bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection
Authors: Sung C. C. , Boomer J. S. , Givens T. S. , DuChateau B. K. , Lepe M. R. , Feller A. , Westerman M. P. , Gilman-Sachs A. , Chedid A. , Beaman K. D.
Published: July 2002

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3+T cells of HIV-seropositive (HIV+) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV+HCV-seropositive (HCV+), HIV- and HCV-seropositive (HIV+HCV+), and HIV- and HCV-seronegative (HIV−HCV−) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV+individuals is upregulated and that its expression on T cells from HCV+individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3+T cells was seen in both HIV+and HIV+HCV+individuals compared to HIV−HCV−individuals. HCV+individuals had lower levels of RTF expression than HIV−HCV−individuals (P< 0.005 for CD4+;P< 0.0005 for CD8+). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV+> HIV+HCV+> HIV−HCV−> HCV+. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.
WdChs2p, a Class I Chitin Synthase, Together with WdChs3p (Class III) Contributes to Virulence in
Wangiella
(
Exophiala
)
dermatitidis
Authors: Wang Zheng , Zheng Li , Liu Hongbo , Wang Qingfeng , Hauser Melinda , Kauffman Sarah , Becker Jeffery M. , Szaniszlo Paul J.
Published: July 2002

The chitin synthase structural gene
WdCHS2
was isolated by screening a subgenomic DNA library of
Wangiella dermatitidis
by using a 0.6-kb PCR product of the gene as a probe. The nucleotide sequence revealed a 2,784-bp open reading frame, which encoded 928 amino acids, with a 59-bp intron near its 5′ end. Derived protein sequences showed highest amino acid identities with those derived from the
CiCHS1
gene of
Coccidioides immitis
and the
AnCHSC
gene of
Aspergillus nidulans
. The derived sequence also indicated that WdChs2p is an orthologous enzyme of Chs1p of
Saccharomyces cerevisiae
, which defines the class I chitin synthases. Disruptions of
WdCHS2
produced strains that showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms. However, assays showed that membranes of
wdchs2
Δ mutants were drastically reduced in chitin synthase activity. Other assays of membranes from a
wdchs1
Δ
wdchs3
Δ
wdchs4
Δ triple mutant showed that their residual chitin synthase activity was extremely sensitive to trypsin activation and was responsible for the majority of zymogenic activity. Although no loss of virulence was detected when
wdchs2
Δ strains were tested in a mouse model of acute infection,
wdchs2
Δ
wdchs3
Δ disruptants were considerably less virulent in the same model, even though
wdchs3
Δ strains also had previously shown no loss of virulence. This virulence attenuation in the
wdchs2
Δ
wdchs3
Δ mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice. The importance of WdChs2p and WdChs3p to the virulence of
W. dermatitidis
was then confirmed by reconstituting virulence in the double mutant by the reintroduction of either
WdCHS2
or
WdCHS3
into the
wdchs2
Δ
wdchs3
Δ mutant background.
Further Characterization of Complement Regulator-Acquiring Surface Proteins ofBorrelia burgdorferi
Authors: Kraiczy Peter , Skerka Christine , Brade Volker , Zipfel Peter F.
Published: July 2002

The three genospeciesBorrelia burgdorferi,Borrelia garinii, andBorrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistantB.afzeliiisolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674–1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitiveB. afzeliiandB. burgdorferiisolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived fromB. afzelii, or as BbCRASPs, for proteins identified inB. burgdorferiisolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, twoB. afzeliiproteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and threeB. burgdorferiproteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37°C compared with those cultured at 20°C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.
Functional Activity of Anti-Neisserial Surface Protein A Monoclonal Antibodies against Strains ofNeisseria meningitidisSerogroup B
Authors: Moe G. R. , Zuno-Mitchell P. , Lee S. S. , Lucas A. H. , Granoff D. M.
Published: July 2002

Neisserial surface protein A (NspA) is currently being investigated with humans as a candidate vaccine for the prevention of meningococcal disease. Although NspA is highly conserved, the ability of anti-NspA antibodies to bind to or elicit complement-mediated bactericidal activity against diverseNeisseria meningitidisserogroup B strains is controversial. To evaluate strain differences in NspA surface accessibility and susceptibility to bactericidal activity, we prepared murine immunoglobulin G2a anti-NspA monoclonal antibodies (MAbs) and evaluated their functional activity against 10 genetically diverseN. meningitidisserogroup B strains. By colony Western blot, all 10 strains expressed NspA as detected by one or more MAbs. By flow cytometry, two MAbs were found to bind to the bacterial surface of 6 of the 10 strains. In addition, two strains showed variable NspA surface accessibility for the MAbs despite being uniformly positive for NspA expression by colony Western blotting. Only 4 of the 10 strains were susceptible to anti-NspA complement-mediated bacteriolysis. Passively administered MAb protected infant rats from developing bacteremia after challenge withN. meningitidisserogroup B strain 8047 (surface binding positive, susceptible to anti-NspA bacteriolysis), was poorly protective against strain BZ232 (surface binding variable, resistant to bacteriolysis), and did not protect against strain M986 (surface binding negative, resistant to bacteriolysis). Finally, NspA does not appear to be critical for causing bacteremia, as an NspA knockout from strain 8047 was highly virulent in infant rats. Taken together, these findings suggest that an NspA-based vaccine will need to incorporate additional antigens to elicit broad protection againstN. meningitidisserogroup B.
Pulmonary and Systemic Host Response toStreptococcus pneumoniaeandKlebsiella pneumoniaeBacteremia in Normal and Immunosuppressed Mice
Authors: Wang Erjian , Ouellet Nathalie , Simard Marie , Fillion Isabelle , Bergeron Yves , Beauchamp Denis , Bergeron Michel G.
Published: July 2002

Mortality related to bacteremic pneumonia remains high, and the role of sepsis in inflammation, pulmonary injury, and death remains unclear, mostly in leukopenic states. In the present study, the microbiology, histopathology, and host response toStreptococcus pneumoniaeandKlebsiella pneumoniaeinfection were determined in an experimental model of bacteremia in immunocompetent and leukopenic mice. Leukocyte depletion by cyclophosphamide did not impair the early clearance of pneumococci from blood but facilitated growth in lungs. By contrast, klebsiellae rapidly grew in blood of leukopenic mice. These observations suggest that tissue-based phagocytes and circulating leukocytes, respectively, play prominent roles inS. pneumoniaeandK. pneumoniaeeradication. The kinetics of leukocyte recruitment in lungs duringS. pneumoniaebacteremia suggested early strong inflammation in immunocompetent mice that is associated with tumor necrosis factor alpha release and histological disorders, including cell debris and surfactant in alveolar spaces. Leukocyte depletion further stimulated pulmonary capillary leakage both inS. pneumoniaeandK. pneumoniaebacteremia, which seemed attributable to bacterial virulence factors. Nitric oxide production did not differ significantly among groups. Leukopenia and low platelet counts characterized the late stage of bacteremia for both strains, but onlyK. pneumoniaealtered renal function. Understanding the pathogenesis of bacteremia will help establish beneficial therapies for both sepsis and pneumonia.
Dual Functions of
Streptococcus salivarius
Urease
Authors: Chen Yi-Ywan M. , Weaver Cheryl A. , Burne Robert A.
Published: July 2002

A urease-deficient derivative of
Streptococcus salivarius
57.I was constructed by allelic exchange at the
ureC
locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also,
S. salivarius
could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.
Genes for the Type IV Secretion System in an Intracellular Symbiont,Wolbachia, a Causative Agent of Various Sexual Alterations in Arthropods
Authors: Masui Shinji , Sasaki Tetsuhiko , Ishikawa Hajime
Published: July 2002

Wolbachiaspecies are intracellular bacteria known to cause reproductive abnormalities in their hosts. In this study, we identifiedWolbachiagenes encoding homologs to the type IV secretion system by which many pathogenic bacteria secrete macromolecules. The genes identified encoded most of the essential components of the secretion system and were cotranscribed as an operon.
Hexokinase Regulates Kinetics of Glucose Transport and Expression of Genes Encoding Hexose Transporters in
Saccharomyces cerevisiae
Authors: Petit Thomas , Diderich Jasper A. , Kruckeberg Arthur L. , Gancedo Carlos , Van Dam Karel
Published: July 2002

Glucose transport kinetics and mRNA levels of different glucose transporters were determined in
Saccharomyces cerevisiae
strains expressing different sugar kinases. During exponential growth on glucose, a
hxk2
null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes
HXT2
and
HXT7
, encoding high-affinity transporters, and a diminished expression of the
HXT1
and
HXT3
genes, encoding low-affinity transporters. Deletion of
HXT7
revealed that the high-affinity component is mostly due to
HXT7
; however, a previously unidentified very-high-affinity component (

K
m

= 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from
Schizosaccharomyces pombe
or
Yarrowia lipolytica
in a
hxk1 hxk2 glk1
strain prevented derepression of the high-affinity transport system at high concentrations of glucose.
Mutational Analysis of Differences in Thermostability between Histones from Mesophilic and Hyperthermophilic Archaea
Authors: Li Wen-Tyng , Shriver John W. , Reeve John N.
Published: July 2002

Amino acid residues responsible for the large difference in thermostability between HMfB and HFoB, archaeal histones from the hyperthermophile
Methanothermus fervidus
and the mesophile
Methanobacterium formicicum
, respectively, have been identified by site-specific mutagenesis. The thermal denaturation of ∼70 archaeal histone variants has been monitored by circular dichroism, and the data generated were fit to a two-state unfolding model (dimer→two random coil monomers) to obtain a standard-state (1M) melting temperature for each variant dimer. The results of single-, double-, and triple-residue substitutions reveal that the much higher stability of rHMfB dimers, relative to rHFoB dimers, is conferred predominantly by improved intermolecular hydrophobic interactions near the center of the histone dimer core and by additional favorable ion pairs on the dimer surface.
Analysis of
Escherichia coli
RecA Interactions with LexA, λ CI, and UmuD by Site-Directed Mutagenesis of
recA
Authors: Mustard Julie A. , Little John W.
Published: July 2002

An early event in the induction of the SOS system of
Escherichia coli
is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage λ CI repressor and
E. coli
UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, λ CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.
Two Types of
Bacillus subtilis tetA
(L) Deletion Strains Reveal the Physiological Importance of TetA(L) in K
+
Acquisition as well as in Na
+
, Alkali, and Tetracycline Resistance
Authors: Wang Wei , Guffanti Arthur A. , Wei Yi , Ito Masahiro , Krulwich Terry A.
Published: July 2002

The chromosomally encoded TetA(L) protein of
Bacillus subtilis
is a multifunctional tetracycline-metal/H
+
antiporter that also exhibits monovalent cation/H
+
antiport activity and a net K
+
uptake mode. In this study,
B. subtilis
mutant strains JC112 and JC112C were found to be representative of two phenotypic types of
tetA
(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K
+
, indicating that the net K
+
uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na
+
- or K
+
-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co
+2
as well as Na
+
sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of
czcD-trkA
, a locus with a proposed involvement in K
+
uptake, occurred in both mutants. The increase was highest in the presence of Co
2+
and was higher in JC112 than in JC112C. Deletion of
czcD-trkA
resulted in diminished growth of the wild-type and both mutant strains at low [K
+
], supporting a significant role for this locus in K
+
uptake. Expression of
yheL
, which is a homologue of the Na
+
/H
+
antiporter-encoding
nhaC
gene from
Bacillus firmus
OF4, was also increased in both
tetA
(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of
yheL
were consistent with a modest role for YheL in Na
+
-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na
+
, and alkali resistance and K
+
acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.
Adenylate Kinase as a Virulence Factor ofPseudomonas aeruginosa
Authors: Markaryan Adam , Zaborina Olga , Punj Vasu , Chakrabarty A. M.
Published: July 2002

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg · ATP + AMP ↔ Mg · ADP + ADP. In this study we show that AK secreted by the pathogenic strains ofPseudomonas aeruginosaappears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain ofP. aeruginosa8821 and hyperproduced it as a fusion protein with glutathioneS-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection withP. aeruginosa.Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allowsP. aeruginosato exert its full virulence.
Gene Replacement Analysis of the Butyrolactone Autoregulator Receptor (FarA) Reveals that FarA Acts as a Novel Regulator in Secondary Metabolism ofStreptomyces lavendulaeFRI-5
Authors: Kitani Shigeru , Yamada Yasuhiro , Nihira Takuya
Published: July 2002

IM-2 [(2R,3R,1′R)-2-1′-hydroxybutyl-3-hydroxymethyl γ-butanolide] is a γ-butyrolactone autoregulator which, inStreptomyces lavendulaeFRI-5, switches off the production ofd-cycloserine but switches on the production of a blue pigment and several nucleoside antibiotics. To clarify the in vivo function of an IM-2-specific receptor (FarA) in the IM-2 signaling cascade ofS. lavendulaeFRI-5, afarAdeletion mutant was constructed by means of homologous recombination. On several solid media, no significant difference in morphology was observed between the wild-type strain and thefarAmutant (strain K104), which demonstrated that the IM-2–FarA system does not participate in the morphological control ofS. lavendulaeFRI-5. In liquid media, thefarAmutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the wild-type strain, suggesting that the FarA protein acts primarily as a negative regulator on the biosynthesis of these compounds in the absence of IM-2. However, contrary to the IM-2-dependent suppression ofd-cycloserine production in the wild-type strain, overproduction ofd-cycloserine was observed in thefarAmutant, indicating for the first time that the presence of both IM-2 and intact FarA are necessary for the suppression ofd-cycloserine biosynthesis.
Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of
Saccharomyces cerevisiae
Authors: Distler MacKenzie , Kulkarni Ajit , Rai Rajendra , Cooper Terrance G.
Published: July 2002

Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in
Saccharomyces cerevisiae
. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the
S. cerevisiae
genome, raising the possibility that Dal80p might be able to “stain” DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4′,6′-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.
Regulation of Transcription of the
Bacillus subtilis pyrG
Gene, Encoding Cytidine Triphosphate Synthetase
Authors: Meng Qi , Switzer Robert L.
Published: July 2002

The
B. subtilis pyrG
gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated. The
pyrG
promoter and 5′ leader were fused to
lacZ
and integrated into the chromosomes of several
B. subtilis
strains having mutations in genes of pyrimidine biosynthesis and salvage. These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium. These experiments indicated that
pyrG
expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides. The start of
pyrG
transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon. A factor-independent termination hairpin lying between the
pyrG
promoter and its coding region is essential for regulation of
pyrG
expression. Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator. Furthermore, deletion of the terminator from
pyrG-lacZ
fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine. We suggest that cytidine repression of
pyrG
expression is mediated by an antitermination mechanism in which antitermination by a putative
trans
-acting protein is reduced by elevated levels of cytidine nucleotides. Conservation of sequences and secondary structural elements in the
pyrG
5′ leaders of several other gram-positive bacteria indicates that their
pyrG
genes are regulated by a similar mechanism.
Role of
Agrobacterium
VirB11 ATPase in T-Pilus Assembly and Substrate Selection
Authors: Sagulenko Evgeniy , Sagulenko Vitaliya , Chen Jun , Christie Peter J.
Published: July 2002

The VirB11 ATPase is a subunit of the
Agrobacterium tumefaciens
transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of
virB11
mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing
virB11
dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of
virB11
mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i)
virB11/virB11
∗ merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.
The PPP-Family Protein Phosphatases PrpA and PrpB of
Salmonella enterica
Serovar Typhimurium Possess Distinct Biochemical Properties
Authors: Shi Liang , Kehres David G. , Maguire Michael E.
Published: July 2002

Salmonella enterica
serovar Typhimurium requires Mn
2+
, but only a few Mn
2+
-dependent enzymes have been identified from bacteria. To characterize Mn
2+
-dependent enzymes from serovar Typhimurium, two putative PPP-family protein phosphatase genes were cloned from serovar Typhimurium and named
prpA
and
prpB
. Their DNA-derived amino acid sequences showed 61% identity to the corresponding
Escherichia coli
proteins and 41% identity to each other. Each phosphatase was expressed in
E. coli
and purified to near electrophoretic homogeneity. Both PrpA and PrpB absolutely required a divalent metal for activity. As with other phosphatases of this class, Mn
2+
had the highest affinity and stimulated the greatest activity. The apparent

K
a

of PrpA for Mn
2+
of 65 μM was comparable to that for other bacterial phosphatases, but PrpB had a much higher affinity for Mn
2+
(1.3 μM). The pH optima were pH 6.5 for PrpA and pH 8 for PrpB, while the optimal temperatures were 45 to 55°C for PrpA and 30 to 37°C for PrpB. Each phosphatase could hydrolyze phosphorylated serine, threonine, or tyrosine residues, but their relative specific activities varied with the specific substrate tested. These differences suggest that each phosphatase is used by serovar Typhimurium under different growth or environmental conditions such as temperature or acidity.
Molecular and Phenotypic Analysis ofCaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter
Authors: Nishikawa Akiko , Poster Jay B. , Jigami Yoshifumi , Dean Neta
Published: July 2002

Cell surface mannan is implicated in almost every aspect of pathogenicity ofCandida albicans. InSaccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation inC. albicans,we isolated theCaVRG4gene and analyzed the effects of loss of its function.CaVRG4encodes a functional homologue of theS. cerevisiaeGDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm ofC. albicansin a pattern reminiscent of localization of Vrg4p in the Golgi apparatus inS. cerevisiae. Like partial loss ofScVRG4function, partial loss ofCaVRG4function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy ofCaVRG4under control of the methionine-repressibleMET3promoter, did not grow in the presence of methionine and cysteine, demonstrating thatCaVRG4is essential for viability. MutantCandida vrg4strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because theVRG4gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.
Multistate Outbreak of
Salmonella
Serovar Muenchen Infections Associated with Alfalfa Sprouts Grown from Seeds Pretreated with Calcium Hypochlorite
Authors: Proctor Mary E. , Hamacher Marge , Tortorello Mary Lou , Archer John R. , Davis Jeffrey P.
Published: July 2002

During September 1999, a multistate outbreak of
Salmonella
serovar Muenchen infection associated with eating raw alfalfa sprouts was identified in Wisconsin. Despite use of a calcium hypochlorite sanitizing procedure to pretreat seeds before sprouting, at least 157 outbreak-related illnesses were identified in seven states having sprouters who received alfalfa seed from a specific lot. The continued occurrence of sprout-related outbreaks despite presprouting disinfection supports the concern that no available treatment will eliminate pathogens from seeds before sprouting and reinforces the need for additional safeguards to protect the public. A lack of consumer knowledge regarding exposure to sprouts documented in this investigation suggests that more-targeted outreach to high-risk individuals may be needed to reduce their risk.
Performance of Eight Methods, Including Two New Rapid Methods, for Detection of Oxacillin Resistance in a Challenge Set of
Staphylococcus aureu
s Organisms
Authors: Swenson Jana M. , Williams Portia P. , Killgore George , O'Hara Caroline Mohr , Tenover Fred C.
Published: July 2002

Using a set of 55
Staphylococcus aureus
challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of
S. aureus
to oxacillin. The methods were evaluated by using the presence of the
mecA
gene, as detected by PCR, as the “gold standard.” The strains included 19
mecA
-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 μg/ml) and 36
mecA
-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 μg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the
mecA-
negative strains increased to 4 to 8 μg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.
Clustering of South African
Helicobacter pylori
Isolates from Peptic Ulcer Disease Patients Is Demonstrated by Repetitive Extragenic Palindromic-PCR Fingerprinting
Authors: Kidd M. , Atherton J. C. , Lastovica A. J. , Louw J. A.
Published: July 2002

The present report assesses the association between clonal groupings, disease, and the virulence fingerprint of 76 South African

Helicobacter pylori cagA
+

strains isolated from 57 Cape-colored subjects. Two methods, repetitive extragenic palindromic (REP)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to generate DNA fingerprints, and computer-assisted analysis was used to derive clusters. The two PCR techniques were only partially complementary (48%). REP-PCR fingerprints identified a distinct pathological cluster consisting of strains from 63% of the patients and was strongly associated with both disease (
P
< 0.00001) and the vacuolating cytotoxin A (
vacA
) signal sequence type (
P
< 0.003). RAPD-PCR fingerprinting was not associated with disease and was less strongly associated with
vacA
(
P
< 0.05) than REP-PCR was. Hierarchical analysis indicated that isolates from patients with peptic ulcer disease tended to cluster differently than isolates from patients with gastritis alone or gastric adenocarcinoma. These relationships are consistent with a loosely clonal population structure associated with disease for
H. pylori
in the Cape-colored population in South Africa.
Possible Approach for Serodiagnosis of Ascariasis by Evaluation of Immunoglobulin G4 Response UsingAscaris lumbricoidesSomatic Antigen
Authors: Bhattacharyya Tanusree , Santra Amal , Majumder Debendra N. Guha , Chatterjee Bishnu P.
Published: July 2002

Somatic antigen ofAscaris lumbricoideswas purified to homogeneity (molecular mass, 34 kDa) by ammonium sulfate fractionation and successive chromatographic procedures, namely, gel permeation, ion exchange, and high-performance gel permeation liquid chromatographies. The antigen showed strong binding with immunoglobulin G (IgG) inAscaris-infested patients and was cross-reactive with IgE and IgG in patients infested with other nematodes. It reacted specifically with IgG4 (P< 0.001) in 63Ascaris-infested patients, which represented 65% of the total IgG response, though cross-reactivity with IgG1, IgG2, and IgG3 subclasses was observed, indicating the unique specificity of this test system and its potential utility in the serodiagnosis of ascariasis.
Disseminated Toxoplasmosis, Resulting from Infection of Allograft, after Orthotopic Liver Transplantation: Usefulness of Quantitative PCR
Authors: Botterel F. , Ichai P. , Feray C. , Bouree P. , Saliba F. , Tur Raspa R. , Samuel D. , Romand S.
Published: July 2002

Disseminated toxoplasmosis is a life-threatening disease in liver transplant recipients that can result from an organ-transmitted infection. We report here a case of fatal disseminated toxoplasmosis after orthotopic liver transplantation from a seropositive donor (immunoglobulin G [IgG]
+
and IgM
−
) in a patient who was nonimmune for toxoplasmosis prior to transplantation. Quantitative PCR analyses of various clinical specimens, including serum samples, appeared retrospectively to be a valuable diagnostic tool that might guide therapeutic attitudes.
Human Papillomavirus Type 33 E7 Peptides Presented by HLA-DR*0402 to Tumor-Infiltrating T Cells in Cervical Cancer
Authors: Höhn Hanni , Pilch Henryk , Günzel Susanne , Neukirch Claudia , Freitag Kirsten , Necker Antje , Maeurer Markus J.
Published: July 2002

Several characteristics make human papillomavirus (HPV) amenable to vaccination. Anti-HPV-directed vaccines are based on the observation that HPV E6 and E7 oncoproteins are constitutively expressed in HPV-positive cervical cancer and may serve as tumor rejection antigens. Five HPV types (16, 18, 31, 33, and 45) account for 80% of cervical cancer. Until now, the type of immune response capable of mediating an effective antitumor response has not been defined. In order to define the anticancer-directed immune response in situ, we characterized CD4+and CD8+sorted T cells from peripheral blood lymphocytes, freshly harvested tumor tissue, and tumor-infiltrating lymphocytes (TIL) from a patient with cervical cancer. The HLA-DR-restricted CD4+T-cell receptor VB16-, VA10-, VA21-, and VA22-positive CD4+T-cell line derived from TIL recognizes autologous HLA-DR*0402+(HPV33+) cervical cancer cells, as determined by gamma interferon secretion. Testing of different peptides spanning the E7 gene revealed that the HPV3373–87peptide ASDLRTIQQLLMGTV represents the immunodominant epitope which can also be presented by the DR*0401 allele to TIL. Such major histocompatibility complex class II-presented peptides represent attractive candidates to augment T-cell responses directed against autologous tumor cells.
Simian-Human Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Subtype-E Envelope Gene: Persistent Infection, CD4
+
T-Cell Depletion, and Mucosal Membrane Transmission in Macaques
Authors: Himathongkham Sunee , Halpin Nancy S. , Li Jinling , Stout Michael W. , Miller Christopher J. , Luciw Paul A.
Published: July 2002

The envelope (
env
) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on
env
have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze
env
genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the
env
ectodomain of the SHIV-33 clone with the
env
ectodomain from the subtype-E strain HIV-1
CAR402
, which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4
+
T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E
env
genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1
env
antigens.
Influence of the Theiler's Virus L∗ Protein on Macrophage Infection, Viral Persistence, and Neurovirulence
Authors: van Eyll Olivier , Michiels Thomas
Published: July 2002

The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L∗. We confirmed previous work showing that the L∗ ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L∗ protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L∗ ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L∗ AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L∗ initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605–8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.
Rescue of Multiple Viral Functions by a Second-Site Suppressor of a Human Immunodeficiency Virus Type 1 Nucleocapsid Mutation
Authors: Cimarelli Andrea , Sandin Sara , Höglund Stefan , Luban Jeremy
Published: July 2002

Human immunodeficiency type 1 (HIV-1) bearing the nucleocapsid (NC) mutation R10A/K11A is replication defective. After serial passage of the mutant virus in tissue culture, we isolated a revertant that retained the original mutation. It had acquired, in addition, a new mutation (E21K) that was formally demonstrated to be sufficient for restoration of viral replication. Detailed analysis of the replication defect of R10A/K11A revealed a threefold reduction in virion yield and a fivefold reduction in packaging of viral genomic RNA. Real-time PCR was then used to quantitate viral DNA synthesis following infection of Jurkat T cells. After adjustment for the assembly and packaging defects, a minor (twofold) reduction in synthesis of either strong-stop, full-length linear DNA or 2-LTR circles was observed with R10A/K11A virions, indicating that reverse transcription and nuclear transport of the viral genome were largely intact. However, after adjustment for the amounts of full-length or 2-LTR circles produced, R10A/K11A virions were at least 10-fold less infectious than wild type, indicating that viral DNA produced by the R10A/K11A mutant failed to integrate. Each of the above-mentioned defects was corrected by introduction of the second-site compensatory mutation E21K. These results demonstrate that the replication defect of mutant R10A/K11A can be explained by impairment at multiple steps in the viral life cycle, most important among them being integration and RNA packaging. The E21K mutation is predicted to restore positive charge to the face of the R10A/K11A mutant NC protein that interacts with the HIV-1 SL3 RNA stem-loop, emphasizing the importance of NC basic residues for HIV-1 replication.
ATP-Dependent Simian Virus 40 T-Antigen–Hsc70 Complex Formation
Authors: Sullivan Christopher S. , Gilbert Susan P. , Pipas James M.
Published: July 2002

Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen–Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 μM), but T-antigen–Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen–Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.
Expression of Major Capsid Protein VP-1 in the Absence of Viral Particles in Thymomas Induced by Murine Polyomavirus
Authors: Sanjuan Norberto , Porrás Analı́a , Otero Javier , Perazzo Sofı́a
Published: July 2002

Thymomas induced by polyomavirus strain PTA in mice are known to express the major capsid protein VP-1. Since the expression of a late structural protein such as VP-1 is considered a sign of virus replication, the present work attempted to clarify the implication of the presence of this protein in tumor cells. Electron microscopy of tumors showed a striking absence of viral particles in the vast majority of the cells. However, immunoelectron microscopy of the same samples demonstrated intranuclear VP-1 in most cells despite the absence of viral particles. Very little infectious virus was recovered from tumors. A change in the electrophoretic mobility of VP-1 from thymomas was detected compared with VP-1 from productively infected cells. The data presented in this work prove that the expression of VP-1 in polyomavirus-induced tumors is not synonymous with the presence of infectious virus, suggesting a possible defect in viral encapsidation.
Downregulation of IRF-3 Levels by Ribozyme Modulates the Profile of IFNA Subtypes Expressed in Infected Human Cells
Authors: Yeow Wen-Shuz , Au Wei-Chun , Lowther William J. , Pitha Paula M.
Published: July 2002

As an early response to viral infection, cells express a number of cellular genes that play a role in innate immunity, including alpha/beta interferons (IFN). IFN-α/β are encoded by a single IFNB gene and multiple, closely related IFNA genes. The induction of these IFN genes in infected cells occurs at the transcriptional level, and two transcription factors of the IRF family, IRF-3 and IRF-7, were shown to play a role in their activation. While the expression of IRF-3 alone was shown to be sufficient for induction of the IFNB gene, induction of all the IFNA subtypes in human cells required the presence of IRF-7. Since IRF-3 is expressed constitutively in all cells examined, the role of IRF-3 in the induction of IFNA genes has not been clarified. Using ribozyme targeted to IRF-3 mRNA, we found that the downregulation of IRF-3 levels in the infected cells inhibited not only the induction of IFNB gene but also the expression of IFNA genes. Furthermore, downmodulation of IRF-3 levels altered the expression profile of IFNA subtypes induced by viral infection. These studies suggest that the ratio between the relative levels of IRF-3 and IRF-7 is a critical determinant for the induction of the individual IFNA subtypes in infected cells.