bluuebunny/crossref_metadata_2025 huggingface dataset:
The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis
Authors: Reichmann Gaby , Walker William , Villegas Eric N. , Craig Linden , Cai Guifang , Alexander James , Hunter Christopher A.
Published: July 2002

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance toToxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice withT. gondiiresults in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected withT. gondiifor 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/−mice infected withT. gondiiproduced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/−mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication ofT. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance toT. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.
ankA
: an
Ehrlichia phagocytophila
Group Gene Encoding a Cytoplasmic Protein Antigen with Ankyrin Repeats
Authors: Caturegli Patrizio , Asanovich Kristin M. , Walls Jennifer J. , Bakken Johan S. , Madigan John E. , Popov Vsevolod L. , Dumler J. Stephen
Published: July 2002

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to
Ehrlichia phagocytophila
. To identify and characterize
E. phagocytophila
group-specific protein antigen genes, we prepared and screened HGE agent and
Ehrlichia equi
genomic DNA expression libraries using polyclonal equine
E. equi
antibodies. Two clones, one each from HGE agent and
E. equi
, that were recognized specifically by antibodies to the
E. phagocytophila
group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named
ank
(for ankyrin). When the encoded protein, named AnkA, was expressed in
Escherichia coli
, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and
E. equi
infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in
E. phagocytophila
group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this
Ehrlichia
protein may influence host cell gene expression.
The
toxR
Gene of
Vibrio
(
Listonella
)
anguillarum
Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model
Authors: Okuda Jun , Nakai Toshihiro , Chang Park Se , Oh Takanori , Nishino Takeshi , Koitabashi Tsutomu , Nishibuchi Mitsuaki
Published: July 2002

To examine the hypothesis that the ancestral role of the
toxR
gene in the family
Vibrionaceae
is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the
toxR
gene in
Vibrio anguillarum
, an important fish pathogen. The
toxR
gene of
V. angullarum
(Va-
toxR
) was cloned from strain PT-87050 isolated from diseased ayu (
Plecoglossus altivelis
), and the sequence was analyzed. The
toxR
sequence was 63 to 51% identical to those reported for other species of the family
Vibrionaceae
. Distribution of the Va-
toxR
gene sequence in
V. anguillarum
strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic
toxR
mutant of
V. anguillarum
PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the
toxR
mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the
toxR
mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the
toxR
mutant. For the
toxR
mutant, the MICs of two β-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the
toxR
-regulated major OMPs of the family
Vibrionaceae
. The results indicate that the
toxR
gene is not involved in virulence expression in
V. anguillarum
PT-24 and that
toxR
regulation of major OMPs is universal in the family
Vibrionaceae
. These results support the hypothesis that the ancestral role of the
toxR
gene is regulation of OMP gene expression and that only in some
Vibrio
species has ToxR been appropriated for the regulation of a virulence gene(s).
Macrophage Nitric Oxide Synthase Associates with Cortical Actin but Is Not Recruited to Phagosomes
Authors: Webb J. L. , Harvey M. W. , Holden David W. , Evans T. J.
Published: July 2002

Nitric oxide (NO) produced from inducible NO synthase (iNOS) is an important component of host defense against intracellular pathogens. To understand how phagocytes deliver NO to ingested microorganisms while avoiding cytotoxicity, we set out to study the subcellular localization of iNOS within macrophages following phagocytosis. Confocal microscopy of immunostained cells showed that iNOS was located not only diffusely within cytoplasm but also in vesicles, as well as immediately adjacent to the peripheral cell membrane. This peripheral iNOS colocalized with the cortical actin cytoskeleton and was removed by the actin-depolymerizing drug cytochalasin B. Biochemical fractionation of RAW 264 macrophages showed that 32.75% (±5.11%;
n
= 3) of iNOS was present in a particulate fraction, which cosedimented with low-density cellular vesicles. Following phagocytosis of latex beads, zymosan, immunoglobulin G-coated beads, or complement-coated zymosan, submembranous cortical iNOS was not recruited to phagosomes, nor was there any relocalization of intracellular iNOS. Similarly, following phagocytosis of
Salmonella enterica
serovar Typhimurium there was no recruitment of iNOS to the
Salmonella
vacuole at any stage after internalization. NO mediated significant killing of intracellular
S. enterica
serovar Typhimurium in RAW macrophages treated with lipopolysaccharide and gamma interferon; this was evident 4 h after infection. Although not recruited to phagosomes, iNOS association with the submembranous cortical actin cytoskeleton is ideally suited to deliver NO to microbes in contact with the cell surface and may contribute to early killing of ingested
Salmonella.
Intranasal Immunization with SAG1 and Nontoxic Mutant Heat-Labile Enterotoxins Protects Mice againstToxoplasma gondii
Authors: Bonenfant C. , Dimier-Poisson I. , Velge-Roussel F. , Buzoni-Gatel D. , Del Giudice G. , Rappuoli R. , Bout D.
Published: July 2002

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested withToxoplasma gondiiSAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with aT. gondiilysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain ofT. gondiiwas obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.
Reduced Virulence of aBordetella bronchisepticaSiderophore Mutant in Neonatal Swine
Authors: Register Karen B. , Ducey Thomas F. , Brockmeier Susan L. , Dyer David W.
Published: July 2002

One means by whichBordetella bronchisepticascavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene intoalcA, one of the genes essential for alcaligin biosynthesis. The virulence of thealcAmutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production byB. bronchisepticais not essential for colonization of swine but is required for maximal virulence.B. bronchisepticamutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.
Protective Efficacy of H Antigen fromHistoplasma capsulatumin a Murine Model of Pulmonary Histoplasmosis
Authors: Deepe George S. , Gibbons Reta
Published: July 2002

We previously reported that immunization with H antigen fromHistoplasma capsulatumdid not protect mice against an intravenous challenge with yeasts. Here, we investigated the utility of H antigen to protect mice in a model of pulmonary histoplasmosis. Mice immunized with H antigen and challenged intranasally 4 weeks postvaccination were protected against sublethal and lethal challenges withH. capsulatumyeasts. If the challenge was performed 3 months after vaccination, there was a reduction in fungal burden following sublethal challenge and a modest delay in mortality in mice given a lethal inoculum. Vaccination was associated with production of gamma interferon, granulocyte-macrophage colony-stimulating factor, interleukin-4, and interleukin-10 by splenocytes. Vaccination with H antigen was not accompanied by a major expansion of CD4+or CD8+cells in spleens of mice. These results demonstrate that H antigen may be useful as a protective immunogen against pulmonary exposure toH. capsulatum.
Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent
Streptococcus pneumoniae
Transformed with a Novel Gram-Positive
lux
Transposon
Authors: Francis Kevin P. , Yu Jun , Bellinger-Kawahara Carolyn , Joh Danny , Hawkinson Matthew J. , Xiao Grace , Purchio Tony F. , Caparon Michael G. , Lipsitch Marc , Contag Pamela R.
Published: July 2002

Animal studies with
Streptococcus pneumoniae
have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive
lux
transposon cassette, Tn
4001 luxABCDE
Km
r
, that allows random integration of
lux
genes onto the bacterial chromosome was constructed. The cassette was designed so that the
luxABCDE
and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome.
S. pneumoniae
D39 was transformed with plasmid pAUL-A Tn
4001 luxABCDE
Km
r
, and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical
S. pneumoniae
isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the
lux
construct was highly stable even after several days in vivo. We believe that this
lux
transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.
Enterotoxin Plasmid from
Clostridium perfringens
Is Conjugative
Authors: Brynestad Sigrid , Sarker Mahfuzur R. , McClane Bruce A. , Granum Per Einar , Rood Julian I.
Published: July 2002

Clostridium perfringens
enterotoxin is the major virulence factor involved in the pathogenesis of
C. perfringens
type A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene,
cpe
, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the
cpe
plasmid encodes its own conjugative transfer, a
C. perfringens
strain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to the
cpe
gene had been replaced by the chloramphenicol resistance gene
catP
, was used as a donor in matings with several
cpe
-negative
C. perfringens
isolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 × 10
−2
to 4.6 × 10
−4
transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype
C. perfringens
tetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the
cpe
plasmid occurred in vivo, it would have the potential to convert
cpe
-negative
C. perfringens
strains in normal intestinal flora into strains capable of causing gastrointestinal disease.
EspG, a Novel Type III System-Secreted Protein from Enteropathogenic
Escherichia coli
with Similarities to VirA of
Shigella flexneri
Authors: Elliott Simon J. , Krejany Efrosinia O. , Mellies Jay L. , Robins-Browne Roy M. , Sasakawa Chihiro , Kaper James B.
Published: July 2002

The function of the
rorf2
gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic
Escherichia coli
(EPEC) has not been described. We report that
rorf2
encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with
Shigella flexneri
protein VirA, and the cloned
espG
(
rorf2
) gene can rescue invasion in a
Shigella virA
mutant, indicating that these proteins are functionally equivalent in
Shigella
. An EPEC
espG
mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic
E. coli
strain carrying a mutant
espG
showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned
orf3
gene could also rescue invasion in a
Shigella virA
mutant, but an EPEC
espG orf3
double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.
Antigens of
Mycobacterium tuberculosis
Expressed during Preclinical Tuberculosis: Serological Immunodominance of Proteins with Repetitive Amino Acid Sequences
Authors: Singh K. K. , Zhang X. , Patibandla A. S. , Chien P. , Laal S.
Published: July 2002

Four antigens of
Mycobacterium tuberculosis
that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of
M. tuberculosis
genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system,
mtrA
-
mtrB,
of
M. tuberculosis
. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.
Differences in Components at Delayed-Type Hypersensitivity Reaction Sites in Mice Immunized with Either a Protective or a Nonprotective Immunogen ofCryptococcus neoformans
Authors: Nichols Kasie L. , Bauman Sean K. , Schafer Fredda B. , Murphy Juneann W.
Published: July 2002

Cell-mediated immunity is the major protective mechanism againstCryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-α) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-α is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-α activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.
Immunogenicity and Safety Testing of a Group B Intranasal Meningococcal Native Outer Membrane Vesicle Vaccine
Authors: Katial Rohit K. , Brandt Brenda L. , Moran Ellen E. , Marks Stephen , Agnello Victor , Zollinger Wendell D.
Published: July 2002

The presently licensed meningococcal vaccine is a tetravalent capsular polysaccharide vaccine that induces immunity to serogroups A, C, Y, and W-135 but not to group B, which causes nearly half of the meningitis cases in the United States. The purpose of this study was to evaluate the safety and immunogenicity of an intranasal native outer membrane vesicle (NOMV) vaccine prepared from a capsule negative strain of group B ofNeisseria meningitidis.In this study all volunteers received the same dose of vaccine, but we evaluated two different immunization schedules and the oropharyngeal and intranasal routes of vaccine delivery, assessed nasal cytology for cellular infiltration, and measured antibody-secreting cells (enzyme-linked immunospot assay [ELISPOT]) as an early marker for systemic immune response. Additionally, both intranasal and serum vaccine-specific antibodies were measured as well as serum bactericidal activity. Four groups with a total of 42 subjects were immunized on days 0, 28, and 56. Group 3 received an additional dose on day 7. Group 2 subjects were immunized both intranasally and oropharyngeally. Group 4 received a different lot of vaccine. All groups received approximately 1,200 μg of vaccine per subject. Patients were evaluated for side effects. The vaccine was well tolerated without evidence of inflammation on nasal cytology. The group receiving the extra vaccine dose showed the maximum increase in bactericidal activity. Thirty of 42 subjects demonstrated an increase in meningococcus-specific intranasal immunoglobulin A (IgA) titers, while 23 of 42 demonstrated an increase in specific IgG titers. The group receiving vaccine intranasally and oropharyngeally showed the highest rise in intranasal titers for both IgA and IgG. Groups 1, 3, and 4 showed a significant increase in antibody-secreting cells on ELISPOT. Eighteen of 42 volunteers demonstrated a fourfold or greater rise in bactericidal titers, with 81% showing an increase over baseline. We have demonstrated the immunogenicity and safety of a group B lipopolysaccharide-containing, intranasal, NOMV vaccine.
Modulation of Mouse Endotoxic Fever by Complement
Authors: Li S. , Holers V. M. , Boackle S. A. , Blatteis C. M.
Published: July 2002

It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS). The present study was designed to identify which component(s) of the complement cascade may be specifically critical. To this end, we used mice with C3, C5, and CR2 gene deletions. To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs. Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli, 1 μg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously [i.v.]). These animals did not develop fever, whereas the core temperature (Tc) of CVF vehicle-treated controls rose ∼1°C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p. LPS injection and the suitability of this species for these studies. C3- and C5-sufficient (C3+/+and C5+/+) mice also developed 1°C fevers within 80 min after i.p. LPS (1 or 2 μg/mouse) injection. These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment. C3- and C5-deficient (C3−/−and C5−/−) mice were also unable to developTcrises after i.p. LPS. Both CR2+/+and CR2−/−mice responded normally to i.p. LPS (1 μg/mouse). These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p. LPS.
Malaria-Induced Acquisition of Antibodies toPlasmodium falciparumVariant Surface Antigens
Authors: Ofori Michael F. , Dodoo Daniel , Staalsoe Trine , Kurtzhals Jørgen A. L. , Koram Kwadwo , Theander Thor G. , Akanmori Bartholomew D. , Hviid Lars
Published: July 2002

In areas of intensePlasmodium falciparumtransmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area, clinical disease is caused mainly by parasites expressing VSA not recognized by preexisting VSA-specific antibodies in that child. Such malaria episodes are known to cause an increase in agglutinating antibodies specifically recognizing VSA expressed by the parasite isolate causing the illness, whereas antibody responses to other parasite isolates are relatively unaffected. However, the detailed kinetics of this VSA antibody acquisition are unknown and hence were the aim of this study. We show thatP. falciparummalaria in Ghanaian children generally caused a rapid and sustained increase in variant-specific VSA antibody levels, while more transient and limited increases in levels of antibodies to VSA expressed by other parasite isolates were also seen. Plasma VSA antibody levels were positively correlated with the age of the healthy plasma donors but negatively correlated with the age of the parasite donors (the malaria patient). The data from this first detailed longitudinal study of acquisition of VSA antibodies support the hypothesis that naturally acquired protective immunity toP. falciparummalaria is mediated, at least in part, by VSA-specific antibodies.
Conversion of 4-Hydroxyacetophenone into 4-Phenyl Acetate by a Flavin Adenine Dinucleotide-Containing Baeyer-Villiger-Type Monooxygenase
Authors: Tanner Adam , Hopper David J.
Published: July 2002

An arylketone monooxygenase was purified from
Pseudomonas putida
JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an
M
r
of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent

K
m

values of 47 μM for 4-hydroxyacetophenone, 384 μM for acetophenone, and 23 μM for 4-hydroxypropiophenone. The apparent

K
m

value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 μM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an
Acinetobacter
sp.
Metabolic Signals Trigger Glucose-Induced Inactivation of Maltose Permease in
Saccharomyces
Authors: Jiang Hua , Medintz Igor , Zhang Bin , Michels Corinne A.
Published: July 2002

Organisms such as
Saccharomyces
capable of utilizing several different sugars selectively ferment glucose when less desirable carbon sources are also available. This is achieved by several mechanisms. Glucose down-regulates the transcription of genes involved in utilization of these alternate carbon sources. Additionally, it causes posttranslational modifications of enzymes and transporters, leading to their inactivation and/or degradation. Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease. Pathway 1 uses Rgt2p as a sensor of extracellular glucose and causes degradation of maltose permease protein. Pathway 2 is dependent on glucose transport and stimulates degradation of permease protein and very rapid inactivation of maltose transport activity, more rapid than can be explained by loss of protein alone. In this report, we characterize signal generation through pathway 2 using the rapid inactivation of maltose transport activity as an assay of signaling activity. We find that pathway 2 is dependent on
HXK2
and to a lesser extent
HXK1
. The correlation between pathway 2 signaling and glucose repression suggests that these pathways share common upstream components. We demonstrate that glucose transport via galactose permease is able to stimulate pathway 2. Moreover, rapid transport and fermentation of a number of fermentable sugars (including galactose and maltose, not just glucose) are sufficient to generate a pathway 2 signal. These results indicate that pathway 2 responds to a high rate of sugar fermentation and monitors an intracellular metabolic signal. Production of this signal is not specific to glucose, glucose catabolism, glucose transport by the Hxt transporters, or glucose phosphorylation by hexokinase 1 or 2. Similarities between this yeast glucose sensing pathway and glucose sensing mechanisms in mammalian cells are discussed.
Glutathione Is Involved in Environmental Stress Responses in
Rhizobium tropici
, Including Acid Tolerance
Authors: Riccillo Pablo M. , Muglia Cecilia I. , de Bruijn Frans J. , Roe Andrew J. , Booth Ian R. , Aguilar O. Mario
Published: July 2002

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance.
Rhizobium tropici
strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of
R. tropici
CIAT899 mutants affected in acid tolerance using Tn
5-luxAB
mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn
5-luxAB
insertion in a gene showing a high degree of similarity with the
Escherichia coli gshB
gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in
R. tropici
the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.
Proteins ofMycobacterium bovisBCG Induced in the Wayne Dormancy Model
Authors: Boon Calvin , Li Rong , Qi Robert , Dick Thomas
Published: July 2002

Oxygen starvation triggers the shiftdown of the obligate aerobeMycobacterium bovisBCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the α-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.
Transcription Analysis of the
Bacillus subtilis
PucR Regulon and Identification of a
cis
-Acting Sequence Required for PucR-Regulated Expression of Genes Involved in Purine Catabolism
Authors: Beier Lars , Nygaard Per , Jarmer Hanne , Saxild Hans H.
Published: July 2002

The PucR protein of
Bacillus subtilis
has previously been suggested to regulate the expression of 15 genes,
pucABCDE
,
pucFG
,
pucH
,
pucI
,
pucJKLM
,
pucR
, and
gde
, all of which encode proteins involved in purine catabolism. When cells are grown under nitrogen-limiting conditions, the expression of these genes is induced and intermediary compounds of the purine catabolic pathway affect this expression. By using
pucR
deletion mutants, we have found that PucR induces the expression of
pucFG
,
pucH
,
pucI
,
pucJKLM
, and
gde
while it represses the expression of
pucR
and
pucABCDE
. Deletions in the promoters of the five induced operons and genes combined with bioinformatic analysis suggested a conserved upstream activating sequence, 5′-WWWCNTTGGTTAA-3′, now named the PucR box. Potential PucR boxes overlapping the −35 and −10 regions of the
pucABCDE
promoter and located downstream of the
pucR
transcription start point were also found. The positions of these PucR boxes are consistent with PucR acting as a negative regulator of
pucABCDE
and
pucR
expression. Site-directed mutations in the PucR box upstream of
pucH
and
pucI
identified positions that are essential for the induction of
pucH
and
pucI
expression, respectively. Mutants with decreased
pucH
or increased
pucR
expression obtained from a library of clones containing random mutations in the
pucH
-to-
pucR
intercistronic region all contained mutations in or near the PucR box. The induction of
pucR
expression under nitrogen-limiting conditions was found to be mediated by the global nitrogen-regulatory protein TnrA. In other gram-positive bacteria, we have found open reading frames that encode proteins similar to PucR located next to other open reading frames encoding proteins with similarity to purine catabolic enzymes. Hence, the PucR homologues are likely to exert the same function in other gram-positive bacteria as PucR does in
B. subtilis
.
Gas Channels for NH
3
: Proteins from Hyperthermophiles Complement an
Escherichia coli
Mutant
Authors: Soupene Eric , Chu Tony , Corbin Rebecca W. , Hunt Donald F. , Kustu Sydney
Published: July 2002

Ammonium transport (Amt) proteins appear to be bidirectional channels for NH
3
. The
amt
genes of the hyperthermophiles
Aquifex aeolicus
and
Methanococcus jannaschii
complement enteric
amtB
mutants for growth at 25 nM NH
3
at 37°C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged
Aquifex
Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in
Escherichia coli
.
Correlation between Genotype and Phenotypic Categorization of Staphylococci Based on Methicillin Susceptibility and Resistance
Authors: Gradelski Elizabeth , Valera Lourdes , Aleksunes Lauren , Bonner Daniel , Fung-Tomc Joan
Published: July 2002

Positive correlation between methicillin and oxacillin susceptibility test results and the detection of the
mecA
gene was observed for
Staphylococcus
aureus, S. epidermidis,
and
S. haemolyticus
as well as among
mecA
+
strains of other species of coagulase-negative staphylococci (CNS). However, at least 50% of the
mecA
-negative strains of these other species of CNS were falsely classified as methicillin and oxacillin resistant.
Molecular Analysis of Bacterial Species Associated with Childhood Caries
Authors: Becker Mitzi R. , Paster Bruce J. , Leys Eugene J. , Moeschberger Melvin L. , Kenyon Sarah G. , Galvin Jamie L. , Boches Susan K. , Dewhirst Floyd E. , Griffen Ann L.
Published: July 2002

Although substantial epidemiologic evidence links
Streptococcus
mutans
to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species:
S
.
sanguinis
was associated with health and, in order of decreasing cell numbers,
Actinomyces
gerencseriae
,
Bifidobacterium
,
S
.
mutans
,
Veillonella
,
S
.
salivarius
,
S
.
constellatus
,
S
.
parasanguinis
, and
Lactobacillus
fermentum
were associated with caries. These data suggest that
A
.
gerencseriae
and other
Actinomyces
species may play an important role in caries initiation and that a novel
Bifidobacterium
may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.
Integrated Human Papillomavirus Type 16 Is Frequently Found in Cervical Cancer Precursors as Demonstrated by a Novel Quantitative Real-Time PCR Technique
Authors: Peitsaro Panu , Johansson Bo , Syrjänen Stina
Published: July 2002

In contrast to cervical cancer, integration of human papillomavirus (HPV) DNA into the host genome has been considered a rare event in cancer precursor lesions (cervical intraepithelial neoplasia [CIN]). With our new real-time PCR method, we demonstrated that integrated HPV type 16 (HPV16) is already present in CIN lesions. The physical state of HPV16 and the viral load were simultaneously detected. A unique region of the E2 open reading frame (ORF) that is most often deleted during HPV16 integration is targeted by one set of PCR primers and a probe, and another set targets the E6 ORF. In episomal form, both targets should be equivalent, while in integrated form, the copy numbers of E2 would be less than those of E6. The method was tested with DNAs from 31 cervical lesions (non-CIN to CINIII) from 24 women prospectively followed up for 10 years. This report presents viral load and integration results from the largest series of CIN lesions described to date. Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Samples from another patient, with only integrated HPV16, rapidly progressed from CINI to CINIII in 2 years. In all other patients, episomal and integrated forms of HPV16 DNA were found to coexist. Rapid progression of the CIN lesions was closely associated with a heavy load of integrated HPV16. Thus, the method described here is a very sensitive tool with which to assess the physical state of HPV, which is useful in predicting disease progression.
Limited Genetic Diversity of Recent Invasive Isolates of Non-Serotype b Encapsulated
Haemophilus influenzae
Authors: Omikunle Adebomi , Takahashi Shinji , Ogilvie Chrishana L. , Wang Yan , Rodriguez Carina A. , St. Geme Joseph W. , Adderson Elisabeth E.
Published: July 2002

Invasive infections caused by non-type b encapsulated
Haemophilus influenzae
have increased in frequency in the last decade. This change prompted us to characterize the genetic relationships of 48 recently isolated invasive
H. influenzae
type a (Hia), e (Hie), and f (Hif) strains by comparison of restriction digest patterns (RDPs). Recent Hia isolates exhibited moderate genetic diversity, with the majority segregating into two major clonotypes. Recent Hie and, especially, Hif strains displayed considerably restricted genetic diversity. In particular, all but one Hif strain segregated into a single clonotype, and half of these isolates had identical RDPs. These results are consistent with the hypothesis that the increased incidence of disease due to non-type b encapsulated
H. influenzae
reflects the emergence of hypervirulent clones, especially in the case of Hif. Alternatively, it is possible that non-type b encapsulated
H. influenzae
strains have limited overall genetic diversity.
Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of
Candida albicans
Directly from Blood Culture Bottles
Authors: Rigby Susan , Procop Gary W. , Haase Gerhard , Wilson Deborah , Hall Geraldine , Kurtzman Cletus , Oliveira Kenneth , Von Oy Sabina , Hyldig-Nielsen Jens J. , Coull James , Stender Henrik
Published: July 2002

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of
Candida albicans
directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the
C. albicans
PNA FISH method) is based on a fluorescein-labeled PNA probe that targets
C. albicans
26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including
C. albicans
(
n
= 72),
C. dubliniensis
(
n
= 58),
C. glabrata
(
n
= 5),
C. krusei
(
n
= 2),
C. parapsilosis
(
n
= 4), and
C. tropicalis
(
n
= 3). The performance of the
C. albicans
PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of
C. albicans
directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.
Evidence for a Stable Interaction of gp41 with Pr55Gagin Immature Human Immunodeficiency Virus Type 1 Particles
Authors: Wyma Donald J. , Kotov Alexander , Aiken Christopher
Published: July 2002

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55Gagin infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55Gagin HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55Gagin HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55Gagat the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55Gagand the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55Gagwith gp41 may regulate the function of the envelope proteins during HIV-1 maturation.
Role of the Pseudorabies Virus gI Cytoplasmic Domain in Neuroinvasion, Virulence, and Posttranslational N-Linked Glycosylation
Authors: Tirabassi R. S. , Enquist L. W.
Published: July 2002

The glycoproteins I and E of pseudorabies virus are important mediators of cell-to-cell spread and virulence in all animal models tested. Although these two proteins form a complex with one another, ascribing any function to the individual proteins has been difficult. We have shown previously, using nonsense mutations, that the N-terminal ectodomain of the gE protein is sufficient for gE-mediated transsynaptic spread whereas the cytoplasmic domain of the protein is required for full expression of virulence. These same studies demonstrated that the cytoplasmic domain of gE is also required for endocytosis of the protein. In this report, we describe the construction of viruses with nonsense mutations in gI that allowed us to determine the contributions of the gI cytoplasmic domain to protein expression as well as virus neuroinvasion and virulence after infection of the rat eye. We also constructed double mutants with nonsense mutations in both gE and gI so that the contributions of both the gE and gI cytoplasmic domains could be determined. We observed that the gI cytoplasmic domain is required for efficient posttranslational modification of the gI protein. The gE cytoplasmic domain has no effect on gE posttranslational glycosylation. In addition, we found that infection of all gE-gI-dependent anterograde circuits projecting from the rat retina requires both ectodomains and at least one of the cytoplasmic domains of the proteins. The gI cytoplasmic domain promotes transsynaptic spread of virus better than the gE cytoplasmic domain. Interestingly, both gE and gI cytoplasmic tails are required for virulence; lack of either one or both results in an attenuated infection. These data suggest that gE and gI play differential roles in mediating directional neuroinvasion of the rat; however, the gE and gI cytoplasmic domains most likely function together to promote virulence.
Identification and Characterization of Novel Human Endogenous Retrovirus Families by Phylogenetic Screening of the Human Genome Mapping Project Database
Authors: Tristem Michael
Published: July 2002

Human endogenous retroviruses (HERVs) were first identified almost 20 years ago, and since then numerous families have been described. It has, however, been difficult to obtain a good estimate of both the total number of independently derived families and their relationship to each other as well as to other members of the family
Retroviridae
. In this study, I used sequence data derived from over 150 novel HERVs, obtained from the Human Genome Mapping Project database, and a variety of recently identified nonhuman retroviruses to classify the HERVs into 22 independently acquired families. Of these, 17 families were loosely assigned to the class I HERVs, 3 to the class II HERVs and 2 to the class III HERVs. Many of these families have been identified previously, but six are described here for the first time and another four, for which only partial sequence information was previously available, were further characterized. Members of each of the 10 families are defective, and calculation of their integration dates suggested that most of them are likely to have been present within the human lineage since it diverged from the Old World monkeys more than 25 million years ago.