bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immune Function in Healthy Inner-City Children
Authors: Bartlett Jacqueline A. , Goldklang Arielle R. , Schleifer Steven J. , Keller Steven E. 
Published: July 2002

ABSTRACTThe importance of investigating immunity in healthy children has been underscored in the last few years by studies of the immune pathology of childhood illnesses, including human immunodeficiency virus. This study reports both ennumerative and functional immune measures in healthy inner city children. A total of 152 of 207 children studied were completely heathy at the time of venipuncture and were included in this study. Laboratory immune batteries were completed (or begun) the same day as venipuncture. Relationships between age, gender, ethnicity, and immunity were then analyzed. We found that gender predicted both the absolute number and the percentage of T cells and helper cells and the percentage of natural killer cells. Total leukocyte counts and percentages of lymphocytes and granulocytes were related to ethnicity, as was the response to mitogen stimulation (concanavalin A and pokeweed mitogen) and phagocytic ability. In conclusion, age, gender, and ethnicity factors were found to contribute to differences in various immune measures in children and require further investigation.
Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays
Authors: Schmitz John L. , Denny Thomas N. , Garcia Ambrosia , Lathey Janet L. 
Published: July 2002

ABSTRACT

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (
gag
) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm
3
, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.
Plasmid-Encoded Toxin of Enteroaggregative
Escherichia coli
is Internalized by Epithelial Cells
Authors: Navarro-Garcı́a Fernando , Canizalez-Roman Adrián , Luna José , Sears Cynthia , Nataro James P. 
Published: July 2002

ABSTRACT

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative
Escherichia coli
(EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet (J. M. Villaseca, F. Navarro-Garcı́a, G. Mendoza-Hernández, J. P. Nataro, A. Cravioto, and C. Eslava, Infect. Immun. 68:5920–5927, 2000).
Escherichia coliCdtB Mediates Cytolethal Distending Toxin Cell Cycle Arrest
Authors: Elwell Cherilyn , Chao Kinlin , Patel Kamlesh , Dreyfus Lawrence 
Published: July 2002

ABSTRACTWe previously reported that the CdtB polypeptide ofEscherichia colicytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with anE. coliextract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtBH154Alacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.
vspGene Expression byGiardia lambliaClone GS/M-83-H7 during Antigenic Variation In Vivo and In Vitro
Authors: Bienz M. , Siles-Lucas M. , Wittwer P. , Müller N. 
Published: July 2002

ABSTRACTGiardia lambliainfections are associated with antigenic variation of the parasite, which is generated by a continuous change of the variant-specific surface proteins (VSPs). Many investigations on the process of antigenic variation were based on the use ofG. lambliaclone GS/M-83-H7, which expresses VSP H7 as its major surface antigen. In the present study, mice were infected with the aforementioned clonal line to investigatevspgene expression during the complex process of antigenic variation of the parasite. Trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for theirvspgene expression patterns, i.e., without cultivating the recovered parasites in vitro. Because few trophozoites were recovered at late time points p.i., a combined 5′ rapid amplification of cDNA ends–reverse transcription-PCR approach was utilized. This allowed detection and subsequent sequence analysis ofvspgene transcripts upon generation of amplified cDNA analogues. The same PCR approach was applied for analysis ofvspgene expression in variants obtained after negative selection of axenic GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view of the entire panel of in vivo- and in vitro-derived parasite populations, expression of 29 differentvspgene sequences could be demonstrated. In vivo antigenic variation ofG. lambliaclone GS/M-83-H7 was shown to be a continuous process involving the consecutive appearance of relatively distinct sets ofvsptranscripts. During the 42-day infection period investigated, this process activated at least 22 differentvspgenes. Comparative molecular analyses of the amino acid level demonstrated that all cDNA segments identified encode structural elements typical of the terminal segment ofGiardiaVSP. The similarity of most of the GS/M-83-H7 VSP sequences identified in the present study supports previous suggestions thatvspgene diversification inG. lambliais the result of ancestral gene duplication, mutation, and/or recombination events.
Identification and Characterization of Mycobacterial Proteins Differentially Expressed under Standing and Shaking Culture Conditions, Including Rv2623 from a Novel Class of Putative ATP-Binding Proteins
Authors: Florczyk Matthew A. , McCue Lee Ann , Stack Robert F. , Hauer Charles R. , McDonough Kathleen A. 
Published: July 2002

ABSTRACT

The environmental signals that affect gene regulation in
Mycobacterium tuberculosis
remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including
acr
(also known as
hspX
) (α-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of
M. bovis
BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified
M. tuberculosis
Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in
M. tuberculosis
's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.
Transfer of the Core Region Genes of the
Yersinia enterocolitica
WA-C Serotype O:8 High-Pathogenicity Island to
Y. enterocolitica
MRS40, a Strain with Low Levels of Pathogenicity, Confers a Yersiniabactin Biosynthesis Phenotype and Enhanced Mouse Virulence
Authors: Pelludat Cosima , Hogardt Michael , Heesemann Jürgen 
Published: July 2002

ABSTRACT

The high-pathogenicity island (HPI) of yersiniae encodes an iron uptake system represented by its siderophore yersiniabactin (Ybt). The HPI is present in yersiniae with high levels of pathogenicity—i.e.,
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
biogroup (BG) 1B—but absent in
Y. enterocolitica
strains with low (BG 2 to 5) and no (BG 1A) levels of pathogenicity and has been shown to be an important virulence factor. Comparison of the HPI in
Y. enterocolitica
(Yen-HPI) and that in
Y. pestis
and
Y. pseudotuberculosis
revealed that, in contrast to genes of the variable region, genes of the core region (genes
irp9
to
fyuA
) are highly homologous. In the present work the Yen-HPI core genes were rescued from the chromosome of
Y. enterocolitica
WA-C (BG 1B, serotype O:8) using the
FRT
-FLP recombinase system. Transfer of the resulting plasmid pCP1 into the siderophore-deficient strain
Y. enterocolitica
NF-O (BG 1A) led to no halo on siderophore indicator chrome azurol S (CAS) agar. Transfer of pCP1 into the
Y. enterocolitica
strain MRS40 (serotype O:9, BG 2; phenotype, CAS negative) led to a CAS halo larger than that of parental strain WA-C, indicating high Ybt production. pCP1 was highly unstable in iron-deficient medium, and no enhanced mouse virulence conferred by MRS40 carrying pCP1 could be detected. To overcome the problem of instability, pCP1 was integrated into the chromosome of MRS40, leading to the formation of a CAS halo comparable to that seen with WA-C and correspondingly to increased mouse virulence. Thus, the core genes of Yen-HPI are sufficient to confer a positive CAS phenotype and mouse virulence to
Y. enterocolitica
MRS40, BG 2, but are insufficient to confer this phenotype to
Y. enterocolitica
NF-O, BG 1A.
Role of Gamma Interferon in Chemokine Expression in the Ileum of Mice and in a Murine Intestinal Epithelial Cell Line afterCryptosporidium parvumInfection
Authors: Lacroix-Lamandé Sonia , Mancassola Roselyne , Naciri Muriel , Laurent Fabrice 
Published: July 2002

ABSTRACTCryptosporidium parvumis a protozoan parasite that infects intestinal epithelial cells and induces inflammation of the intestine. To better understand the inflammatory process occurring during cryptosporidiosis, we investigated in this study the kinetics of chemokine expression in the mucosa of mice by quantitative reverse transcription-PCR. Our results demonstrate that among the chemokine mRNAs studied, gamma interferon (IFN-γ)-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), i-TAC, lymphotactin, macrophage inflammatory protein 1β (MIP-1β), and RANTES mRNAs were strongly up-regulated in infected neonate mice, which correlated with the immunofluorescence staining results showing T-cell and macrophage infiltration in the mucosa. Our in vitro data showed that intestinal epithelial cells infected byC. parvumor stimulated by the proinflammatory cytokines (IFN-γ, interleukin-1β, and tumor necrosis factor alpha) produce a pattern of chemokine secretion similar to that observed in vivo, suggesting that these cells may take part in the initial production of chemokines. In order to identify the chemokines responsible for the recruitment of the inflammatory cells leading to a protective immune response, we compared the patterns of chemokine expression in a healing neonate mouse model and a nonhealing IFN-γ knockout (GKO) mouse model of cryptosporidiosis. In the absence of IFN-γ, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1β mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2α, and KC mRNAs) were strongly overexpressed. These results are consistent with the neutrophil recruitment observed in the lamina propria of GKO mice at day 9 postinfection but are not consistent with the hypothesis that these cells play an important role in the resolution of the infection. On the contrary, the altered response of chemokines responsible for the recruitment of macrophages and T cells in GKO mice suggests that these two populations may be critical in the development of a protective immune response.
The Promoter of the Yeast
INO4
Regulatory Gene: a Model of the Simplest Yeast Promoter
Authors: Robinson Kelly A. , Lopes John M. 
Published: July 2002

ABSTRACT

In
Saccharomyces cerevisiae
, the phospholipid biosynthetic genes are transcriptionally regulated in response to inositol and choline. This regulation requires the transcriptional activator proteins Ino4p and Ino2p, which form a heterodimer that binds to the UAS

INO

element. We have previously shown that the promoters of the
INO4
and
INO2
genes are among the weakest promoters characterized in yeast. Because little is known about the promoters of weakly expressed yeast genes, we report here the analysis of the constitutive
INO4
promoter. Promoter deletion constructs scanning 1,000 bp upstream of the
INO4
gene identified a small region (−58 to −46) that is absolutely required for expression. S1 nuclease mapping shows that this region contains the transcription start sites for the
INO4
gene. An additional element (−114 to −86) modestly enhances
INO4
promoter activity (fivefold). Thus, the region required for
INO4
transcription is limited to 68 bp. These studies also found that
INO4
gene expression is not autoregulated by Ino2p and Ino4p, despite the presence of a putative UAS

INO

element in the
INO4
promoter. We further report that the
INO4
steady-state transcript levels and Ino4p levels are regulated twofold in response to inositol and choline, suggesting a posttranscriptional mechanism of regulation.
Cholate Resistance in
Lactococcus lactis
Is Mediated by an ATP-Dependent Multispecific Organic Anion Transporter
Authors: Yokota Atsushi , Veenstra Marloes , Kurdi Peter , van Veen Hendrik W. , Konings Wil N. 
Published: July 2002

ABSTRACT

The cholate-resistant
Lactococcus lactis
strain C41-2, derived from wild-type
L. lactis
MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However,
L. lactis
C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in
L. lactis
MG1363. The cholate efflux activity in
L. lactis
C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in
L. lactis
C41-2 was inhibited by
ortho
-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in
L. lactis
, one for the artificial pH probe 2′,7′-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe
N
-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in
L. lactis
C41-2. Further experiments with
L. lactis
C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in
L. lactis.
Effects of Nonpolar Mutations in Each of the SevenBacillus subtilis mrpGenes Suggest Complex Interactions among the Gene Products in Support of Na+and Alkali but Not Cholate Resistance
Authors: Ito Masahiro , Guffanti Arthur A. , Wang Wei , Krulwich Terry A. 
Published: July 2002

ABSTRACTTheBacillus subtilis mrp(multiple resistance and pH) operon supports Na+and alkali resistance via an Na+/H+antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the sevenmrpgenes, only themrpFmutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted genein trans. Expression ofmrpFin themrpnull (VKN1) strain also restored cholate transport and increased Na+efflux, indicating that MrpF does not require even low levels of othermrpgene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of othermrpgenes, i.e.,mrpArestored Na+resistance and efflux to strain VK6 (a polarmrpAmutant which expresses low levels ofmrpBto -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individualmrpgenes caused profound Na+sensitivity at both pH 7.0 and 8.3. ThemrpAandmrpDmutants were particularly sensitive to alkaline pH even without added Na+. While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiplemrpgene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships amongmrpgenes as well. All themrpmutants, especially themrpA,-B,-D,-E, and-Gmutants, had elevated levels ofmrpRNA relative to the wild type. Expression of an upstream gene,maeN, that encodes an Na+/malate symporter, was coordinately regulated withmrp, although it is not part of the operon.
The N-Terminal Region of the
Oenococcus oeni
Bacteriophage fOg44 Lysin Behaves as a Bona Fide Signal Peptide in
Escherichia coli
and as a
cis
-Inhibitory Element, Preventing Lytic Activity on Oenococcal Cells
Authors: São-José Carlos , Parreira Ricardo , Vieira Graça , Santos Mário A. 
Published: July 2002

ABSTRACT

The function of the N-terminal region of the
Oenococcus oeni
phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in
Escherichia coli
, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in
E. coli
was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from
O. oeni
-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known
O. oeni
and
Lactococcus lactis
phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.
Regulation of the
glv
Operon in
Bacillus subtilis
: YfiA (GlvR) Is a Positive Regulator of the Operon That Is Repressed through CcpA and
cre
Authors: Yamamoto Hiroki , Serizawa Masakuni , Thompson John , Sekiguchi Junichi 
Published: July 2002

ABSTRACT

Maltose metabolism and the regulation of the
glv
operon of
Bacillus subtilis
, comprising three genes,
glvA
(6-phospho-α-glucosidase),
yfiA
(now designated
glvR
), and
glvC
(EIICB transport protein), were investigated. Maltose dissimilation was dependent primarily upon the
glv
operon, and insertional inactivation of either
glvA, glvR
, or
glvC
markedly inhibited growth on the disaccharide. A second system (MalL) contributed to a minor extent to maltose metabolism. Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of
glvA
and a polycistronic mRNA of
glvA-glvR-glvC
. Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5′ end of
glvA
. When
glvR
was placed under control of the
spac
promoter, expression of the
glv
operon was dependent upon the presence of isopropyl-β-
d
-thiogalactopyranoside (IPTG). In regulatory studies, the promoter sequence of the
glv
operon was fused to
lacZ
and inserted into the
amyE
locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled
glvR
plasmid, pHYCM2VR. When cultured in Difco sporulation medium containing citrate, this transformant [AMGLV(pHYCM2VR)] expressed LacZ activity, but synthesis of LacZ was repressed by glucose. In an isogenic strain, [AMGLVCR(pHYCM2VR)], except for a mutation in the sequence of a catabolite-responsive element (
cre
), LacZ activity was expressed in the presence of citrate and glucose. Insertion of a citrate-controlled
glvR
plasmid at the
amyE
locus of
ccpA
+
and
ccpA
mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively. In the presence of both glucose and citrate, AMCMVR failed to express the
glv
operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC. Collectively, our findings suggest that GlvR (the product of the
glvR
gene) is a positive regulator of the
glv
operon and that glucose exerts its effect via catabolite repression requiring both CcpA and
cre
.