bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi:
Peniophora lycii, Agrocybe pediades
, a
Ceriporia
sp., and
Trametes pubescens
Authors: Lassen Søren F. , Breinholt Jens , Østergaard Peter R. , Brugger Roland , Bischoff Andrea , Wyss Markus , Fuglsang Claus C. 
Published: July 2002

ABSTRACT

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (
myo
-inositol hexakisphosphate), thereby creating lower forms of
myo
-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (
Peniophora lycii, Agrocybe pediades
, a
Ceriporia
sp., and
Trametes pubescens
) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the
Ceriporia
sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in
Aspergillus oryzae
, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the
P. lycii
phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein
−1
and were capable of hydrolyzing phytate down to
myo
-inositol monophosphate. Surprisingly,
1
H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC
3.1.3.26
).
Methyl
t
-Butyl Ether Mineralization in Surface-Water Sediment Microcosms under Denitrifying Conditions
Authors: Bradley Paul M. , Chapelle Francis H. , Landmeyer James E. 
Published: July 2002

ABSTRACT

Mineralization of [U-
14
C]methyl
t
-butyl ether (MTBE) to
14
CO
2
without accumulation of
t
-butyl alcohol (TBA) was observed in surface-water sediment microcosms under denitrifying conditions. Methanogenic activity and limited transformation of MTBE to TBA were observed in the absence of denitrification. Results indicate that bed sediment microorganisms can effectively degrade MTBE to nontoxic products under denitrifying conditions.
NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction
Authors: Riefler R. Guy , Smets Barth F. 
Published: July 2002

ABSTRACT

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH,

K
N

, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.
PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between
Cryptosporidium parvum
and
C. meleagridis
and between
C. parvum
Isolates of Human and Animal Origin
Authors: Guyot K. , Follet-Dumoulin A. , Recourt C. , Lelièvre E. , Cailliez J. C. , Dei-Cas E. 
Published: July 2002

ABSTRACT

Genomic DNAs from human
Cryptosporidium
isolates previously typed by analysis of the 18S ribosomal DNA locus (
Cryptosporidium parvum
bovine genotype,
C. parvum
human genotype,
Cryptosporidium meleagridis
, and
Cryptosporidium felis
) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous
Cryptosporidium wrairi
sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate
Cryptosporidium
species and
C. parvum
genotypes.
Gastrointestinal Tract Location of
Escherichia coli
O157:H7 in Ruminants
Authors: Grauke Luke J. , Kudva Indira T. , Yoon Jang Won , Hunt Carl W. , Williams Christopher J. , Hovde Carolyn J. 
Published: July 2002

ABSTRACT

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of
Escherichia coli
O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with
E. coli
O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of
E. coli
O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum.
E. coli
O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of
E. coli
O157:H7 persistence was the lower GIT.
E. coli
O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for
E. coli
O157:H7 persistence and proliferation in mature ruminant animals.
Conservation of Plasmid-Encoded Traits among Bean-Nodulating
Rhizobium
Species
Authors: Brom Susana , Girard Lourdes , García-de los Santos Alejandro , Sanjuan-Pinilla Julio M. , Olivares José , Sanjuan Juan 
Published: July 2002

ABSTRACT

Rhizobium etli
type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (
Rhizobium etli
,
Rhizobium gallicum
,
Rhizobium giardinii
,
Rhizobium leguminosarum
, and
Sinorhizobium fredii
). Our results indicate that independent of geographic origin,
R. etli
strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from
R. etli
.
Increased Anionic Peptide Distribution and Intensity during Progression and Resolution of Bacterial Pneumonia
Authors: Fales-Williams Amanda J. , Gallup Jack M. , Ramírez-Romero Rafael , Brogden Kim A. , Ackermann Mark R. 
Published: July 2002

ABSTRACT

Anionic peptides (APs) are small anionic antimicrobial peptides composed of 7 aspartic acid residues and are produced in the lungs of humans, sheep, and cattle. Although expression by epithelial cells of some antimicrobial peptides (e.g., β-defensins) of humans and ruminants is increased in response to acute infection, AP expression is not increased during acute infection, which suggests that the expression of the latter peptide is constitutive. In this study, the degree of AP expression during the progression (acute, subacute, and chronic) of bronchopneumonia was determined.
Mannheimia
(
Pasteurella
)
haemolytica
, a known inducer of bovineβ-defensins, was inoculated intrabronchially with a fiber-optic bronchoscope in nine 3-month-old sheep, and tissues were collected at 1, 15, and 45 days postinoculation (p.i.); nine control animals received pyrogen-free saline by the same procedure and were killed at the same time points. In the acute group (1 day p.i.), the lungs had lesions typical of bronchopneumonia and the distribution and intensity of AP immunoreactivity (AP-IR) were similar to those of previous studies (minimal intensity and distribution of AP-IR in bronchiolar epithelial cells). In the subacute group (15 days p.i.), there was prominent hyperplasia of bronchiolar and alveolar epithelial cells, and the chronic group (45 days p.i.) had yet more pronounced hyperplasia. In the subacute and chronic groups, the intensity and distribution of AP-IR in the cytoplasm of hyperplastic bronchiolar and type II alveolar cells were significantly increased compared to those of saline-inoculated and contralateral (noninoculated) lung lobes. Although AP expression appears constitutive, the constitutive production of AP is higher in hyperplastic, less differentiated cells than in fully differentiated, mature cells of the respiratory airways. The increased intensity and distribution of AP-IR in immature (hyperplastic) epithelial cells may be a mechanism by which production of a noninducible antimicrobial is increased temporarily during lesion progression and repair. This increased production of AP by hyperplastic cells may protect the lung against further infection until new, fully differentiated epithelial cells are capable of expressing their own inducible array of antimicrobial peptides.
Identification of Constituents of Human Neutrophil Azurophil Granules That Mediate Fungistasis againstHistoplasma capsulatum
Authors: Newman Simon L. , Gootee Lisa , Gabay Joelle E. , Selsted Michael E. 
Published: July 2002

ABSTRACTPreviously we demonstrated that human neutrophils mediate potent and long-lasting fungistasis againstHistoplasma capsulatumyeasts and that all of the fungistatic activity resides in the azurophil granules. In the present study, specific azurophil granule constituents with fungistatic activity were identified by incubation withH. capsulatumyeasts for 24 h and by quantifying the subsequent growth of yeasts via the incorporation of [3H]leucine. Human neutrophil defensins HNP-1, HNP-2, and HNP-3 inhibited the growth ofH. capsulatumyeasts in a concentration-dependent manner with maximum inhibition at 8 μg/ml. At a concentration of 4 μg/ml, all possible paired combinations of defensins exhibited additive fungistatic activity againstH. capsulatumyeasts. Cathepsin G and bactericidal-permeability-increasing protein (BPI) also mediated fungistasis againstH. capsulatumin a concentration-dependent manner. The fungistatic activities of combinations of cathepsin G and BPI were additive, as were those of combinations of cathepsin G or BPI with HNP-1, HNP-2, and HNP-3. Lysozyme and elastase exhibited modest antifungal activity, and azurocidin and proteinase 3 exhibited no significant fungistasis againstH. capsulatumyeasts. Thus, defensins, cathepsin G, and BPI are the major anti-H. capsulatumeffector molecules in the azurophil granules of human neutrophils.
Candida albicans RIM101
pH Response Pathway Is Required for Host-Pathogen Interactions
Authors: Davis Dana , Edwards John E. , Mitchell Aaron P. , Ibrahim Ashraf S. 
Published: July 2002

ABSTRACT

The ability of
Candida albicans
to respond to diverse environments is critical for its success as a pathogen. The
RIM101
pathway controls gene expression and the yeast-to-hyphal transition in
C. albicans
in response to changes in environmental pH in vitro. In this study, we found that the
RIM101
pathway is necessary in vivo for pathogenesis. First, we show that

rim101
−
/rim101
−

and

rim8
−
/rim8
−

mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, the

rim101
−
/rim101
−

and

rim8
−
/rim8
−

mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of
RIM101
,
RIM101-405
, is a suppressor of the

rim8
−

mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the

rim8
−
/rim8
−

mutant. These results demonstrate that the
RIM101
pathway is required for
C. albicans
virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.
Human Antibodies against a Purified Glucosylceramide fromCryptococcus neoformansInhibit Cell Budding and Fungal Growth
Authors: Rodrigues Marcio L. , Travassos Luiz R. , Miranda Kildare R. , Franzen Anderson J. , Rozental Sonia , de Souza Wanderley , Alviano Celuta S. , Barreto-Bergter Eliana 
Published: July 2002

ABSTRACTA major ceramide monohexoside (CMH) was purified from lipidic extracts ofCryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a β-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types ofC. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the β-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains ofC. neoformansinhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal β-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.
Genital Antibody Responses in Mice after Intranasal Infection with an Attenuated Candidate Vector Strain of
Bordetella pertussis
Authors: Mielcarek Nathalie , Nordström Inger , Menozzi Franco D. , Locht Camille , Holmgren Jan 
Published: July 2002

ABSTRACT

Intranasal administration of live attenuated
Bordetella pertussis
, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of
B. pertussis
. By comparing the immune responses induced after infection with wild-type virulent
B. pertussis
with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of
B. pertussis
-infected mice. The intranasal infection with either the attenuated or the virulent
B. pertussis
strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated
B. pertussis
could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.
Defective Hyphal Development and Avirulence Caused by a Deletion of the
SSK1
Response Regulator Gene in
Candida albicans
Authors: Calera José Antonio , Zhao Xiao-Jiong , Calderone Richard 
Published: July 2002

ABSTRACT

In a previous study, we reported the isolation and characterization of the two-component response regulator
SSK1
gene of
Candida albicans
. This gene is a structural but not a functional homolog of the
SSK1
and
mcs4
+
genes of
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
, respectively. In the present study, we have constructed and phenotypically characterized Δ
ssk1
mutants of
C. albicans
. The results confirmed our previous observation that
CaSSK1
, unlike
SSK1
or
mcs4
+
, does not regulate cellular responses to either osmotic or oxidative stress. Instead, Δ
ssk1
null strains showed severely reduced hyphal formation on serum agar and were totally defective in hyphal development on other solid media, such as medium 199 (pH 7.5) and Spider medium. In contrast, under conditions of low nitrogen availability on solid media, Δ
ssk1
null strains dramatically hyperinvaded the agar. However, while forming germ tubes and hyphae in liquid media similar to those of the wild type, Δ
ssk1
null strains flocculated in a manner similar to that of Δ
chk1
two-component histidine kinase mutants, which we have previously described. Finally, virulence studies indicated that
SSK1
is essential for the pathogenesis of
C. albicans
, suggesting that the Ssk1p response regulator could be a good target for antifungal therapy.
Effects of the Immunoglobulin A1 Protease on
Neisseria gonorrhoeae
Trafficking across Polarized T84 Epithelial Monolayers
Authors: Hopper Sylvia , Vasquez Brandi , Merz Alex , Clary Susan , Wilbur J. Scott , So Magdalene 
Published: July 2002

ABSTRACT

We previously demonstrated that the
Neisseria
IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of
Neisseria gonorrhoeae
across polarized T84 epithelial monolayers. We report that
N. gonorrhoeae
infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that
iga
mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by
N. gonorrhoeae.
Induction of Emetic, Pyrexic, and Behavioral Effects of
Staphylococcus aureus
Enterotoxin B in the Ferret
Authors: Wright Angela , Andrews Paul L. R. , Titball Richard W. 
Published: July 2002

ABSTRACT

Ferrets which had been orally dosed with 5 mg of Staphylococcal enterotoxin B (SEB) responded with an increase in subcutaneous temperature. At 75 min, the subcutaneous temperature was significantly higher (+ 0.9°C ± 0.38°C,
P
< 0.007) than in control animals. Animals dosed with 1 or 2 mg of SEB responded with a small, but not significant, increase in subcutaneous temperature. All of the animals dosed with 5 mg of SEB retched and vomited. The mean latency for the onset of retching was 105 ± 36 min, and the mean latency for the onset of vomiting was 106 ± 34 min. The mean number of retches was 17.8 ± 19.6, and the mean number of vomits was 2.0 ± 1.5. These findings indicate that ferrets can be used as alternatives to primates for the study of the biological activities of SEB.
Tetanus Toxin Fragment C Expressed in LiveSalmonellaVaccines Enhances Antibody Responses to Its Fusion PartnerSchistosoma haematobiumGlutathioneS-Transferase
Authors: Lee Jeong Jin , Sinha Katharine A. , Harrison Julia A. , de Hormaeche Raquel Demarco , Riveau Gilles , Pierce Raymond J. , Capron Andre , Wilson R. Alan , Khan C. M. Anjam 
Published: July 2002

ABSTRACTTetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed inSalmonellavaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathioneS-transferase (GST) fromSchistosoma haematobium(Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulentaroA-attenuated strain ofSalmonella entericaserovar Typhimurium. The Sh28GST proteins were soluble and stably expressed inSalmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinantSalmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.
The Collagen-Binding Adhesin Is a Virulence Factor in
Staphylococcus aureus
Keratitis
Authors: Rhem Marcus N. , Lech Elizabeth M. , Patti Joseph M. , McDevitt Damien , Höök Magnus , Jones Dan B. , Wilhelmus Kirk R. 
Published: July 2002

ABSTRACT

A collagen-binding strain of
Staphylococcus aureus
produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the
cna
gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased
S. aureus
adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of
S. aureus
infection of the cornea.