bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the LDL‐A module mutants of Tva, the subgroup A Rous sarcoma virus receptor, and the implications in protein folding
Authors: Wang Qing‐Yin , Manicassamy Balaji , Yu Xuemei , Dolmer Klavs , Gettins Peter G.W. , Rong Lijun 
Published: October 2002

AbstractTva is the cellular receptor for subgroup A Rous sarcoma virus (RSV‐A), and the viral receptor function is solely determined by a 40‐residue motif called the LDL‐A module of Tva. In this report, an integral approach of molecular, biochemical, and biophysical techniques was used to examine the role of a well‐conserved tryptophan of the LDL‐A module of Tva in protein folding and ligand binding. We show that substitution of tryptophan by glycine adversely affected the correct folding of the LDL‐A module of Tva, with only a portion giving a calcium‐binding conformation. Furthermore, we show that the misfolded LDL‐A conformations of Tva could not efficiently bind to its ligand. These results indicate that this conserved tryptophan in the LDL‐A module of Tva plays an important role in correct protein folding and ligand recognition. Furthermore, these results suggest that the familial hypercholesterolemia (FH) French Canadian‐4 mutation is likely caused by protein misfolding of low‐density lipoprotein receptor, thus explaining the defect for this class of FH.
The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers
Authors: Ottosen Mette Brimheim , Björnberg Olof , Nørager Sofie , Larsen Sine , Palfey Bruce Allan , Jensen Kaj Frank 
Published: October 2002

AbstractThe flavoenzyme dihydroorotate dehydrogenase A from Lactococcus lactis is a homodimeric protein of 311 residues/subunit, and the two active sites are positioned at a distance from the dimer interface. To promote formation of the monomeric form of the enzyme, we changed the residues involved in formation of two salt bridges formed between the residues Glu206 of the one polypeptide and Lys296 of the other polypeptide. The mutant enzymes formed inactive precipitates when cells were grown at 37°C, but remained soluble and active when cells were grown at 25°C. The salt bridges were not needed for activity, because the mutant enzymes in which one of the residues was converted to an alanine (E206A or K296A) retained almost full activity. The mutant enzymes in which the charge of one of the residues of the salt bridge was inverted (i.e., E206K or K296E) were severely impaired. The double mutant E206K‐K296E, which has the possibility of forming salt bridges in the opposite orientation of the wild type, was fully active in concentrated solutions, but dissociated into inactive monomers upon dilution. The KD for the dimer to monomer dissociation reaction was 12 μM, and dimer formation was favored by the product, orotate, or by high ionic strength, indicating that the hydrophobic interactions are important for the subunit contacts. Wild‐type dihydroorotate dehydrogenase A was similarly found to dissociate into inactive monomers, but with a KD for dissociation equal to 0.12 μM. These results imply that the dimeric state is necessary for activity of the enzyme.
Role of
Vibrio cholerae
O139 Surface Polysaccharides in Intestinal Colonization
Authors: Nesper Jutta , Schild Stefan , Lauriano Crystal M. , Kraiss Anita , Klose Karl E. , Reidl Joachim 
Published: October 2002

ABSTRACT

Since the first occurrence of O139
Vibrio cholerae
as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a ≈30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.
Expression ofAnaplasma marginaleMajor Surface Protein 2 Operon-Associated Proteins during Mammalian and Arthropod Infection
Authors: Löhr Christiane V. , Brayton Kelly A. , Shkap Varda , Molad Thea , Barbet Anthony F. , Brown Wendy C. , Palmer Guy H. 
Published: October 2002

ABSTRACTThe antigenically variant major surface protein 2 (MSP2) ofAnaplasma marginaleis expressed from a 3.5-kb operon that contains, in a 5′-to-3′ direction, four open reading frames,opag3,opag2,opag1, andmsp2. This operon structure was shown to be conserved among genotypically and phenotypically distinctA. marginale,A. ovis, andA. centralestrains.The individual OpAG amino acid sequences are highly conserved amongA. marginalestrains, with identities ranging from 95 to 99%. OpAG2 and OpAG3 were expressed by all examinedA. marginalestrains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface. OpAG2 and OpAG3 were also expressed in an infectedIxodes scapularistick cell line. In contrast, the sameA. marginalestrains expressed only OpAG2 in two differentDermacentorspp. during transmission feeding. OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infectedDermacentorticks. The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition.
Nature and Specificity of the Required Protective Immune Response That Develops Postchallenge in Mice Vaccinated with the 19-Kilodalton Fragment ofPlasmodium yoeliiMerozoite Surface Protein 1
Authors: Wipasa Jiraprapa , Xu Huji , Makobongo Morris , Gatton Michelle , Stowers Anthony , Good Michael F. 
Published: October 2002

ABSTRACTImmunity induced by the 19-kDa fragment ofPlasmodium yoeliimerozoite surface protein 1 (MSP119) is dependent on high titers of specific antibodies present at the time of challenge and a continuing active immune response postinfection. However, the specificity of the active immune response postinfection has not been defined. In particular, it is not known whether anti-MSP119antibodies that arise following infection alone are sufficient for protection. We developed systems to investigate whether an MSP119-specific antibody response alone both prechallenge and postchallenge is sufficient for protection. We were able to exclude antibodies with other specificities, as well as any contribution of MSP119-specific CD4+T cells acting independent of antibody, and we concluded that an immune response focused solely on MSP119-specific antibodies is sufficient for protection. The data imply that the ability of natural infection to boost an MSP119-specific antibody response should greatly improve vaccine efficacy.
The Polysaccharide Portion Plays an Indispensable Role inSalmonellaLipopolysaccharide-Induced Activation of NF-κB through Human Toll-Like Receptor 4
Authors: Muroi Masashi , Tanamoto Ken-ichi 
Published: October 2002

ABSTRACTThe lipid A portion has been identified as the active center responsible for lipopolysaccharide (LPS)-induced macrophage activation. However, we found thatSalmonella(Salmonella entericaserovars Abortusequi, Minnesota, and Typhimurium) lipid A is inactive in human macrophages, despite its LPS being highly active. Thus we investigated the critical role of polysaccharide inSalmonellaLPS-induced activation of NF-κB. In human monocytic cell line THP-1,Salmonellalipid A and syntheticSalmonella-type lipid A (516) did not induce NF-κB-dependent reporter activity up to 1 μg/ml, whereas strong activation was observed in response toSalmonellaLPS. The difference in activity between this lipid A and LPS was further examined by using 293 cells expressing human CD14/Toll-like receptor 4 (TLR4)/MD-2, and similar results were obtained in these cells as well. A polysaccharide preparation obtained fromSalmonellaLPS was inactive in 293 cells expressing human CD14/TLR4/MD-2 even in combination with 516.Salmonella entericaserovar Minnesota Re LPS, whose structure consists of lipid A and two molecules of 2-keto-3-deoxyoctonic acid, but not its lipid A exhibited strong activity in THP-1 cells and 293 cells expressing human CD14/TLR4/MD-2. These results indicate that the polysaccharide portion covalently bound to lipid A plays the principal role inSalmonellaLPS-induced activation of NF-κB through human CD14/TLR4/MD-2.
Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge withLeptospira borgpeterseniiSerovar Hardjo: Involvement of WC1+γδ and CD4 T Cells
Authors: Naiman Brian M. , Blumerman Seth , Alt David , Bolin Carole A. , Brown Rachel , Zuerner Richard , Baldwin Cynthia L. 
Published: October 2002

ABSTRACTOrganisms within the Hardjo serovar ofLeptospiraspecies are harbored in cattle throughout the world, causing abortion in pregnant animals as well as being shed in the urine, thereby providing sources of zoonotic infection for humans. We recently showed that sterile immunity in vaccinated cattle is associated with induction of a type 1 (Th1) cell-mediated immune response. Here naïve and previously vaccinated pregnant cattle were challenged with a virulent strain of serovar Hardjo and subsequently evaluated for expression of a type 1 immune response. Lymphocytes that responded in a recall response to antigen by undergoing blast transformation were evident in cultures of peripheral blood mononuclear cells (PBMC) from vaccinated cattle throughout the postchallenge test period while those from naïve cattle were evident at one time point only. Nevertheless, beginning at 2 weeks after challenge, gamma interferon (IFN-γ) was measured in supernatants of antigen-stimulated PBMC cultures from nonvaccinated animals although the amount produced was always less than that in cultures of PBMC from vaccinated animals. IFN-γ+cells were also evident in antigen-stimulated cultures of PBMC from vaccinated but not from nonvaccinated animals throughout the postchallenge period. The IFN-γ+cells included CD4+and WC1+γδ T cells, and a similar proportion of these two subpopulations were found among the dividing cells in antigen-stimulated cultures as ascertained by carboxyfluorescein succinimidyl ester loading. Finally, while naïve and vaccinated animals had similar levels of antigen-specific immunoglobulin G1 (IgG1) following challenge, vaccinated animals had twofold-more IgG2. In conclusion, while infection may induce a type 1 response we suggest that it is too weak to prevent establishment of chronic infection.
Postexposure Prophylaxis against Anthrax: Evaluation of Various Treatment Regimens in Intranasally Infected Guinea Pigs
Authors: Altboum Zeev , Gozes Yehoshua , Barnea Ada , Pass Avi , White Moshe , Kobiler David 
Published: October 2002

ABSTRACTThe efficiency of postexposure prophylaxis againstBacillus anthracisinfection was tested in guinea pigs infected intranasally with either Vollum or strain ATCC 6605 spores (75 times the 50% lethal dose [LD50] and 87 times LD50,respectively). Starting 24 h postinfection, animals were treated three times per day for 14 days with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole (TMP-SMX). Administration of cefazolin and TMP-SMX failed to protect the animals, while ciprofloxacin, tetracycline, and erythromycin prevented death. Upon cessation of treatment all erythromycin-treated animals died; of the tetracycline-treated animals, two of eight infected with Vollum and one of nine infected with ATCC 6605 survived; and of the ciprofloxacin group injected with either 10 or 20 mg/kg of body weight, five of nine and five of five animals, respectively, survived. To test the added value of extending the treatment period, Vollum-infected (46 times the LD50) animals were treated for 30 days with ciprofloxacin or tetracycline, resulting in protection of eight of nine and nine of nine animals, respectively. Once treatment was discontinued, only four of eight and five of nine animals, respectively, survived. Following rechallenge (intramuscularly) of the survivors with 30 times the LD50of Vollum spores, all ciprofloxacin-treated animals were protected while none of the tetracycline-treated animals survived. In an attempt to confer protective immunity lasting beyond the termination of antibiotic administration, Vollum-infected animals were immunized with a protective antigen (PA)-based vaccine concurrently with treatment with either ciprofloxacin or tetracycline. The combined treatment protected eight of eight and nine of nine animals. Following cessation of antibiotic administration seven of eight and eight of eight animals survived, of which six of seven and eight of eight resisted rechallenge. These results indicate that a combined treatment of antibiotics together with a PA-based vaccine could provide long-term protection to prevent reoccurrence of anthrax disease.
Divergent Role of Gamma Interferon in a Murine Model of Pulmonary versus SystemicKlebsiella pneumoniaeInfection
Authors: Moore Thomas A. , Perry Michelle L. , Getsoian Andrew G. , Newstead Michael W. , Standiford Theodore J. 
Published: October 2002

ABSTRACTKlebsiella pneumoniaeis a leading cause of both community-acquired and nosocomial gram-negative-bacterial pneumonia. A further clinical complication of pulmonaryK. pneumoniaeinfection is dissemination of bacteria from the lung into the peripheral blood, resulting in bacteremia concurrent with the localized pulmonary infection. Here, we report studies detailing the divergent role of gamma interferon (IFN-γ) in pulmonary versus systemicK. pneumoniaeinfection. Intratracheal inoculation of IFN-γ knockout mice resulted in significantly increased mortality compared to that observed for wild-type infected animals. Increased mortality correlated with a 100-fold increase in pulmonary bacteria within 2 days postinfection and upregulation of lung-associated interleukin-10 (IL-10) mRNA. Interestingly, IFN-γ knockout mice had a twofold reduction in plasma aminospartate transferase activity, indicating diminished liver injury following peripheral blood bacterial dissemination. To study the host response towards blood-borne bacteria in the absence of the ongoing pulmonary infection, intravenous inoculation studies were initiated. IFN-γ knockout mice were no more susceptible to intravenous infection than their wild-type counterparts. The consistent observation in IFN-γ knockout mice was for improved survival correlating with increased clearance of blood- and liver-associated bacteria. Intravenous inoculation resulted in a two- to threefold increase in hepatic IL-10 production 24 and 48 h postinfection. Liver injury was also significantly reduced in IFN-γ knockout mice. These data indicate that IFN-γ secretion is a critical mediator in the resolution of localized gram-negative pulmonary pneumonia. Surprisingly, host responses towards systemic infection with the same bacteria appear to be IFN-γ independent.
Human Leukocytes Adhere to, Penetrate, and Respond to
Staphylococcus aureus
Biofilms
Authors: Leid Jeff G. , Shirtliff Mark E. , Costerton J. W. , Stoodley and Paul 
Published: October 2002

ABSTRACT

Staphylococcus aureus
is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms.
S. aureus
bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured
S. aureus
biofilm.
Transcriptional Analysis of
Rickettsia prowazekii
Invasion Gene Homolog (
invA
) during Host Cell Infection
Authors: Gaywee Jariyanart , Radulovic Suzana , Higgins James A. , Azad Abdu F. 
Published: October 2002

ABSTRACT

An invasion gene homolog,
invA
, of
Rickettsia prowazekii
has recently been identified to encode a member of the Nudix hydrolase subfamily which acts specifically on dinucleoside oligophosphates (Np

n

N;
n
≥ 5), a group of cellular signaling molecules known as alarmones. InvA is thought to enhance intracellular survival by regulating stress-induced toxic nucleotide levels during rickettsial infection. To further characterize the physiological function of InvA, the gene expression pattern during various stages of rickettsial intracellular growth was investigated. Using semiquantitative reverse transcription-PCR (RT-PCR) and real-time fluorescent probe-based quantitative RT-PCR, a differential expression profile of
invA
during rickettsial host cell infection was examined. The
invA
transcript temporarily increased during the early period of infection. Expression of rickettsial
groEL
, a molecular indicator of cellular stresses, was also shown to be upregulated during the early period of infection. Furthermore,
invA
was cotranscribed in a polycistronic message with
rrp
, a gene encoding the response regulator protein homolog, which is a part of a two-component signal transduction system. These results support our earlier findings that under such stress conditions dinucleoside oligophosphate pyrophosphatase may function as a buffer, enhancing rickettsial survival within the cytoplasm of a eukaryotic cell. The expression of rickettsial dinucleoside oligophosphate pyrophosphatase may be regulated by a part of the two-component signal transduction system similar to that described for response regulators in other bacterial systems.
Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II
Authors: Demuth Donald R. , Irvine Douglas C. 
Published: October 2002

ABSTRACT

Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with
Streptococcus mutans
requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two
Streptococcus gordonii
M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related
S. mutans
antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact
S. gordonii
and
S. mutans
cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly α-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced α-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost α-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2- to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340.
Evidence Inconsistent with a Negative Influence of T Helper 2 Cells on Protection Afforded by a Dominant T Helper 1 Response againstMycobacterium tuberculosisLung Infection in Mice
Authors: Jung Yu-Jin , LaCourse Ronald , Ryan Lynn , North Robert J. 
Published: October 2002

ABSTRACTMice incapable of generating an efficient Th2 response because of functional deletion of the genes for signal transducer and activation of transcription 6 (Stat6), interleukin-4 receptor alpha chain (IL-4Rα), or IL-4 plus IL-13 (IL-4/IL-13) were no more resistant than wild-type (WT) mice to airborne infection with virulentMycobacterium tuberculosis. WT mice were able to control infection and hold it at a stationary level following 20 days of log linearM. tuberculosisgrowth. Likewise, infection was kept under control and was held at the same stationary level in IL-4/IL-13−/−mice but progressed to a slightly higher level in Stat6−/−and IL-4Rα−/−mice. The onset of stationary-level infection in WT mice was associated with the expression of Th1-mediated immunity, as evidenced by an approximately 100- to 1,000-fold increase in the lungs in the synthesis of mRNA for IL-12, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) that was sustained for at least 100 days. IL-12 is essential for the induction of Th1 immunity, IFN-γ is a key Th1 cytokine involved in mediation of immunity, and NOS2 is an inducible enzyme of macrophages and is needed by these cells to express immunity. In response to infection, the lungs of Stat6−/−mice showed increases in synthesis of mRNA for IL-12, IFN-γ, and NOS2 similar to that seen in WT mice. In IL-4/IL-13−/−mice, however, synthesis of mRNA for IFN-γ and NOS2 reached higher levels than in WT mice. These results argue against the notion that a Th2 response is partly or wholly responsible for the inability of Th1-mediated immunity to resolve infection with a virulent strain ofM. tuberculosis.
A Comparison of Murine and Human Immunoproteomes of
Helicobacter pylori
Validates the Preclinical Murine Infection Model for Antigen Screening
Authors: Bumann Dirk , Holland Petra , Siejak Frank , Koesling Jan , Sabarth Nicolas , Lamer Stefanie , Zimny-Arndt Ursula , Jungblut Peter R. , Meyer Thomas F. 
Published: October 2002

ABSTRACT

Preclinical mouse infection models are widely used for
Helicobacter
vaccine development, but how well such models mimic important aspects of human infections is unknown. A comparison of
Helicobacter pylori
immunoproteomes of infected mice with previously reported patient data reveals a high agreement in the antigens recognized, suggesting that
H. pylori
in vivo protein composition and recognition by the host immune system are comparable in mice and humans. Murine
Helicobacter
models may thus be valid to screen antigens for human vaccination.