bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helicobacter pyloriActivates the Cyclin D1 Gene through Mitogen- Activated Protein Kinase Pathway in Gastric Cancer Cells
Authors: Hirata Yoshihiro , Maeda Shin , Mitsuno Yuzo , Akanuma Masao , Yamaji Yutaka , Ogura Keiji , Yoshida Haruhiko , Shiratori Yasushi , Omata Masao 
Published: July 2002

ABSTRACTHelicobacter pyloriinduces cellular proliferation in host cells, but the mechanism remains unclear. Thus, we examined the effect ofH. pylorion cyclin D1, an important regulator of the cell cycle, especially in relation to intracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS) cells was enhanced by coculture withH. pyloristrain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form ofvacA also increased cyclin D1 transcription, but mutant forms ofcagEor the entirecagpathogenicity island did not enhance cyclin D1 transcription. These effects were confirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation byH. pyloriwas inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporter plasmid having any point mutations of the NF-κB binding sites in the promoter (pD1-κB1M, pD1-κB2M, or pD1-κB1/2M) or cotransfection of dominant negative IκBα did not affect cyclin D1 activation byH. pylori. In conclusion,H. pyloriactivates cyclin D1 through the mitogen-activated protein kinase pathway and not through NF-κB activation in AGS cells. This activation of cyclin D1 is partly dependent on thecagpathogenicity island but not onvacA.
Immune Response Induction and New Effector Mechanisms Possibly Involved in Protection Conferred by the Cuban Anti-Meningococcal BC Vaccine
Authors: Pérez Oliver , Lastre Miriam , Lapinet José , Bracho Gustavo , Dı́az Miriam , Zayas Caridad , Taboada Carlos , Sierra Gustavo 
Published: July 2002

ABSTRACTThis report explores the participation of some afferent mechanisms in the immune response induced by the Cuban anti-meningococcal vaccine VA-MENGOC-BC. The induction of delayed-type hypersensitivity in nursing babies and lymphocyte proliferation after immunization is demonstrated. The presence of gamma interferon IFN-γ and interleukin-2 (IL-2) mRNAs but absence of IL-4, IL-5, and IL-10 mRNAs were observed in peripheral blood mononuclear cells from immunized subjects after in vitro challenge with outer membrane vesicles. In addition, some effector functions were also explored. The presence of opsonic activity was demonstrated in sera from vaccinees. The role of neutrophils as essential effector cells was shown. In conclusion, we have shown that, at least in the Cuban adult population, VA-MENGOC-BC induces mechanisms with a T-helper 1 pattern in the afferent and effector branches of the immune response.
Leishmania donovanip36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis
Authors: Melby Peter C. , Yang Jue , Zhao Weiguo , Perez Luis E. , Cheng Jun 
Published: July 2002

ABSTRACTThe acquisition of immunity following subclinical or resolved infection with the intracellular parasiteLeishmania donovanisuggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmaniahomolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murineL. majorinfection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneousL. majorinfection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of theLeishmaniap36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other thanL. majorhas not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-γ but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemicL. donovanichallenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.
Gamma Interferon-Induced Inhibition ofToxoplasma gondiiin Astrocytes Is Mediated by IGTP
Authors: Halonen Sandra K. , Taylor Gregory A. , Weiss Louis M. 
Published: July 2002

ABSTRACTToxoplasma gondiiis an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-γ) is the main cytokine preventing reactivation ofToxoplasmaencephalitis in the brain. Microglia are important IFN-γ-activated effector cells controlling the growth ofT. gondiiin the brain via a nitric oxide (NO)-mediated mechanism. IFN-γ can also activate astrocytes to inhibit the growth ofT. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasmamechanisms. In this study we investigated the role ofIGTP, a recently identified IFN-γ-regulated gene, in IFN-γ inhibition ofT. gondiiin murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-γ, and then tested for anti-Toxoplasmaactivity. In wild-type astrocytesT. gondiigrowth was significantly inhibited by IFN-γ, whereas in astrocytes from IGTP-deficient mice IFN-γ did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-γ induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-γ-induced inhibition ofT. gondiiin murine astrocytes.
Visualization of
Proteus mirabilis
within the Matrix of Urease-Induced Bladder Stones during Experimental Urinary Tract Infection
Authors: Li Xin , Zhao Hui , Lockatell C. Virginia , Drachenberg Cinthia B. , Johnson David E. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

The virulence of a urease-negative mutant of uropathogenic
Proteus mirabilis
and its wild-type parent strain was assessed by using a CBA mouse model of catheterized urinary tract infection. Overall, catheterized mice were significantly more susceptible than uncatheterized mice to infection by wild-type
P. mirabilis
. At a high inoculum, the urease-negative mutant successfully colonized bladders of catheterized mice but did not cause urolithiasis and was still severely attenuated in its ability to ascend to kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we demonstrated the presence of
P. mirabilis
within the urease-induced stone matrix. Alizarin red S staining was used to detect calcium-containing deposits in bladder and kidney tissues of
P. mirabilis
-infected mice.
Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras
Authors: Jobling Michael G. , Holmes Randall K. 
Published: July 2002

ABSTRACTVariants of cholera toxin B subunit (CTB) were made by bisulfite- and oligonucleotide-directed mutagenesis of thectxBgene. Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC). Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM1in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies. Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB. RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 + 54, 91, and 95. Tyrosine-12 was identified as a novel residue important for GM1binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM1in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM1binding, based on the crystal structure of CTB and the oligosaccharide of GM1, were not appreciably altered in their ability to bind GM1in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC. Hybrid B genes made by fusingctxBand the relatedEscherichia coliheat-labile enterotoxineltBgenes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM1as well as wild-type CTB. This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs). Each MAb had a unique pattern of reactivity with the panel of CTB variants. Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope. The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers. Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.
Bicarbonate Ion Stimulates the Expression of Locus of Enterocyte Effacement-Encoded Genes in Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Abe Hiroyuki , Tatsuno Ichiro , Tobe Toru , Okutani Akiko , Sasakawa Chihiro 
Published: July 2002

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) strains adhere to the intestinal mucosa and produce an attaching and effacing (A/E) lesion. Most of the genes required to produce A/E lesions are thought to be encoded by the 36-kb pathogenicity island termed the locus for enterocyte effacement (LEE). Although the mechanisms underlying the bacterial adherence, including the genes involved, are still poorly understood, the preferential adherence phenotype of EHEC is thought to depend on the nature of the genes and/or the response of these genes to changes in environmental conditions. To explore the environmental factors affecting EHEC adherence, we used an O157:H7 strain and investigated the optimal growth conditions for its adherence to Caco-2 cells. We observed that EHEC grown in Dulbecco's modified Eagle's medium (DMEM) adhered more efficiently to Caco-2 cells than EHEC grown in Luria-Bertani (LB) broth. Among the components of DMEM, only NaHCO
3
was found to remarkably stimulate bacterial adherence. When bacteria were grown in LB broth containing NaHCO
3
, the production of intimin, Tir, EspA, and EspB was greatly enhanced compared with the production in LB broth. Indeed, the transcription of
ler
required for LEE-encoded gene expression was promoted in response to the concentration of NaHCO
3
in LB broth. Since the concentration of NaHCO
3
in the lower intestinal tract has been shown to be relatively high compared with that in the upper small intestine, our results may imply that NaHCO
3
is an important signaling factor for promoting colonization of EHEC in the lower intestinal tract in humans.
In Vivo Splicing and Functional Characterization of
Mycobacterium leprae
RecA
Authors: Frischkorn Klaus , Springer Burkhard , Böttger Erik C. , Davis Elaine O. , Colston M. Joseph , Sander Peter 
Published: July 2002

ABSTRACT

The RecA proteins from
Mycobacterium tuberculosis
and
Mycobacterium leprae
contain inteins. In contrast to the
M. tuberculosis
RecA, the
M. leprae
RecA is not spliced in
Escherichia coli
. We demonstrate here that
M. leprae
RecA is functionally spliced in
Mycobacterium smegmatis
and produces resistance toward DNA-damaging agents and homologous recombination.
Septal Localization of the Membrane-Bound Division Proteins of
Bacillus subtilis
DivIB and DivIC Is Codependent Only at High Temperatures and Requires FtsZ
Authors: Katis V. L. , Wake R. G. , Harry E. J. 
Published: July 2002

ABSTRACT

Using immunofluorescence microscopy, we have examined the dependency of localization among three
Bacillus subtilis
division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.
Marinomonas mediterranea
MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant
Authors: Solano Francisco , Lucas-Elío Patricia , Fernández Eva , Sanchez-Amat Antonio 
Published: July 2002

ABSTRACT

Marinomonas mediterranea
is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to
M. mediterranea
by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn
10
derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability.
Sorting Out Bacterial Viability with Optical Tweezers
Authors: Ericsson M. , Hanstorp D. , Hagberg P. , Enger J. , Nyström T. 
Published: July 2002

ABSTRACT

We have developed a method, using laser, optical tweezers and direct microscopic analysis of reproductive potential and membrane integrity, to assess single-cell viability in a stationary-phase
Escherichia coli
population. It is demonstrated here that a reduction in cell integrity, determined by using the fluorescent nucleic acid stain propidium iodide, correlated well with a reduction in cell proliferating potential during the stationary-phase period studied. Moreover, the same cells that exhibited reduced integrity were found to be the ones that failed to divide upon nutrient addition. A small but significant number of the intact cells (496 of 7,466 [6.6%]) failed to replicate. In other words, we did not find evidence for the existence of a large population of intact but nonculturable cells during the stationary-phase period studied but it is clear that reproductive ability can be lost prior to the loss of membrane integrity. In addition, about 1% of the stationary-phase cells were able to divide only once upon nutrient addition, and in a few cases, only one of the two cells produced by division was able to divide a second time, indicating that localized cell deterioration, inherited by only one of the daughters, may occur. The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary-phase-induced bacterial death and population heterogeneity is discussed.
Characterization of PrpC fromBacillus subtilis, a Member of the PPM Phosphatase Family
Authors: Obuchowski M. , Madec E. , Delattre D. , Boël G. , Iwanicki A. , Foulger D. , Séror S. J. 
Published: July 2002

ABSTRACTWe cloned theyloOgene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate,p-nitrophenol phosphate, and endogenousBacillus subtilisproteins. TheprkCandprpCgenes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.
Activation of the Multiple Drug Resistance Gene
MDR1
in Fluconazole-Resistant, Clinical
Candida albicans
Strains Is Caused by Mutations in a
trans
-Regulatory Factor
Authors: Wirsching Stephanie , Michel Sonja , Köhler Gerwald , Morschhäuser Joachim 
Published: July 2002

ABSTRACT

Resistance of
Candida albicans
against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant
C. albicans
isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the
MDR1
gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for
MDR1
activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable
MDR1
activation were analyzed. Sequence analysis of the
MDR1
regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of
trans
-regulatory factors, a
GFP
reporter gene was placed under the control of the
MDR1
promoter from the fluconazole-susceptible
C. albicans
strain CAI4, which does not express the
MDR1
gene in vitro. This
MDR1P-GFP
fusion was integrated into the genome of the clinical
C. albicans
isolates with the help of the dominant selection marker
MPA
R
developed for the transformation of
C. albicans
wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident
MDR1
promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the
GFP
gene. These results demonstrate that the
MDR1
promoter was activated by a
trans
-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive
MDR1
expression.
Degradation of Heme in Gram-Negative Bacteria: the Product of the
hemO
Gene of Neisseriae Is a Heme Oxygenase
Authors: Zhu Wenming , Wilks Angela , Stojiljkovic Igor 
Published: July 2002

ABSTRACT

A full-length heme oxygenase gene from the gram-negative pathogen
Neisseria meningitidis
was cloned and expressed in
Escherichia coli
. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the
E. coli
cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen
Corynebacterium diphtheriae
and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXα and carbon monoxide as the final products. Homologs of the
hemO
gene were identified and characterized in six commensal
Neisseria
isolates,
Neisseria lactamica
,
Neisseria subflava
,
Neisseria flava
,
Neisseria polysacchareae
,
Neisseria kochii
, and
Neisseria cinerea
. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.
Mutations in
catB
, the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distal
ben
Genes and Contribute to a Complex Regulatory Circuit in
Acinetobacter
sp. Strain ADP1
Authors: Cosper Nathaniel J. , Collier Lauren S. , Clark Todd J. , Scott Robert A. , Neidle Ellen L. 
Published: July 2002

ABSTRACT

Mutants of the bacterium
Acinetobacter
sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate and
cis
,
cis
-muconate to activate expression of the
benABCDE
operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by
cat
gene-encoded enzymes. In this report, four spontaneous mutants were found to carry
catB
mutations that enabled BenM-independent growth on benzoate.
catB
encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate,
cis
,
cis
-muconate, is enzymatically produced from catechol by the
catA
-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the
catB
mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomal
benA
::
lacZ
transcriptional fusion were constructed and used to investigate how
catB
mutations affect growth on benzoate. All of the
catB
mutations increased
cis
,
cis
-muconate-activated
ben
gene expression in strains lacking BenM. A model is presented in which the
catB
mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular
cis
,
cis
-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate
ben
gene transcription. CatM normally responds to
cis
,
cis
-muconate to activate
cat
gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown
catB
mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels of
cis
,
cis
-muconate and increasing
catA
expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.