI) and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled to tetanus toxoid (MCPS-TT), elicits a thymus-dependent (TD) response. In order to understand the influence of the form of a vaccine (TI versus TD) on the Ab repertoire, we generated monoclonal antibody (MAb) panels from mice immunized and boosted with MCPS or MCPS-TT in different ways. The panels of MAbs were examined for isotype, fine specificity, affinity, and VHgene family usage. The use of MCPS-TT resulted in a shift in the isotype from immunoglobulin M (IgM) and IgG3 elicited in response to the MCPS to primarily IgG1. This isotype shift was accompanied by a change in the fine specificity of the response to the conjugate compared to that of PS. New fine specificities and increased affinity were observed in response to the TD antigen (Ag). Dot blot and Northern analyses of MCPS MAbs revealed that VHgene family usage is dominated by VHJ558, used by 23 of 39 MAbs. VH3609 was seen in three MAbs of restricted fine specificity. VHQ52, VH7183, and VHVGAM3-8 were seen in more than one MAb across these panels, while VH10 and VHX24 were detected only once in response to the TI-2 Ag. All MAbs in the panels utilized kappa light chains, and all functional Jκgenes were expressed.
Delivery of CD8+T-Cell Epitopes into Major Histocompatibility Complex Class I Antigen Presentation Pathway byBordetella pertussisAdenylate Cyclase: Delineation of Cell Invasive Structures and Permissive Insertion Sites
Authors: Osička Radim , Osičková Adriana , Basar Tümay , Guermonprez Pierre , Rojas Marie , Leclerc Claude , Šebo Peter
Published: May 2009

Bordetella pertussisadenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8+T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8+T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8+T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.
Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis inHaemophilus somnus
Authors: Wu Yanping , McQuiston Jennifer H. , Cox Andrew , Pack Todd D. , Inzana Thomas J.
Published: May 2009

Haemophilus somnusundergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-1) containing repetitive 5′-CAAT-3′ sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675–4681, 1997). We have now identified another putative gene (lob-2A) immediately upstream fromlob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the familyPasteurellaceaeand the LgtB and LgtE galactosyltransferases ofNeisseria meningitidisandN. gonorrhoeae. Unlikelob-1,lob-2Acontained 18 to 20 5′-GA-3′ repeats 141 bp upstream of the termination codon as determined by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5′-GA-3′ repeats were present a stop codon would occur 1 bp after the last 5′-GA-3′ repeat. A 630-bpSalI-BsgI fragment withinlob-2Awas deleted, and a kanamycin resistance (Kmr) gene was inserted into this site to create pCAATΔlob2A. Following electroporation of pCAATΔlob2A intoH. somnus738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1::Km, was altered, and the phase variation rate was reduced but phase variation was not eliminated. A variant with 19 5′-GA-3′ repeats inlob-2Ahad an LOS profile similar to that of 738-lob2A1::Km, suggesting thatlob-2Awas turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal βGal(1-3)βGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. WhenH. somnus129Pt was electrotransformed with shuttle vector pLS88 containinglob-2A, its LOS electrophoretic profile was modified and additionalN-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated thatlob-2Amay be anN-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important toH. somnusvirulence.
Interleukin-8 Secretion of Cortical Tubular Epithelial Cells Is Directed to the Basolateral Environment and Is Not Enhanced by Apical Exposure toEscherichia coli
Authors: Krüger Sabine , Brandt Ernst , Klinger Matthias , Krüger Stefan , Kreft Burkhard
Published: May 2009

In upper urinary tract infections, tubular epithelial cells (TEC) may play a pivotal role in the initiation of the renal inflammatory response. They exert crucial immunological functions such as processing and presentation of foreign antigen, secretion of proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha) and chemokines (IL-8, MCP-1, ENA-78, and RANTES). Since monolayer cultures are a limited model for polarized tubular epithelial cells, we studied the side-dependent IL-8 secretion of TEC by using cell culture inserts as a basement membrane imitation. Primary cultures of proximal TEC were stimulated with differently fimbriated mutants ofEscherichia coli,E. coliLPS, S-fimbria isolates, and IL-1α. IL-8 protein was measured by enzyme-linked immunosorbent assay, and IL-8-like biological activity was tested by measuring elastase release from polymorphonuclear cells in supernatants of the upper and lower compartments. IL-8 mRNA was compared by competitive PCR. IL-8 secretion by TEC into the basolateral environment was significantly higher than secretion into the apical compartment, representing the tubular lumen. However, stimulation of IL-8 secretion by TEC was restricted to IL-1α and was not inducible byE. colimutants, S fimbriae, or lipopolysaccharide. With this in vitro model of polarized TEC, we show that luminal contact of TEC with uropathogenicE. colidoes not result in enhanced IL-8 secretion. The basolaterally directed production of the neutrophil chemotactic factor IL-8 by TEC after stimulation with IL-1α might play an important role in the initiation of inflammatory cell influx into the renal parenchyma.
A P5 Peptide That Is Homologous to Peptide 10 of OprF from
Pseudomonas aeruginosa
Enhances Clearance of Nontypeable
Haemophilus influenzae
from Acutely Infected Rat Lung in the Absence of Detectable Peptide-Specific Antibody
Authors: Webb Dianne C. , Cripps Allan W.
Published: May 2009

Nontypeable
Haemophilus influenzae
(NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from
Pseudomonas aeruginosa
(MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.
DNA Vaccination with Genes EncodingToxoplasma gondiiAntigens GRA1, GRA7, and ROP2 Induces Partially Protective Immunity against Lethal Challenge in Mice
Authors: Vercammen M. , Scorza T. , Huygen K. , De Braekeleer J. , Diet R. , Jacobs D. , Saman E. , Verschueren H.
Published: May 2009

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encodingToxoplasma gondiiantigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals.T. gondiilysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-γ) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-γ. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two differentT. gondiistrains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethalT. gondiidose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination againstT. gondii.
Evaluation of
Mycobacterium tuberculosis
Genes Involved in Resistance to Killing by Human Macrophages
Authors: Miller Barbara H. , Shinnick Thomas M.
Published: May 2009

A coinfection assay was developed to examine
Mycobacterium tuberculosis
genes suspected to be involved in resistance to killing by human macrophages. THP-1 macrophages were infected with a mixture of equal numbers of recombinant
Mycobacterium smegmatis
LR222 bacteria expressing an
M. tuberculosis
gene and wild-type
M. smegmatis
LR222 bacteria expressing the
xylE
gene. At various times after infection, the infected macrophages were lysed and the bacteria were plated. The resulting colonies were sprayed with catechol to determine the number of recombinant colonies and the number of
xylE
-expressing colonies.
M. smegmatis
bacteria expressing the
M. tuberculosis
glutamine synthetase A (
glnA
) gene or open reading frame
Rv2962c
or
Rv2958c
demonstrated significantly increased survival rates in THP-1 macrophages relative to those of
xylE
-expressing bacteria.
M. smegmatis
bacteria expressing
M. tuberculosis
genes for phospholipase C (
plcA
and
plcB
) or for high temperature requirement A (
htrA
) did not.
Inhibition of
Borrelia burgdorferi
Migration from the Midgut to the Salivary Glands following Feeding by Ticks on OspC-Immunized Mice
Authors: Gilmore Robert D. , Piesman Joseph
Published: May 2009

Borrelia burgdorferi
-infected ticks were fed on either OspC-immunized mice or normal, nonimmunized mice. After 72 h, the ticks were detached, followed by dissection and subsequent culturing in Barbour-Stoenner-Kelley II medium of the salivary glands from each tick to determine the presence of borreliae. Forty percent (10 of 25) of salivary glands from ticks that had fed on nonimmunized mice were culture positive, while only 7.4% (2 of 27) of salivary glands from ticks that had fed on OspC-immunized mice were culture positive, thus indicating a much reduced borrelial migration from the midgut when the bloodmeal contained anti-OspC antibodies. Fluorescent antibody staining of the corresponding midguts from ticks that had fed on the OspC-immunized mice showed that borreliae were present but did not produce OspC. In contrast, borreliae in midguts from ticks that had fed on normal mice demonstrated substantial
ospC
expression. This study provides evidence that, during tick feeding on an OspC-immunized host, transmission of borreliae from the tick is prevented; it also suggests that OspC functions in a tick-to-host transmission mechanism.
Lymphocyte Function-Associated Antigen 1 Is a Receptor forPasteurella haemolyticaLeukotoxin in Bovine Leukocytes
Authors: Jeyaseelan S. , Hsuan S. L. , Kannan M. S. , Walcheck B. , Wang J. F. , Kehrli M. E. , Lally E. T. , Sieck G. C. , Maheswaran S. K.
Published: May 2009

Pasteurella (Mannheimia) haemolyticaleukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate thatP. haemolyticaLkt binds to bovine CD18, the common subunit of all β2 integrins. We designed experiments with the following objectives: to identify which member of the β2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced β2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml;P< 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P< 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.
Hydrogen-bond self-assembly of DNA-base analogues — Experimental results
Authors: Rakotondradany Felaniaina , Sleiman Hanadi , Whitehead M. A.
Published: May 2009

A novel biomimetic DNA analogue with fluorescence has been synthesized to generate functional supramolecular architectures. Experimental studies show that triaminopyrimidine nucleoside (2) undergoes a sterically controlled self-assembly into hydrogen-bonded linear tapes and hexameric rosettes. Self-association of the hydrogen-bonded triaminopyrimidine–cyanuric acid complex into elongated, rodlike nanostructures was shown by dynamic light scattering and transmission electron microscopy, suggesting hierarchical formation of higher-order, π-stacked assemblies. The hydrogen-bond self-assembly of the DNA analogue decreased the fluorescence of the nucleosides. This guest-induced fluorescence quenching can be used to develop DNA-hybridization probes. MM+ molecular modelling and semi-empirical molecular orbital PM3 calculations (1) predicted the incorporation of triaminopyrimidine nucleoside into new types of artificial DNA strands and triplex formation with natural, complementary DNA strands containing thymine (1).
8-Isocyanoamphilecta-11(20),15-diene, a new antimalarial isonitrile diterpene from the sponge Ciocalapata sp.
Authors: Wattanapiromsakul Chatchai , Chanthathamrongsiri Naphatson , Bussarawit Somchai , Yuenyongsawad Supreeya , Plubrukarn Anuchit , Suwanborirux Khanit
Published: May 2009

A new isonitrile diterpene of the amphilectane family, 8-isocyanoamphilecta-11(20),15-diene (4), was isolated from the sponge Ciocalapata sp., along with three known isonitriles, 8,15-diisocyano-11(20)-amphilectene (1), 7-isocyanoamphilecta-11(20),15-diene (2), and 8-isocyanoamphilecta-11(20),14-diene (3), and two steroidal peroxides, ergosterol peroxide (5) and 5α,9α-epidioxy-8α,14α-epoxy-(22E)-ergosta-6,22-dien-3β-ol (6). The structure of the new isonitrile was elucidated spectroscopically. In addition, anomalous multiplicities in the NMR spectra of some isolated isonitriles were observed and are reported here. The four isonitriles were strongly active against Plasmodium falciparum K1 with IC50 in a range of 0.09–1.07 μmol/L. Except for 1, which was cytotoxic against both MCF-7 and fibroblast cell lines, the other three diterpenes showed no significant cytotoxicity against either targeted cell lines. On the other hand, the steroidal peroxides 5 and 6, which were less active in the antimalarial bioassay (IC50 values of 6.28 and 7.13 µmol/L, respectively), were strongly cytotoxic against MCF-7 (IC50 values of 0.025 and 0.003 µmol/L, respectively), with very little toxicity against human fibroblasts.
Controlling the regioselectivity of the di-π-methane rearrangements of 1,2-naphtho-annelated barrelene derivatives — Solution versus solid-state photochemistry
Authors: Luo Jia , Ihmels Heiko , Deiseroth Hans-Jörg , Schlosser Marc
Published: May 2009

The synthesis of 1,2-naphtho-annelated barrelene derivatives, namely dimethyl-7,12-dihydro-7,12-ethenobenzo[a]anthracene-13,14-dicarboxylate (4a) and dimethyl-7,14-dihydro-7,14-ethenodibenzo[a,j]-anthracene-15,16-dicarboxylate (4b), and the investigation of their photoreactivity in solution and in the solid state is reported. The irradiation of 4a and 4b resulted in regioselective di-π-methane rearrangements to give semibullvalene products; however, the product distribution is inverted upon changing the reaction medium from solution to the solid state. In solution, an α-naphtho–vinyl bridging predominates as the initial photochemical step because of the selective stabilization of the corresponding transition state, as discussed on the basis of the structures of the related biradical intermediates, whereas the solid-state photoreaction is significantly influenced by the steric constraints within the crystal lattice that only allow a β-naphtho–vinyl bridging.
Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin
Authors: Perry M. D. , Sandle G. I.
Published: May 2009

High-conductance apical K+(BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K+losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+enrichment. We found that PKA activation using 50 μmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 μmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 μmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 μmol/l Na+orthovanadate and 10 μmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.
Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development
Authors: Vidrich Alda , Buzan Jenny M. , Brodrick Brooks , Ilo Chibuzo , Bradley Leigh , Fendig Kirstin Skaar , Sturgill Thomas , Cohn Steven M.
Published: May 2009

Fibroblast growth factor receptor 3 (FGFR-3) is expressed in the lower crypt epithelium, where stem cells of the intestine reside. The role of FGFR-3 signaling in regulating features of intestinal morphogenesis was examined in FGFR-3-null (FGFR-3−/−) mice. FGFR-3−/−mice had only about half the number of intestinal crypts and a marked decrease in the number of functional clonogenic stem cells, as assessed by an in vivo microcolony-forming assay, compared with wild-type littermates. A marked deficit in allocation of progenitor cells to Paneth cell differentiation was noted, although all the principal epithelial lineages were represented in FGFR-3−/−mice. The total cellular content and nuclear localization of β-catenin protein were reduced in FGFR-3−/−mice, as was expression of cyclin D1 and matrix metalloproteinase-7, major downstream targets of β-catenin/T cell factor-4 (Tcf-4) signaling. Activation of FGFR-3 in Caco-2 cells, an intestinal epithelial cell line, abrogated the fall in β-catenin/Tcf-4 signaling activity that is normally observed in these cells as cultures become progressively more confluent. These findings are consistent with the hypothesis that, during intestinal development, FGFR-3 signaling regulates crypt epithelial stem cell expansion and crypt morphogenesis, as well as Paneth cell lineage specification, through β-catenin/Tcf-4-dependent and -independent pathways.
Discrete responses of myenteric neurons to structural and functional damage by neurotoxins in vitro
Authors: Lourenssen Sandra , Miller Kurtis G. , Blennerhassett Michael G.
Published: May 2009

Damage to the enteric nervous system is implicated in human disease and animal models of inflammatory bowel disease, diabetes, and Parkinson's disease, but the mechanism of death and the response of surviving neurons are poorly understood. We explored this in a coculture model of myenteric neurons, glia, and smooth muscle during exposure to the established or potential neurotoxins botulinum A, hydrogen peroxide, and acrylamide. Neuronal survival, axonal degeneration and regeneration, and neurotransmitter release were assessed during acute exposure (0–24 h) to neurotoxin and subsequent recovery (96–144 h). Unique and selective responses to each neurotoxin were found with acrylamide (0.5–2.0 mM) causing a 30% decrease in axon number without neuronal loss, whereas hydrogen peroxide (1–200 μM) caused a parallel loss in both axon and neuron number. Immunoblotting identified the loss of synaptic vesicle proteins that paralleled axon damage and was associated with marked suppression of depolarization-induced release of acetylcholine (ACh). The caspase inhibitor zVAD, but not DEVD, significantly prevented neuronal death, implying a largely caspase-3/7-independent mechanism of apoptotic death that was supported by staining for annexin V and cleaved caspase-3. In contrast, botulinum A (2 μg/ml) caused a 40% decrease in ACh release without effect on neuronal survival or axon structure. By 96 h after exposure to acrylamide or hydrogen peroxide, axon number was restored to or even surpassed the level of time-matched controls, regardless of partial neuronal loss, but ACh release remained markedly suppressed. Neural responses to toxic factors are initially unique but then converge upon robust axonal regeneration, whereas neurotransmitter release is both vulnerable to damage and slow to recover.
Hyperinflation-induced cardiorespiratory failure in rats
Authors: Simpson Jeremy A. , Brunt Keith R. , Collier Christine P. , Iscoe Steve
Published: May 2009

We previously showed that severe inspiratory resistive loads cause acute (<1 h) cardiorespiratory failure characterized by arterial hypotension, multifocal myocardial infarcts, and diaphragmatic fatigue. The mechanisms responsible for cardiovascular failure are unknown, but one factor may be the increased ventricular afterload caused by the large negative intrathoracic pressures generated when breathing against an inspiratory load. Because expiratory threshold loads increase intrathoracic pressure and decrease left ventricular afterload, we hypothesized that anesthetized rats forced to breathe against such a load would experience only diaphragmatic failure. Loading approximately doubled end-expiratory lung volume, halved respiratory frequency, and caused arterial hypoxemia and hypercapnia, respiratory acidosis, and increased inspiratory drive. Although hyperinflation immediately reduced the diaphragm's mechanical advantage, fatigue did not occur until near load termination. Mean arterial pressure progressively fell, becoming significant (cardiovascular failure) midway through loading despite tachycardia. Loading was terminated (endurance 125 ± 43 min; range 82–206 min) when mean arterial pressure dropped below 50 mmHg. Blood samples taken immediately after load termination revealed hypoglycemia, hyperkalemia, and cardiac troponin T, the last indicating myocardial injury that was, according to histology, mainly in the right ventricle. This damage probably reflects a combination of decreased O2delivery (decreased venous return and arterial hypoxemia) and greater afterload due to hyperinflation-induced increase in pulmonary vascular resistance. Thus, in rats breathing at an increased end-expiratory lung volume, cardiorespiratory, not just respiratory, failure still occurred. Right heart injury and dysfunction may contribute to the increased morbidity and mortality associated with acute exacerbations of obstructive airway disease.
Myc Regulates Embryonic Vascular Permeability and Remodeling
Authors: Kokai Enikö , Voss Florian , Fleischer Frank , Kempe Sybille , Marinkovic Dragan , Wolburg Hartwig , Leithäuser Frank , Schmidt Volker , Deutsch Urban , Wirth Thomas
Published: May 2009

Previous work has shown that c-Myc is required for adequate vasculogenesis and angiogenesis. To further investigate the contribution of Myc to these processes, we conditionally expressed c-Myc in embryonic endothelial cells using a tetracycline-regulated system. Endothelial Myc overexpression resulted in severe defects in the embryonic vascular system. Myc-expressing embryos undergo widespread edema formation and multiple hemorrhagic lesions. They die between embryonic days 14.5 and 17.5. The changes in vascular permeability are not caused by deficiencies in vascular basement membrane composition or pericyte coverage. However, the overall turnover of endothelial cells is elevated as is revealed by increased levels of both proliferation and apoptosis. Whole-mount immunohistochemical analysis revealed alterations in the architecture of capillary networks. The dermal vasculature of Myc-expressing embryos is characterized by a reduction in vessel branching, which occurs despite upregulation of the proangiogenic factors vascular endothelial growth factor-A and angiopoietin-2. Thus, the net outcome of an excess of vascular endothelial growth factor-A and angiopoietin-2 in the face of an elevated cellular turnover appears to be a defect in vascular integrity.
Causes and Severity of Ischemic Stroke in Patients With Symptomatic Intracranial Arterial Stenosis
Authors: Famakin Bolanle M. , Chimowitz Marc I. , Lynn Michael J. , Stern Barney J. , George Mary G.
Published: May 2009

Background and Purpose—

There are limited data on the causes and severity of subsequent stroke in patients presenting initially with TIA or stroke attributed to intracranial arterial stenosis.

Methods—

We evaluated the location, type (lacunar vs nonlacunar), cause, and severity of stroke in patients who had an ischemic stroke endpoint in the Warfarin Aspirin Symptomatic Intracranial Disease (WASID) trial.

Results—

Of the 569 patients enrolled in the WASID trial, 106 patients (18.6%) had an ischemic stroke during a mean follow-up of 1.8 years. Stroke occurred in the territory of the symptomatic artery in 77 (73%) of 106 patients. Among the 77 strokes in the territory, 70 (91%) were nonlacunar and 34 (44%) were disabling. Stroke out of the territory of the symptomatic artery occurred in 29 (27%) of 106 patients. Among these 29 strokes, 24 (83%) were nonlacunar, 14 (48%) were attributed to previously asymptomatic intracranial stenosis, and 9 (31%) were disabling.

Conclusions—

Most subsequent strokes in patients with symptomatic intracranial artery stenosis are in the same territory and nonlacunar, and nearly half of the strokes in the territory are disabling. The most commonly identified cause of stroke out of the territory was a previously asymptomatic intracranial stenosis. Penetrating artery disease was responsible for a low number of strokes.
Clinical–Diffusion Mismatch and Benefit From Thrombolysis 3 to 6 Hours After Acute Stroke
Authors: Ebinger Martin , Iwanaga Takeshi , Prosser Jane F. , De Silva Deidre A. , Christensen Soren , Collins Marnie , Parsons Mark W. , Levi Christopher R. , Bladin Christopher F. , Barber P. Alan , Donnan Geoffrey A. , Davis Stephen M.
Published: May 2009

Background and Purpose—

The clinical-diffusion mismatch (CDM) model has been proposed as a simpler tool than perfusion-diffusion mismatch (PDM) to select acute ischemic stroke patients for thrombolytic therapy. We hypothesized that in the 3- to 6-hour time window, the effect of tPA was significantly greater in patients with CDM than in patients without CDM.

Methods—

This is a substudy of EPITHET, a double-blind multi-center study of 100 patients randomized to tPA or placebo 3 to 6 hours after stroke onset. MRI was obtained before treatment, and at 3 to 5 days and 90 days after treatment. Presence of PDM (perfusion deficit/DWI
volume
>1.2 and perfusion deficit at least 10 mL>DWI
volume
) and CDM (NIHSS ≥8 and DWI
volume
≤25 mL) was determined for each patient. We assessed lesion growth and neurological improvement (decrease in NIHSS ≥8 points between baseline and 90 days, or a 90-day NIHSS ≤1).

Results—

86% of the patients had PDM, but only 41% had CDM. CDM detected PDM with a sensitivity of 46% and a specificity of 86%. We found statistically significant effects of reperfusion on the rate of neurological improvement (OR 9.92, 95% CI 1.91 to 51.64;
P
<0.01) and on absolute growth (difference: −59.60 mL, 95% CI −95.40 mL to −23.81 mL;
P
<0.01). Neither treatment with tPA nor reperfusion had a significantly different impact on lesion growth or clinical course in CDM patients compared to patients without CDM.

Conclusions—

There was no increased benefit from tPA in patients with CDM. The beneficial effects of reperfusion were similar in patients with and without CDM.
Beyond Circularity and Normativity: Measurement and Progress in Behavioral Economics
Authors: Nagatsu Michiru
Published: May 2009

This article assesses two major conceptual arguments against theories of choice.The first argument concerns the circularity of belief-desire psychology, on which decision theory is based. The second argument concerns the normativity arising from the concept of rationality. Each argument is evaluated against experimental practice in economics and psychology, and it is concluded that both arguments fail to establish their skeptical conclusion that there can be no science of intentional human actions.
Studies on Sugarcane Bagasse Fiber—Thermoplastics Composites
Authors: Youssef H.A. , Ismail M.R. , Ali M.A.M. , Zahran A.H.
Published: May 2009

The influence of thermoplastic/bagasse fiber ratio, as well as electron beam irradiation on the physicomechanical properties of low-density polyethylene (LDPE) and high-density polyethylene (HDPE) composites has been investigated. The concentration of bagasse fibers ranges from 20 to 80 wt%. The results indicate that there is deterioration in the properties as fiber concentration increased; however, it becomes significant only beyond 50% (w/w) bagasse fibers in both LDPE and HDPE composites. Also, the data show that this decrease in the properties is more pronounced in HDPE rather than LDPE composites. It can be noticed that enhancing adhesion between fibers and thermoplastics can be achieved by irradiation of the thermoplastic phase prior to mixing at 10 up to 60 kGy; a maximum improvement in the properties is observed at 40 and 10 kGy for LDPE and HDPE, respectively. From the data it can be seen that the increase in the flexural properties is higher than 100%, while it does not exceed 65% for impact toughness. On the other hand, the reduction in water absorption is not more than 5.5%. Also, examining SEM micrographs reveals that there is some sort of adhesion between fibers and thermoplastics phases.