bluuebunny/crossref_metadata_2025 huggingface dataset:
Movement of Retinal Along the Visual Transduction Path
Authors: Borhan Babak , Souto Maria L. , Imai Hiroo , Shichida Yoshinori , Nakanishi Koji
Published: July 2002

Movement of the ligand/receptor complex in rhodopsin (Rh) has been traced. Bleaching of diazoketo rhodopsin (DK-Rh) containing 11-
cis
-3-diazo-4-oxo-retinal yields batho-, lumi-, meta-I-, and meta-II-Rh intermediates corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates shows that the ionone ring cross-links to tryptophan-265 on helix F in DK-Rh and batho-Rh, and to alanine-169 on helix D in lumi-, meta-I-, and meta-II-Rh intermediates. It is likely that these movements involving a flip-over of the chromophoric ring trigger changes in cytoplasmic membrane loops resulting in heterotrimeric guanine nucleotide–binding protein (G protein) activation.
Discovery of a High-Energy Gamma-Ray-Emitting Persistent Microquasar
Authors: Paredes Josep M. , Martı́ Josep , Ribó Marc , Massi Maria
Published: July 2002

Microquasars are stellar x-ray binaries that behave as a scaled-down version of extragalactic quasars. The star LS 5039 is a new microquasar system with apparent persistent ejection of relativistic plasma at a 3-kiloparsec distance from the sun. It may also be associated with a γ-ray source discovered by the Energetic Gamma Ray Experiment Telescope (EGRET) on board the COMPTON–Gamma Ray Observatory satellite. Before the discovery of LS 5039, merely a handful of microquasars had been identified in the Galaxy, and none of them was detected in high-energy γ-rays.
Coordination of a Transcriptional Switch by HMGI(Y) Acetylation
Authors: Munshi Nikhil , Agalioti Theodora , Lomvardas Stavros , Merika Menie , Chen Guoying , Thanos Dimitris
Published: July 2002

Dynamic control of interferon-β (IFN-β) gene expression requires the regulated assembly and disassembly of the enhanceosome, a higher-order nucleoprotein complex formed in response to virus infection. The enhanceosome activates transcription by recruiting the histone acetyltransferase proteins CREB binding protein (CBP) and
p
300/
C
BP-
a
ssociated
f
actors (PCAF)/GCN5, which, in addition to modifying histones, acetylate HMGI(Y), the architectural component required for enhanceosome assembly. We show that the accurate execution of the IFN-β transcriptional switch depends on the ordered acetylation of the high-mobility group I protein HMGI(Y) by PCAF/GCN5 and CBP, which acetylate HMGI(Y) at distinct lysine residues on endogenous promoters. Whereas acetylation of HMGI(Y) by CBP at lysine-65 destabilizes the enhanceosome, acetylation of HMGI(Y) by PCAF/GCN5 at lysine-71 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CBP.
Influence of Human Sera on the In Vitro Activity of the Echinocandin Caspofungin (MK-0991) against
Aspergillus fumigatus
Authors: Chiller Tom , Farrokhshad Kouros , Brummer Elmer , Stevens David A.
Published: July 2002

There have been several reports that the activity of echinocandin antifungal agents is not affected or decreased in the presence of human sera. It is known that these drugs are bound >80% in animal and human sera. The activity of the echinocandin caspofungin (MK-0991), a 1,3-β-
d
-glucan synthase inhibitor, against
Aspergillus fumigatus
with and without human sera was studied. Conidia of
A. fumigatus
in microtest plate wells formed germlings after overnight culture in RPMI 1640. Caspofungin was then added with or without serum, and the germlings were incubated at 37°C for 24 h. Human serum (5%) in RPMI 1640 alone did not significantly inhibit the growth of
A. fumigatus
in vitro. Caspofungin in RPMI 1640 exhibited dose-dependent inhibition, with concentrations of 0.1 and 0.05 μg/ml inhibiting 24.9% +/− 10.4% and 11.7% +/− 3.6%, respectively (
n
= 10;
P
< 0.01). The addition of 5% human serum to caspofungin at 0.1 or 0.05 μg/ml increased the inhibition to 78.6% +/− 5.8% or 58.3% +/− 19.2%, respectively (
n
= 10;
P
< 0.01 versus controls and versus the drug without serum). Lower concentrations of serum also potentiated drug activity. The effect of human sera was further seen when using caspofungin that had lost activity (e.g., by storage) against
A. fumigatus
at 0.1 μg/ml. Inactive caspofungin alone demonstrated no significant inhibition of hyphal growth, whereas the addition of 5% human serum to the inactive drug showed 83% +/− 16.5% inhibition (
n
= 5;
P
< 0.01). The restoration of activity of caspofungin was seen at concentrations as low as 0.05% human serum. In contrast to prior reports, this study suggests that human serum acts synergistically with caspofungin to enhance its inhibitory activity in vitro against
A. fumigatus
.
Recombinant Murine Granulocyte-Macrophage Colony-Stimulating Factor Modulates the Course of Pulmonary Histoplasmosis in Immunocompetent and Immunodeficient Mice
Authors: Deepe George S. , Gibbons Reta
Published: July 2002

Several endogenous cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are necessary for eliminatingHistoplasma capsulatumfrom tissues. In this study, we explored the efficacy of recombinant murine GM-CSF in the treatment of pulmonary histoplasmosis. This cytokine significantly reduced fungal burden in a dose-dependent manner. Pretreatment did not consistently produce a better result than treatment started after infection. The biological effectiveness of GM-CSF was not associated with modulation of lung cytokine production or alteration in lung inflammation, but it directly activated a nonadherent lung cell population to exert anti-Histoplasmaactivity. GM-CSF improved survival of T-cell-depleted mice exposed toH. capsulatum. When combined with a suboptimal amount of amphotericin B, GM-CSF enhanced survival of normal or T-cell-depleted mice given a lethal challenge. These results suggest that this cytokine may be useful as an adjunctive treatment for histoplasmosis.
Capnocytophaga ochracea
: Characterization of a Plasmid-Encoded Extended-Spectrum TEM-17 β-Lactamase in the Phylum
Flavobacter-Bacteroides
Authors: Rosenau Agnes , Cattier Blandine , Gousset Nathalie , Harriau Patrick , Philippon Alain , Quentin Roland
Published: July 2002

A plasmid-encoded extended-spectrum TEM β-lactamase with a pI of 5.5 was detected in a
Capnocytophaga ochracea
clinical isolate. The
bla
gene was associated with a strong TEM-2 promoter and was derived from
bla
TEM-1a
with a single-amino-acid substitution: Glu
104
→Lys, previously assigned to TEM-17, which is thus the first TEM β-lactamase to be reported in the phylum
Flavobacter-Bacteroides
.
Nucleotide Sequence of the
bla
RTG-2
(CARB-5) Gene and Phylogeny of a New Group of Carbenicillinases
Authors: Choury Daniele , Szajnert Marie-France , Joly-Guillou Marie-Laure , Azibi Kemal , Delpech Marc , Paul Gérard
Published: July 2002

We determined the nucleotide sequence of the
bla
gene for the
Acinetobacter calcoaceticus
β-lactamase previously described as CARB-5. Alignment of the deduced amino acid sequence with those of known β-lactamases revealed that CARB-5 possesses an RTG triad in box VII, as described for the
Proteus mirabilis
GN79 enzyme, instead of the RSG consensus characteristic of the other carbenicillinases. Phylogenetic studies showed that these RTG enzymes constitute a new, separate group, possibly ancestors of the carbenicillinase family.
Antibacterial Agents and Release of Periplasmic Pertussis Toxin from
Bordetella pertussis
Authors: Craig-Mylius Kathleen A. , Weiss Alison A.
Published: July 2002

Pertussis toxin accumulates in the periplasm of
Bordetella pertussis
prior to secretion, and we examined its fate following treatment with antimicrobial agents. Both antibiotics that inhibit protein synthesis (erythromycin and chloramphenicol), transcription (rifampin), or cell wall biosynthesis (cefoperazone and piperacillin) and magnesium sulfate (which inhibits transcription of pertussis toxin, but not bacterial growth) did not prevent release of preformed toxin. In contrast, agents that affect bacterial membranes, such as polymyxin B, lidocaine, procaine, and ethanol, inhibited release of preformed pertussis toxin. These results suggest new protein synthesis is not required for pertussis toxin secretion, but a functional membrane complex is required.
IBC-1, a Novel Integron-Associated Class A β-Lactamase with Extended-Spectrum Properties Produced by an
Enterobacter cloacae
Clinical Strain
Authors: Giakkoupi Panagiota , Tzouvelekis Leonidas S. , Tsakris Athanassios , Loukova Veneta , Sofianou Danai , Tzelepi Eva
Published: July 2002

A transferable β-lactamase produced by a multidrug-resistant clinical isolate of
Enterobacter cloacae
was studied. The
bla
gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including
aac
(
6
′)
-Ib
. The encoded β-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with β-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of
Yersinia enterocolitica
, a carbenicillinase from
Proteus mirabilis
GN79, the species-specific β-lactamases of
Klebsiella oxytoca
, and the carbapenemase Sme-1. However, a phylogenetic association with established β-lactamase clusters could not be conclusively shown.
In Vitro Activity of Trovafloxacin against
Bacteroides fragilis
in Mixed Culture with either
Escherichia coli
or a Vancomycin- Resistant Strain of
Enterococcus faecium
Determined by an Anaerobic Time-Kill Technique
Authors: Stearne Lorna E. T. , Kooi Clarissa , Goessens Wil H. F. , Bakker-Woudenberg Irma A. J. M. , Gyssens Inge C.
Published: July 2002

To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of
Bacteroides fragilis
in pure culture or in mixed culture with either
Escherichia coli
or a vancomycin-resistant strain of
Enterococcus faecium
(VREF). With inocula of 5 × 10
5
CFU/ml and trovafloxacin concentrations of ≤2 μg/ml, a maximum observed effect (
E
max
) of ≥6.1 (log
10
CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10
8
CFU/ml, a similar
E
max
and a similar concentration to produce 50% of
E
max
(EC
50
) for
B. fragilis
were found in both pure cultures and mixed cultures with
E. coli
. However, to produce a similar killing of
B. fragilis
in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of
E. faecium
and a trovafloxacin-resistant strain of
E. coli
were also found to confer a similar “protective” effect on
B. fragilis
against the activity of trovafloxacin. Using inocula of 10
9
CFU/ml, the activity of trovafloxacin was retained for
E. coli
and
B. fragilis
and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of
B. fragilis
in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of
B. fragilis
and
E. coli
, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.
Cathelicidin Peptides Inhibit Multiply Antibiotic-Resistant Pathogens from Patients with Cystic Fibrosis
Authors: Saiman Lisa , Tabibi Setareh , Starner Timothy D. , San Gabriel Pablo , Winokur Patricia L. , Jia Hong Peng , McCray Paul B. , Tack Brian F.
Published: July 2002

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant
Pseudomonas aeruginosa
(
n
= 24),
Burkholderia cepacia
(
n
= 5),
Achromobacter xylosoxidans
(
n
= 5), and
Stenotrophomonas maltophilia
(
n
= 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid
P. aeruginosa
strains (MIC, 0.06 to 8 μg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 μg/ml), but CAP18 (MIC, 1.0 to 32 μg/ml), CAP18-18 (MIC, 1.0 to >32 μg/ml), and CAP18-22 (MIC, 0.5 to 32 μg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 μg/ml). Peptides had modest activities against
S. maltophilia
and
A. xylosoxidans
(MIC range, 1.0 to > 32 μg/ml), but none inhibited
B. cepacia
. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed
P. aeruginosa
within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.
Antifungal Activities of Two New Azasordarins, GW471552 and GW471558, in Experimental Models of Oral and Vulvovaginal Candidiasis in Immunosuppressed Rats
Authors: Martinez Antonio , Ferrer Santiago , Santos Inmaculada , Jimenez Elena , Sparrowe John , Regadera Javier , De Las Heras Federico Gomez , Gargallo-Viola Domingo
Published: July 2002

Sordarins constitute a new class of antifungal agents with a novel mechanism of action involving the selective inhibition of fungal protein synthesis. A further evolution of this class of antifungals has led to a new family of sordarin derivatives called azasordarins. The therapeutic efficacies of two new azasordarins, GW471552 and GW471558, were studied in experimental models of oral and vulvovaginal candidiasis in immunosuppressed rats. In all cases rats were immunosuppressed with dexamethasone in the drinking water. Oral candidiasis was established by inoculating 0.1 ml of a yeast suspension containing 5 × 10
8
cells of
Candida albicans
4711E with a cotton swab on three alternate days. Vulvovaginal candidiasis was established in ovariectomized and estrus-induced rats by intravaginal inoculation of 10
7
CFU of
C. albicans
4711E in 0.1 ml of saline. GW471552 and GW471558 were administered at 1, 5, and 10 mg/kg of body weight via the subcutaneous route. In oral candidiasis, azasordarins were administered each 8 h for 7 consecutive days, while in vaginal candidiasis the compounds were given each 4 h for 3 consecutive days. Antifungal activity of azasordarins was assessed by colony counts and by histological examination 1 day after treatment. In the oral infection model, GW471552 and GW471558 administered at 5 mg/kg significantly reduced (
P
< 0.05) the number of CFU of
C. albicans
compared with untreated controls. In addition, GW471552 and GW471558 given at 10 mg/kg eradicated
C. albicans
from the oral cavities of 100% of infected animals. Against vulvovaginal infection, both compounds showed significant therapeutic efficacy. GW471552 was able to eradicate the vaginal fungal burden at a dose of 10 mg/kg, and it significantly reduced the number of CFU of
C. albicans
in vaginas of rats treated with a dose of 5 mg/kg (
P
< 0.05). GW471558 showed greater efficacy, eradicating the fungal burden of 100% of infected rats at a dose of 5 mg/kg and significantly reducing (
P
< 0.05) the
C. albicans
vaginal counts even at a dose of 1 mg/kg. In both therapeutic efficacy studies, the histological findings confirmed the microbiological results. The experimental results presented show that the tested azasordarins are effective against oral and vulvovaginal candidiasis in immunosuppressed rats and could be promising antifungal agents for use in humans.
Activity of Oritavancin (LY333328), an Investigational Glycopeptide, Compared to That of Vancomycin against Multidrug-Resistant
Streptococcus pneumoniae
in an In Vitro Pharmacodynamic Model
Authors: Coyle Elizabeth A. , Rybak Michael J.
Published: July 2002

In the past 2 decades, multidrug-resistant
Streptococcus pneumoniae
has been encountered with increasing frequency around the world. This has led to the need for newer agents in the treatment of
S. pneumoniae
infections. Oritavancin (LY333328) is a new glycopeptide antibiotic with activity against gram-positive organisms; however, there is limited information on the pharmacodynamics of oritavancin with respect to the treatment of
S. pneumoniae
. We utilized an in vitro pharmacodynamic model to compare the activity of oritavancin to that of vancomycin against two penicillin-, macrolide-, and ciprofloxacin-resistant
S. pneumoniae
isolates (R919 and R921) over a 48-h period. Both oritavancin and vancomycin achieved 99.9% (3-log) kill, with oritavancin achieving the limit of detection (10
2
CFU/ml) within 1 h and vancomycin achieving this limit at 24 h for both isolates. Detection of resistance was not observed for oritavancin or vancomycin during the 48-h experiments. The key pharmacodynamic parameter for oritavancin has not been well defined. In our experiment, the ratios of the area under the curve from 0 to 24 h to the MIC of oritavancin, oritavancin plus albumin, and vancomycin for both isolates were greater than 944.5, and the ratios of the maximum concentration of drug in serum to the MIC ranged from 73.7 to 7188.5. T>MIC was 100% for oritavancin and vancomycin for both isolates. Oritavancin is a unique and potent antimicrobial that warrants further investigation against multidrug-resistant
S. pneumoniae
.
Single-Dose Intraperitoneal Magainins Improve Survival in a Gram-Negative-Pathogen Septic Shock Rat Model
Authors: Cirioni Oscar , Giacometti Andrea , Ghiselli Roberto , Mocchegiani Federico , Fineo Anna , Orlando Fiorenza , Del Prete Maria Simona , Rocchi Marco , Saba Vittorio , Scalise Giorgio
Published: July 2002

The therapeutic efficacies of three polycationic peptides selected among the class of the magainins (magainin I, magainin II, and magainin II amide), alone and combined with piperacillin, were investigated in a rat model of septic shock. Rats were given an intraperitoneal injection of 2 × 1010CFU ofEscherichia coliand randomized to receive intraperitoneally isotonic sodium chloride solution, 60 mg of piperacillin per kg of body weight, and 1 mg of each magainin per kg alone and combined with 60 mg of piperacillin per kg. The main outcome measures were bacterial growth in abdominal exudate and plasma, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. Treatments with the magainins achieved significant reductions of bacterial growth and plasma endotoxin and TNF-α concentrations. In general, treatments with the combinations of magainins and piperacillin demonstrated the highest efficacies.
Efficacy of Short-Course Ceftriaxone Therapy for
Borrelia burgdorferi
Infection in C3H Mice
Authors: Pavia Charles , Inchiosa Mario A. , Wormser Gary P.
Published: July 2002

Ceftriaxone is highly effective clinically in patients with Lyme disease. We studied a representative invasive human isolate of
Borrelia burgdorferi
for which the MBC of ceftriaxone was 0.050 μg/ml. A once-per-day dosage regimen of ceftriaxone (50 mg/kg/dose) administered intramuscularly for 5 days was 100% effective in sterilizing tissue samples of C3H mice infected with this strain of
B. burgdorferi
, regardless of whether the mice were being treated concomitantly with a corticosteroid. Administration of the same five doses of ceftriaxone at 6-h intervals over just 24 h was also 100% effective. These experiments suggest that shorter courses of antibiotics than those currently recommended should be considered for study in patients with early uncomplicated Lyme disease.
Differential Expression of Methicillin Resistance by Different Biofilm-Negative
Staphylococcus epidermidis
Transposon Mutant Classes
Authors: Mack Dietrich , Sabottke Axel , Dobinsky Sabine , Rohde Holger , Horstkotte Matthias A. , Knobloch Johannes K.-M.
Published: July 2002

Biofilm formation mediated by polysaccharide intercellular adhesin (PIA) is the major virulence factor of
Staphylococcus epidermidis
and is often associated with methicillin resistance. Transposon Tn
917
insertions leading to a biofilm-negative phenotype in the biofilm-producing
S. epidermidis
strain 1457 (
mecA
-negative) were transferred into the methicillin-resistant, biofilm-producing
S. epidermidis
1057 (
mecA
-positive) by transduction. According to their phenotypes and genotypes, the mutants could be separated into genetic classes I to IV (D. Mack, H. Rohde, S. Dobinsky, J. Riedewald, M. Nedelmann, J. K. M. Knobloch, H.-A. Elsner, and H. H. Feucht, Infect. Immun. 68:3799–3807, 2000). All transductants of
S. epidermidis
1057 had phenotypes for biofilm formation similar to those of the corresponding mutants of
S. epidermidis
1457. With a
mecA
-specific probe, identical hybridization patterns were observed for wild-type
S. epidermidis
1057 and all the transductants. There were minor changes in oxacillin MICs for Class II and III transductants compared to those for wild-type
S. epidermidis
1057. On population analysis,
S. epidermidis
1057 displayed a heterogeneous expression type of resistance with an oxacillin MIC of ≥6 μg/ml for more than 90% of the cells. An almost identical profile was observed with biofilm-negative class I mutants, where the transposon insertions inactivate the
icaADBC
gene locus essential for PIA synthesis. In contrast, class III mutants were more sensitive to oxacillin with a MIC of ≤1 μg/ml for more than 90% of the cells. The class IV mutant displayed homogenous resistance with a MIC of ≥50 μg/ml for more than 90% of the cells. On oxacillin gradient plates, the class II mutant displayed decreased resistance. Apparently, different independent mutations leading to a biofilm-negative phenotype of
S. epidermidis
by influencing expression of
icaADBC
on the level of transcription significantly influence the expression of methicillin resistance. However, transcription of
mecA
was not significantly altered in the different transductants compared to the wild type, independent of
mecA
induction with oxacillin, indicating that other mechanisms influencing phenotypic expression of methicillin resistance are involved.
Molecular Characterization of the
vanE
Gene Cluster in Vancomycin-Resistant
Enterococcus faecalis
N00-410 Isolated in Canada
Authors: Boyd D. A. , Cabral T. , Van Caeseele P. , Wylie J. , Mulvey M. R.
Published: July 2002

The
vanE
operon was characterized from
Enterococcus faecalis
N00-410 (MIC of vancomycin = 24 μg/ml). The organization of the
vanE
operon was identical to that of the
vanC1
operon from
Enterococcus gallinarum
, with protein identities ranging from 46 to 63%. An open reading frame located downstream of the
vanE
operon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene
guaA
.
Detection of a Variant Metallo-β-Lactamase, IMP-10, from Two Unrelated Strains of
Pseudomonas aeruginosa
and an
Alcaligenes xylosoxidans
Strain
Authors: Iyobe Shizuko , Kusadokoro Haruko , Takahashi Ayako , Yomoda Sachie , Okubo Toyoji , Nakamura Akio , O'Hara Koji
Published: July 2002

The gene
bla
IMP-10
of a variant metallo-β-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1. Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.
Molecular Characterization ofYarrowia lipolyticaandCandida zeylanoidesIsolated from Poultry
Authors: Deak T. , Chen J. , Beuchat L. R.
Published: July 2002

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains ofYarrowia lipolyticaand seven strains ofCandida zeylanoides. Digestion of amplicons withHinfI andHaeIII produced two fragments of 200 and 150 bp fromY. lipolyticaand three fragments of 350, 150, and 100 bp fromC. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA ofY. lipolyticainto three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained fromC. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.
Immobilization with Metal Hydroxides as a Means To Concentrate Food-Borne Bacteria for Detection by Cultural and Molecular Methods
Authors: Lucore Lisa A. , Cullison Mark A. , Jaykus Lee-Ann
Published: July 2002

The application of nucleic acid amplification methods to the detection of food-borne pathogens could be facilitated by concentrating the organisms from the food matrix before detection. This study evaluated the utility of metal hydroxide immobilization for the concentration of bacterial cells from dairy foods prior to detection by cultural and molecular methods. Using reconstituted nonfat dry milk (NFDM) as a model, two food-borne pathogens (
Listeria monocytogenes
and
Salmonella enterica
serovar Enteritidis) were concentrated from 25-ml samples by the sequential steps of clarification and high-speed centrifugation (designated primary concentration) and immobilization with zirconium hydroxide and low-speed centrifugation (designated secondary concentration). Sample volume reduction after immobilization with zirconium hydroxide was 50-fold, with total bacterial recoveries ranging from 78 to 96% of input for serovar Enteritidis and 65 to 96% of input for
L. monocytogenes
. Immobilized bacteria remained viable and could be enumerated by standard cultural procedures. When followed by RNA extraction and subsequent detection by reverse transcription (RT)-PCR, detection limits of 10
1
to 10
2
CFU/25 ml of reconstituted NFDM were achieved for both organisms. The bacterial-immobilization step was relatively nonspecific, resulting in recovery of >50% of the input cells when evaluated on a panel of representative bacterial strains of significance to foods. The method could be adapted to more complex dairy products, such as whole milk and ice cream, for which bacterial recoveries after immobilization ranged from 64 to >100%, with subsequent RT-PCR detection limits of ≥10
2
CFU/ml for whole milk and ≥10
1
CFU for ice cream for both serovar Enteritidis and
L. monocytogenes
. The bacterial-immobilization method is easy, rapid, and inexpensive and may have applications for the concentration of a wide variety of food-borne bacteria prior to detection by both conventional and alternative methods.
Isolation and Characterization of 2,3-Dichloro-1-Propanol-Degrading Rhizobia
Authors: Effendi Agus J. , Greenaway Steven D. , Dancer Brian N.
Published: July 2002

2,3-Dichloro-1-propanol is more chemically stable than its isomer, 1,3-dichloro-2-propanol, and is therefore more difficult to degrade. The isolation of bacteria capable of complete mineralization of 2,3-dichloro-1-propanol was successful only from enrichments at high pH. The bacteria thus isolated were found to be members of the α division of the
Proteobacteria
in the
Rhizobium
subdivision, most likely
Agrobacterium
sp. They could utilize both dihaloalcohol substrates and 2-chloropropionic acid. The growth of these strains in the presence of 2,3-dichloro-1-propanol was strongly affected by the pH and buffer strength of the medium. Under certain conditions, a ladder of four active dehalogenase bands could be visualized from this strain in activity gels. The enzyme involved in the complete mineralization of 2,3-dichloro-1-propanol was shown to have a native molecular weight of 114,000 and consisted of four subunits of similar molecular weights.
A Serine Protease-Encoding Gene (
aprII
) of
Alteromonas
sp. Strain O-7 Is Regulated by the Iron Uptake Regulator (Fur) Protein
Authors: Tsujibo Hiroshi , Miyamoto Katsushiro , Okamoto Takashi , Orikoshi Hideyuki , Inamori Yoshihiko
Published: July 2002

The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (
aprII
) from a marine bacterium,
Alteromonas
sp. strain O-7. To clarify whether the production of AprII (the gene product of
aprII
) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting. The production of AprII was significantly repressed under iron-rich conditions. Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription. These results indicate that
aprII
is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the gene encoding Fur was cloned and sequenced. The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.
Proteins Induced during Adaptation of
Acetobacter aceti
to High Acetate Concentrations
Authors: Steiner Peter , Sauer Uwe
Published: July 2002

As a typical product of microbial metabolism, the weak acid acetate is well known for its cytotoxic effects. In contrast to most other microbes, the so-called acetic acid bacteria can acquire significant resistance to high acetate concentrations when properly adapted to such hostile conditions. To characterize the molecular events that are associated with this adaptation, we analyzed global protein expression levels during adaptation of
Acetobacter aceti
by two-dimensional gel electrophoresis. Adaptation was achieved by using serial batch and continuous cultivations with increasing acetate supplementation. Computer-aided analysis revealed a complex proteome response with at least 50 proteins that are specifically induced by adaptation to acetate but not by other stress conditions, such as heat or oxidative or osmotic stress. Of these proteins, 19 were significantly induced in serial batch and continuous cultures and were thus noted as acetate adaptation proteins (Aaps). Here we present first microsequence information on such Aaps from
A. aceti
. Membrane-associated processes appear to be of major importance for adaptation, because some of the Aap bear N-terminal sequence homology to membrane proteins and 11 of about 40 resolved proteins from membrane protein-enriched fractions are significantly induced.
Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis
Authors: Satokari Reetta M. , Vaughan Elaine E. , Akkermans Antoon D. L. , Saarela Maria , de Vos Willem M.
Published: July 2002

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).
Bifidobacterium
genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in
Bifidobacterium
-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces.
Bifidobacterium adolescentis
was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of
B. adolescentis
, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of
B. adolescentis
E-981074
T
exhibited microheterogeneity differing in eight positions over almost the total length of the gene.
Molecular Characterization of Cycloinulooligosaccharide Fructanotransferase from
Bacillus macerans
Authors: Kim Hwa-Young , Choi Yong-Jin
Published: July 2002

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of β-(2→1)-linked
d
-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from
Bacillus macerans
CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated
M
r
of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the
B. macerans
CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from
B. macerans
CFC1. The CFT107 enzyme was produced by
B. macerans
CFC1 but was not detected from the recombinant
Escherichia coli
cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their

K
m

values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (
k
cat
/

K
m

) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.
Succession of Indigenous
Pseudomonas
spp. and Actinomycetes on Barley Roots Affected by the Antagonistic Strain
Pseudomonas fluorescens
DR54 and the Fungicide Imazalil
Authors: Thirup Laila , Johnsen Kaare , Winding Anne
Published: July 2002

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain
Pseudomonas fluorescens
DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous
Pseudomonas
organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (
Pseudomonas
) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting
Streptomyces
and closely related genera. DR54 constituted more than 75% of the
Pseudomonas
population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous
Pseudomonas
organisms negatively, whereas imazalil affected
Pseudomonas
numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for
Pseudomonas
during plant growth, but after plant harvest
Pseudomonas
-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence,
Pseudomonas
was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.
Morphological and Physiological Characterization of
Listeria monocytogenes
Subjected to High Hydrostatic Pressure
Authors: Ritz M. , Tholozan J. L. , Federighi M. , Pilet M. F.
Published: July 2002

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on
Listeria monocytogenes
cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from −86 to −5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.
Specific Identification and Targeted Characterization of
Bifidobacterium lactis
from Different Environmental Isolates by a Combined Multiplex-PCR Approach
Authors: Ventura Marco , Reniero Roberto , Zink Ralf
Published: July 2002

The species
Bifidobacterium lactis
, with its main representative strain Bb12 (DSM 10140)
,
is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29
Bifidobacterium
and 9
Lactobacillus
species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus
Bifidobacterium
and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of
B. lactis
were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.
Single-Base-Pair Discrimination of Terminal Mismatches by Using Oligonucleotide Microarrays and Neural Network Analyses
Authors: Urakawa Hidetoshi , Noble Peter A. , El Fantroussi Said , Kelly John J. , Stahl David A.
Published: July 2002

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (
T
d
) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5′ terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the
T
d
and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the
T
d
and signal intensity, and it decreased the variability of the signal. Although
T
d
s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower
T
d
s than those with mismatches in the first or second position. The trained NNs predicted the
T
d
with high accuracies (
R
2
= 0.93). However, the NNs predicted the signal intensity only moderately accurately (
R
2
= 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in
T
d
s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5′ terminus plays a greater role in determining the
T
d
and signal intensity of duplexes than the type of mismatch.
Lactobacillus bulgaricus
Proteinase Expressed in
Lactococcus lactis
Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins
Authors: Bernasconi Eric , Germond Jacques-Edouard , Delley Michèle , Fritsché Rodolphe , Corthésy Blaise
Published: July 2002

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant
Lactococcus lactis
expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with
Lactobacillus bulgaricus
extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins.
L. lactis
expressing PrtB∂-BLG yielded up to 170 μg per 10
9
CFU in the culture supernatant and 9 μg per 10
9
CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant
L. lactis
and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.
Nonradioactive Techniques for Measurement of In Vitro T-Cell Proliferation: Alternatives to the [3H]Thymidine Incorporation Assay
Authors: Messele Tsehaynesh , Roos Marijke T. L. , Hamann Dorte , Koot Maarten , Fontanet Arnaud L. , Miedema Frank , Schellekens Peter T. A. , Rinke de Wit Tobias F.
Published: July 2002

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [3H]TdR assay as a “gold standard.” As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2′-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [3H]TdR incorporation in both study groups (HIV-1 positives,r= 0.96; HIV-1 negatives,r= 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [3H]TdR incorporation, both in HIV-1-positive (r= 0.96) and HIV-1-negative (r= 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.