bluuebunny/crossref_metadata_2025_split huggingface dataset:
Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria
Authors: Metcalf James S. , Bell Steven G. , Codd Geoffrey A. 
Published: July 2002

ABSTRACT

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp
3
-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (
R
2
= 0.94,
P
< 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.
Role of Dipicolinic Acid in Survival of
Bacillus subtilis
Spores Exposed to Artificial and Solar UV Radiation
Authors: Slieman Tony A. , Nicholson Wayne L. 
Published: July 2002

ABSTRACT

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of
Bacillus subtilis
spore dry weight and has been shown to play a significant role in the survival of
B. subtilis
spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon
dpaAB
were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the
gerA, gerB
, and
gerK
nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.
Growth of Chitinolytic Dune Soil β-Subclass
Proteobacteria
in Response to Invading Fungal Hyphae
Authors: De Boer Wietse , Klein Gunnewiek Paulien J. A. , Kowalchuk George A. , Van Veen Johannes A. 
Published: July 2002

ABSTRACT

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate. However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils. An unidentified group of β-subclass
Proteobacteria
(CβPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils. Here we demonstrate that the CβPs grew at the expense of extending fungal mycelium of three dune soil fungi (
Chaetomium globosum, Fusarium culmorum
, and
Mucor hiemalis
) under nutrient-limiting, soil-like conditions. Aggregates of CβPs were also often found attached to fungal hyphae. The growth of a control group of dominant nonchitinolytic dune soil bacteria (β- and γ-subclass
Proteobacteria
) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae. Therefore, mycolytic activities of CβPs have apparently been involved in allowing them to grow after exposure to living hyphae. The chitinase inhibitor allosamidin did not, in the case of
Mucor
, or only partially, in the cases of
Chaetomium
and
Fusarium
, repress mycolytic growth of the CβPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation. Chitinolytic
Stenotrophomonas
-like and
Cytophaga
-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.
cumA
Multicopper Oxidase Genes from Diverse Mn(II)-Oxidizing and Non-Mn(II)-Oxidizing
Pseudomonas
Strains
Authors: Francis Chris A. , Tebo Bradley M. 
Published: July 2002

ABSTRACT

A multicopper oxidase gene,
cumA,
required for Mn(II) oxidation was recently identified in
Pseudomonas putida
strain GB-1. In the present study, degenerate primers based on the putative copper-binding regions of the
cumA
gene product were used to PCR amplify
cumA
gene sequences from a variety of
Pseudomonas
strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains. The presence of highly conserved
cumA
gene sequences in several apparently non-Mn(II)-oxidizing
Pseudomonas
strains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms. Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters. Overall, our results indicate that both the
cumA
gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse
Pseudomonas
strains.
Characterization of a Class II Defective Transposon Carrying Two Haloacetate Dehalogenase Genes from
Delftia acidovorans
Plasmid pUO1
Authors: Sota Masahiro , Endo Masahiro , Nitta Keiji , Kawasaki Haruhiko , Tsuda Masataka 
Published: July 2002

ABSTRACT

The two haloacetate dehalogenase genes,
dehH1
and
dehH2
, on the 65-kb plasmid pUO1 from
Delftia acidovorans
strain B were found to be located on transposable elements. The
dehH2
gene was carried on an 8.9-kb class I composite transposon (Tn
Had1
) that was flanked by two directly repeated copies of IS
1071
, IS
1071
L and IS
1071
R. The
dehH1
gene was also flanked by IS
1071
L and a truncated version of IS
1071
(IS
1071
N). Tn
Had1
,
dehH1
, and IS
1071
N were located on a 15.6-kb class II transposon (Tn
Had2
) whose terminal inverted repeats and
res
site showed high homology with those of the Tn
21
-related transposons. Tn
Had2
was defective in transposition because of its lacking the transposase and resolvase genes. Tn
Had2
could transpose when the Tn
21
-encoded transposase and resolvase were supplied in
trans
. These results demonstrated that Tn
Had2
is a defective Tn
21
-related transposon carrying another class I catabolic transposon.
Lignocellulose Affects Mn
2+
Regulation of Peroxidase Transcript Levels in Solid-State Cultures of
Pleurotus ostreatus
Authors: Cohen Roni , Yarden Oded , Hadar Yitzhak 
Published: July 2002

ABSTRACT

The effect of Mn
2+
amendment on peroxidase gene expression was studied during
Pleurotus ostreatus
growth on cotton stalks. Four peroxidase-encoding genes were expressed differentially and in a manner different from that observed in defined media. Mn
2+
affects
mnp3
expression even 2 h after its addition to the cultures, suggesting a direct effect of the metal ion on expression.
Trehalose 6,6′-Dimycolate (Cord Factor) ofMycobacterium tuberculosisInduces Corneal Angiogenesis in Rats
Authors: Saita Norio , Fujiwara Nagatoshi , Yano Ikuya , Soejima Kazuhiko , Kobayashi Kazuo 
Published: July 2002

ABSTRACTNeovascularization or angiogenesis is required for the progression of chronic inflammation. The mechanism of inflammatory neovascularization in tuberculosis remains unknown. Trehalose 6,6′-dimycolate (TDM) purified fromMycobacterium tuberculosiswas injected into rat corneas. TDM challenge provoked a local granulomatous response in association with neovascularization. Neovascularization was seen within a few days after the challenge, with the extent of neovascularization being dose dependent, although granulomatous lesions developed 14 days after the challenge. Cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), IL-1β, and vascular endothelial growth factor (VEGF), were found in lesions at the early stage (within a few days after the challenge) and were detectable until day 21. Neovascularization was inhibited substantially by neutralizing antibodies to VEGF and IL-8 but not IL-1β. Treatment with anti-TNF-α antibodies resulted in partial inhibition. TDM possesses pleiotropic activities, and the cytokine network plays an important role in the process of neovascularization.
Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of
Pseudomonas aeruginosa
and
Salmonella enterica
Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor
Authors: Gerçeker A. Alev , Zaidi Tanweer , Marks Peter , Golan David E. , Pier Gerald B. 
Published: July 2002

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry of
Pseudomonas aeruginosa
and
Salmonella enterica
serovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different
P. aeruginosa
and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for
P. aeruginosa
, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that
P. aeruginosa
and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the
P. aeruginosa
and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.
Decreased Production of Local Immunoglobulin A toPneumocystis cariniiin Bronchoalveolar Lavage Fluid from Human Immunodeficiency Virus-Positive Patients
Authors: Jalil A. , Moja P. , Lambert C. , Perol M. , Cotte L. , Livrozet J. M. , Boibieux A. , Vergnon J. M. , Lucht F. , Tran R. , Contini C. , Genin C. 
Published: July 2002

ABSTRACTAn enzyme-linked immunosorbent assay and a Western blot analysis were developed to study the antibody response toPneumocystis cariniiin serum and bronchoalveolar lavage fluid from 27 human immunodeficiency virus 27 (HIV)-infected patients withP. cariniipneumonia (Pcp), 32 patients without Pcp, and 51 HIV-negative controls. Urea was used for the correct dilution of epithelial lining fluid, and albumin was used to evaluate transudation from plasma for the assessment of local production of antibodies toP. carinii. By contrast with those of immunoglobulin G (IgG), IgA responses toP. cariniiwere increased in serum from HIV-positive patients compared to negative controls. Local production of antibodies toP. carinii, especially IgA, was decreased in patients with Pcp. In a study of 10 patients of each group, IgG and IgA responses to gp116 fromP. cariniiwere lower in patients with Pcp than in other groups. These results suggest that, in addition to alveolar macrophages, local antibodies may play a role in host defense againstP. carinii.
Eimeria tenellaInfection Induces Local Gamma Interferon Production and Intestinal Lymphocyte Subpopulation Changes
Authors: Yun Cheol H. , Lillehoj Hyun S. , Choi Kang D. 
Published: July 2002

ABSTRACTThe role of intestinal lymphocytes and gamma interferon (IFN-γ) production in protective immunity toEimeria tenellainfection was evaluated in two inbred strains of chickens (SC and TK) that display different patterns of susceptibility to coccidiosis. Oral inoculation of either strain withE. tenellaled to parasite invasion of the intestinal cecum and cecal tonsils. Greater fecal oocyst shedding was seen in TK chickens. Flow cytometric analyses of cecal tonsil lymphocytes demonstrated greater numbers of CD4+and T-cell receptor γδ-positive (TCR1+) cells in SC chickens and elevated numbers of CD8+and TCR2+cells in TK chickens following primary infection. IFN-γ mRNA expression was significantly increased in cecal tonsil and intraepithelial lymphocytes at days 6 and 8, respectively, after primary infection in SC compared to TK chickens. While no differences were noted between cecal tonsil lymphocytes of the two strains following secondary infection, TK chickens showed elevated IFN-γ transcript levels in intestinal intraepithelial lymphocytes at this time. Selective depletion of CD4+, but not CD8+, cecal tonsil lymphocytes in SC chickens resulted in a reduced IFN-γ mRNA expression, indicating that CD4+cells are the primary source of this cytokine. Collectively, these results indicate that local lymphocyte responses and production of IFN-γ are influenced by host genetic factors.
Trehalose 6,6′-Dimycolate (Cord Factor) Enhances Neovascularization through Vascular Endothelial Growth Factor Production by Neutrophils and Macrophages
Authors: Sakaguchi Ikuyo , Ikeda Norikazu , Nakayama Miki , Kato Yoshiko , Yano Ikuya , Kaneda Kenji 
Published: July 2002

ABSTRACTTrehalose 6,6′-dimycolate (TDM) plays important roles in the development of granulomatous inflammation during infection withMycobacteriumspp.,Rhodococcusspp., etc. To reveal the augmenting effect of TDM on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue air pouches induced byRhodococcussp. strain 4306 TDM dissolved in Freund's incomplete adjuvant (FIA), comparing them to pouches treated with FIA alone. Histologically, granulomatous tissue and new vessel formation, which reached a maximum at day 7, was greatly enhanced by treatment with TDM. At day 1, VEGF-positive neutrophils accumulated in the pouch wall with frequency of 95% of total infiltrating cells, adhering to TDM-containing micelles. By day 3, granulomatous tissue and new vessels started to develop, and VEGF-positive macrophages appeared in a small number and gradually increased in number thereafter. The pouch contents of VEGF, interleukin-1β, tumor necrosis factor alpha, and transforming growth factor β were significantly elevated in TDM-treated pouches, with peaks at days 1, 0.5, 1, and 3, respectively, compared to those of control pouches, while that of basic fibroblast growth factor showed no significant increase. Treatment with anti-VEGF antibody inhibited TDM-induced granulomatous tissue formation and neovascularization, and administration of recombinant VEGF into pouches treated with FIA alone induced neovascularization comparable to that in the TDM-treated pouches. Incubation of neutrophils and macrophages on TDM-coated plastic dishes increased the VEGF release. The present results indicate that TDM augments VEGF production by neutrophils and macrophages and induces neovascularization in the granulomatous tissue.
Salmonella enterica
Serotype Typhimurium Elicits Cross-Immunity against a
Salmonella enterica
Serotype Enteritidis Strain Expressing LP Fimbriae from the
lac
Promoter
Authors: Nicholson Tracy L. , Bäumler Andreas J. 
Published: July 2002

ABSTRACT

The biological significance of fimbrial phase variation in
Salmonella
serotypes is currently unknown. Exposure to long polar (LP) fimbriae of
Salmonella enterica
serotype Typhimurium results in selection against
lpf
phase ON cells of serotype Enteritidis during a subsequent challenge, suggesting that fimbrial phase variation may be a mechanism to evade cross-immunity between
Salmonella
serotypes. This notion was tested by assessing the effect of an immune response against serotype Typhimurium LP fimbriae on colonization of mice with a serotype Enteritidis mutant in which the
lpf
promoter region was replaced with the
Escherichia coli lac
promoter. During a challenge with a serotype Enteritidis mutant carrying the
lac
promoter in front of the
lpf
operon, significantly lower numbers were recovered from organs and feces of mice previously immunized with an
lpf
phase ON culture of serotype Typhimurium than from mice not previously exposed to LP fimbriae. Immunization with the
lpf
phase ON culture of serotype Typhimurium elicited antibodies that cross-reacted with a purified gluthathione-
S
-transferase–LpfA fusion protein of serotype Enteritidis. These data suggested that cross-immunity against LP fimbrial proteins cannot be evaded if phase variation on the transcriptional level is prevented by expressing the
lpf
operon from the
lac
promoter. These data hence support the idea that phase variation of LP fimbriae is a mechanism to evade cross-immunity between serotypes Enteritidis and Typhimurium.
Generation and Characterization of a Defined Mutant of
Streptococcus suis
Lacking Suilysin
Authors: Allen Andrew G. , Bolitho Steven , Lindsay Heather , Khan Shahid , Bryant Clare , Norton Pam , Ward Philip , Leigh James , Morgan Jeremy , Riches Helen , Eastty Sarah , Maskell Duncan 
Published: July 2002

ABSTRACT

A defined allelic-replacement mutant of the
sly
gene, encoding a thiol-activated cytolysin, from a European isolate of
Streptococcus suis
serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection.
Effector Mechanisms of Protection againstPseudomonas aeruginosaKeratitis in Immunized Rats
Authors: Thakur A. , Kyd J. , Xue M. , Willcox M. D. P. , Cripps A. 
Published: July 2002

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen which causes sight-threatening corneal infections in humans. The purpose of this study was to evaluate various immunization routes that may provide protection againstPseudomonaskeratitis and to define the molecular mechanisms involved in the protection. Sprague-Dawley rats (10 to 12 weeks old) were immunized using paraformaldehyde-killedP. aeruginosa(strain 6206) via oral, nasal, and intra-Peyer's patch (IPP) routes followed by an ocular topical booster dose. Scratched corneas were challenged with an infective dose ofP. aeruginosa. Following clinical examination, eyes were enucleated for histology, polymorphonuclear leukocyte (PMN) quantitation, bacterial count, enzyme-linked immunosorbent assay, and RNase protection assay. PMN infiltration was higher early (4 h) during the infection in immunized rats than in nonimmunized rats. Later during the infection, the number of PMNs diminished in immunized rats while in nonimmunized animals the number of PMNs continued to increase. Bacteria were cleared much faster from immunized groups than from the nonimmunized group, and the nasally immunized group had the most efficacious response among the immunized groups. Nasal and IPP immunization groups had increased cytokine expression of interleukin-2 (IL-2) and IL-5 and differed from each other for IL-6. All three immunized groups had significantly reduced IL-1β levels when compared with the nonimmunized rats and a significantly altered profile for CINC-1 expression. This study has shown that the route of immunization modulates the inflammatory response to ocularP. aeruginosainfection, thus affecting the severity of keratitis and adverse pathology, with nasal immunization being the most effective.
DNA Adenine Methylase Overproduction in
Yersinia pseudotuberculosis
Alters YopE Expression and Secretion and Host Immune Responses to Infection
Authors: Julio Steven M. , Heithoff Douglas M. , Sinsheimer Robert L. , Low David A. , Mahan Michael J. 
Published: July 2002

ABSTRACT

Yersinia pseudotuberculosis
mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several
Yersinia
virulence proteins (
Yersinia
outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on
Yersinia
virulence determinant expression and secretion, as well as the host immune response to
Yersinia
antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of
Yersinia
proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.