bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Na‐K‐ATPase is a target for an EDHF displaying characteristics similar to potassium ions in the porcine renal interlobar artery
Authors: Büssemaker Eckhart , Wallner Christian , Fisslthaler Beate , Fleming Ingrid 
Published: October 2002

The present study was performed to determine the characteristics of the endothelium‐derived hyperpolarizing factor (EDHF) that mediates the nitric oxide (NO)‐ and prostacyclin (PGI2)‐independent hyperpolarization and relaxation of porcine renal interlobar arteries.
Bradykinin‐induced changes in isometric force or smooth muscle membrane potential were assessed in rings of porcine renal interlobar artery preconstricted with the thromboxane analogue U46619 in the continuous presence of Nω‐nitro‐L‐arginine and diclofenac to inhibit NO synthases and cyclo‐oxygenases.
Inhibition of NO‐ and PGI2‐production induced a rightward shift in the concentration‐relaxation curve to bradykinin without affecting maximal relaxation. EDHF‐mediated relaxation was abolished by a depolarizing concentration of KCl (40 mM) as well as by a combination of charybdotoxin and apamin (each 100 nM), two inhibitors of calcium‐dependent K+ (K+Ca) channels. Charybdotoxin and apamin also reduced the bradykinin‐induced, EDHF‐mediated hyperpolarization of smooth muscle cells from 13.7±1.3 mV to 5.7±1.2 mV.
In addition to the ubiquitous α1 subunit of the Na‐K‐ATPase, the interlobar artery expressed the γ subunit as well as the ouabain‐sensitive α2, α3 subunits. A low concentration of ouabain (100 nM) abolished the EDHF‐mediated relaxation and reduced the bradykinin‐induced hyperpolarization of smooth muscle cells (13.6±2.8 mV versus 5.20±1.39 mV in the absence and presence of ouabain).
Chelation of K+, using cryptate 2.2.2., inhibited EDHF‐mediated relaxation, without affecting NO‐mediated responses. Elevating extracellular KCl (from 4 to 14 mM) elicited a transient, ouabain‐sensitive hyperpolarization and relaxation that was endothelium‐independent and insensitive to charybdotoxin and apamin.
These results indicate that in the renal interlobar artery, EDHF‐mediated responses display the pharmacological characteristics of K+ ions released from endothelial K+Ca channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of highly ouabain‐sensitive subunits of the Na‐K‐ATPase.

British Journal of Pharmacology (2002) 137, 647–654. doi:10.1038/sj.bjp.0704919
Calmodulin increases transmitter release by mobilizing quanta at the frog motor nerve terminal
Authors: Brailoiu Eugen , Miyamoto Michael D , Dun Nae J 
Published: October 2002

The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal.
Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p).
Administration of CaM, but not heat‐inactivated CaM, encapsulated in liposomes (1000 units ml−1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM.
Administration of antiCaM, but not heat‐inactivated antiCaM, in liposomes (50 μl ml−1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size.
Bath application of the membrane‐permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM.
Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.

British Journal of Pharmacology (2002) 137, 719–727. doi:10.1038/sj.bjp.0704923
Immunochemical pulsed‐labeling characterization of intermediates during hen lysozyme oxidative folding
Authors: Jarrett Nicole M. , Djavadi‐Ohaniance Lisa , Willson Richard C. , Tachibana Hideki , Goldberg Michel E. 
Published: October 2002

AbstractPrevious studies have shown that reduced hen egg white lysozyme refolds and oxidizes according to a linear model, in which the number of disulfide bonds increases sequentially. In this study, we describe the kinetics of native tertiary structure formation during the oxidative‐renaturation of reduced hen egg white lysozyme, as monitored using an immunochemical pulsed‐labeling method based on enzyme‐linked immunosorbent assay (ELISA) in conjuction with two monoclonal antibodies (mAb). Each of these antibodies recognizes a separate face of the native lysozyme surface and, more importantly, each epitope is composed of discontinuous regions of the polypeptide chain. Renaturation kinetics were studied under the same refolding conditions as previous investigations of the kinetics of the regain of far‐UV CD, fluorescence, enzymatic activity, and disulfide bonds. Comparison of our results with the results from those studies showed that the immunoreactivity (i.e., the native fold) of the α‐domain appeared in intermediates containing two SS bonds only (C6–C127 and C30–C115), while the immunoreactivity of the β‐domain appeared together with the formation of the third SS bond (C64–C80). Thus, the α‐domain folds before the β‐domain during the oxidative folding of reduced lysozyme.
Specific Immune Responses and Enhancement of Murine Pulmonary Clearance of
Moraxella catarrhalis
by Intranasal Immunization with a Detoxified Lipooligosaccharide Conjugate Vaccine
Authors: Jiao Xinan , Hirano Takashi , Hou Yingchun , Gu Xin-Xing 
Published: October 2002

ABSTRACT

Moraxella catarrhalis
is an important human mucosal pathogen. This study investigated the effect of intranasal immunization with a detoxified-lipooligosaccharide-cross-reactive mutant of diphtheria toxin (dLOS-CRM) vaccine candidate on pulmonary clearance following an aerosol challenge of mice with
M. catarrhalis
. Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent increase of immunoglobulin A (IgA) and IgG in the nasal wash, lung lavage fluid, saliva, and fecal extract. In addition, serum IgG, IgM, and IgA against LOS of
M. catarrhalis
were detected. LOS-specific antibody-forming cells were found in the nasal passages, spleens, nasally associated lymphoid tissues, cervical lymph nodes, lungs, and Peyer's patches using an enzyme-linked immunospot assay. The dLOS-CRM vaccine induced a significant bacterial clearance (70 to 90%) of both homologous and heterologous strains in the lungs compared to that observed in the controls (
P
< 0.01). Intriguingly, intranasal immunization with dLOS-CRM showed a higher level of bacterial clearance compared with subcutaneous injections with dLOS-CRM. These data indicate that dLOS-CRM induces specific mucosal and systemic immunity through intranasal immunization and also provides effective bacterial clearance. On the basis of these results, we believe that dLOS-CRM should undergo continued testing to determine whether it would induce protective immune response in humans.
Sequential Immunization with Vesicles Prepared from HeterologousNeisseria meningitidisStrains Elicits Broadly Protective Serum Antibodies to Group B Strains
Authors: Moe Gregory R. , Zuno-Mitchell Patricia , Hammond Samantha N. , Granoff Dan M. 
Published: October 2002

ABSTRACTThe capsular polysaccharide ofNeisseria meningitidisgroup B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity againstN. meningitidisstrains. An NspA-based vaccine prepared from protein expressed byNeisseriaalso may be more effective than the corresponding recombinant protein made inEscherichia coli.
Toxoplasma gondiiInduces Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Secretion by Human Fibroblasts: Implications for Neutrophil Apoptosis
Authors: Channon Jacqueline Y. , Miselis Kristin A. , Minns Laurie A. , Dutta Chaitali , Kasper Lloyd H. 
Published: October 2002

ABSTRACTHuman neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derivedToxoplasma gondiitachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived fromT. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor α subunit (IL-3Rα), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, Giproteins, phosphatidylinositol 3-kinase, p44erk1and p42erk2mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted byT. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in theT. gondii-infected host.
Modulation of a Heterologous Immune Response by the Products ofAscaris suum
Authors: Paterson Jacqueline C. M. , Garside Paul , Kennedy Malcolm W. , Lawrence Catherine E. 
Published: October 2002

ABSTRACTHelminth infections are among the most potent stimulators of Th2-type immune responses and have been widely demonstrated to modify responsiveness to both nonparasite antigens and other infectious agents in a nonspecific manner in infected animals. We investigated the immunomodulatory properties of pseudocoelomic body fluid from adultAscaris suumgastrointestinal helminths (ABF) and its defined allergen (ABA-1) by examining their effects on the immune response to a heterologous antigen, ovalbumin. Our results indicate that ABF has potent immunomodulatory activity and that the effects observed are consistent with skewing towards a Th2-type response rather than induction of anergy. Our findings show that the immunomodulatory activities of ABF are associated with components other than the major constituent and putative allergen, ABA-1. Furthermore, the allergic responses to ABA-1 are not a result of an intrinsic allergenicity of the protein but are more a reflection of the wider induction of a Th2 response by the infection. Importantly, the induction of interleukin-10 by ABF also suggests that T regulatory cells may play a role in immunomodulation of immune responses by parasitic helminths.
Corticotropin-Releasing Hormone Augments Proinflammatory Cytokine Production from Macrophages In Vitro and in Lipopolysaccharide-Induced Endotoxin Shock in Mice
Authors: Agelaki Sofia , Tsatsanis Christos , Gravanis Achille , Margioris Andrew N. 
Published: October 2002

ABSTRACTCorticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-α, IL-1β, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.
Responses of Bovine WC1+γδ T Cells to Protein and Nonprotein Antigens ofMycobacterium bovis
Authors: Welsh Michael D. , Kennedy Hilary E. , Smyth Allister J. , Girvin R. Martyn , Andersen Peter , Pollock John M. 
Published: October 2002

ABSTRACTWC1+γδ T cells ofMycobacterium bovis-infected cattle are highly responsive toM. bovissonic extract (MBSE). In mycobacterial infections of other species, γδ T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1+γδ T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1+γδ T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context ofM. bovisinfection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1+γδ T cells in comparison with CD4+αβ T cells. Both cell types proliferated strongly in response to MBSE, with CD4+T cells being the major producers of gamma interferon (IFN-γ). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4+T-cell responses, whereas some WC1+γδ T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-γ secretion in WC1+γδ T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1+γδ T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1+γδ T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1+γδ T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1+γδ T-cell proliferation and IFN-γ secretion inM. bovisinfection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.
Genetic Analysis and Functional Characterization of the
Streptococcus pneumoniae vic
Operon
Authors: Wagner Christian , Saizieu Antoine de , Schönfeld Hans-Joachim , Kamber Markus , Lange Roland , Thompson Charles J. , Page Malcolm G. 
Published: October 2002

ABSTRACT

The
vic
two-component signal transduction system of
Streptococcus pneumoniae
is essential for growth. The
vic
operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the
Bacillus subtilis
genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the
vic
operon was mapped 16 bp upstream of the ATG codon of
vicR
. Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for
vic
might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.
The
Haemophilus ducreyi
Serum Resistance Antigen DsrA Confers Attachment to Human Keratinocytes
Authors: Cole Leah E. , Kawula Thomas H. , Toffer Kristen L. , Elkins Christopher 
Published: October 2002

ABSTRACT

Haemophilus ducreyi
is the etiologic agent of the sexually transmitted genital ulcer disease chancroid.
H. ducreyi
serum resistance protein A (DsrA) is a member of a family of multifunctional outer membrane proteins that are involved in resistance to killing by human serum complement. The members of this family include YadA of
Yersinia
species, the UspA proteins of
Moraxella catarrhalis
, and the Eib proteins of
Escherichia coli.
The role of YadA, UspA1, and UspA2H as eukaryotic cell adhesins and the function of UspA2 as a vitronectin binder led to our investigation of the cell adhesion and vitronectin binding properties of DsrA. We found that DsrA was a keratinocyte-specific adhesin as it was necessary and sufficient for attachment to HaCaT cells, a keratinocyte cell line, but was not required for attachment to HS27 cells, a fibroblast cell line. We also found that DsrA was specifically responsible for the ability of
H. ducreyi
to bind vitronectin. We then theorized that DsrA might use vitronectin as a bridge to bind to human cells, but this hypothesis proved to be untrue as eliminating HaCaT cell binding of vitronectin with a monoclonal antibody specific to integrin α
v
β
5
did not affect the attachment of
H. ducreyi
to HaCaT cells. Finally, we wanted to examine the importance of keratinocyte adhesion in chancroid pathogenesis so we tested the wild-type and
dsrA
mutant strains of
H. ducreyi
in our swine models of chancroid pathogenesis. The
dsrA
mutant was less virulent than the wild type in both the normal and immune cell-depleted swine models of chancroid infection.
Transfer of the Cholera Toxin A1 Polypeptide from the Endoplasmic Reticulum to the Cytosol Is a Rapid Process Facilitated by the Endoplasmic Reticulum-Associated Degradation Pathway
Authors: Teter Ken , Allyn Rebecca L. , Jobling Michael G. , Holmes Randall K. 
Published: October 2002

ABSTRACT
The active pool of internalized cholera toxin (CT) moves from the endosomes to the Golgi apparatus en route to the endoplasmic reticulum (ER). The catalytic CTA1 polypeptide is then translocated from the ER to the cytosol, possibly through the action of the ER-associated degradation (ERAD) pathway. Translocation was previously measured indirectly through the downstream effects of CT action. We have developed a direct biochemical assay for CTA1 translocation that is independent of toxin activity. Our assay is based upon the farnesylation of a CVIM motif-tagged CTA1 polypeptide (CTA1-CVIM) after it enters the cytosol. When expressed from a eukaryotic vector in transfected CHO cells, CTA1-CVIM was targeted to the ER, but was not secreted. Instead, it was translocated into the cytosol and degraded in a proteosome-dependent manner. Translocation occurred rapidly and was monitored by the appearance of farnesylated CTA1-CVIM in the detergent phase of cell extracts generated with Triton X-114. Detergent-phase partitioning of CTA1-CVIM resulted from the cytoplasmic addition of a 15-carbon fatty acid farnesyl moiety to the cysteine residue of the CVIM motif. Our use of the CTA1-CVIM translocation assay provided supporting evidence for the ERAD model of toxin translocation and generated new information on the timing of CTA1 translocation.
Acquisition of Regulators of Complement Activation byStreptococcus pyogenesSerotype M1
Authors: Pandiripally Vinod , Gregory Eugene , Cue David 
Published: October 2002

ABSTRACTOpsonization of bacteria by complement proteins is an important component of the immune response. The pathogenic bacteriumStreptococcus pyogeneshas evolved multiple mechanisms for the evasion of complement-mediated opsonization. One mechanism involves the binding of human regulators of complement activation such as factor H (FH) and FH-like protein 1 (FHL-1). Acquisition of these regulatory proteins can limit deposition of the opsonin C3b on bacteria, thus decreasing the pathogen's susceptibility to phagocytosis. Binding of complement regulatory proteins byS. pyogeneshas previously been attributed to the streptococcal M and M-like proteins. Here, we report that theS. pyogenescell surface protein Fba can mediate binding of FH and FHL-1. We constructed mutant derivatives ofS. pyogenesthat lack Fba, M1 protein, or both proteins and assayed the strains for FH binding, susceptibility to phagocytosis, and C3 deposition. Fba expression was found to be sufficient for binding of purified FH as well as for binding of FH and FHL-1 from human plasma. Plasma adsorption experiments also revealed that M1+Fba+streptococci preferentially bind FHL-1, whereas M1−Fba+streptococci have similar affinities for FH and FHL-1. Fba was found to contribute to the survival of streptococci incubated with human blood and to inhibit C3 deposition on bacterial cells. Streptococci harvested from log-phase cultures readily bound FH, but binding was greatly reduced for bacteria obtained from stationary-phase cultures. Bacteria cultured in the presence of the protease inhibitor E64 maintained FH binding activity in stationary phase, suggesting that Fba is removed from the cell surface via proteolysis. Western analyses confirmed that E64 stabilizes cell surface expression of Fba. These data indicate that Fba is an antiopsonic, antiphagocytic protein that may be regulated by cell surface proteolysis.