bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Acid Trihydrate (NAT) in Polar Stratospheric Clouds
Authors: Voigt Christiane , Schreiner Jochen , Kohlmann Andreas , Zink Peter , Mauersberger Konrad , Larsen Niels , Deshler Terry , Kröger Chris , Rosen Jim , Adriani Alberto , Cairo Francesco , Donfrancesco Guido Di , Viterbini Maurizio , Ovarlez Joelle , Ovarlez Henri , David Christine , Dörnbrack Andreas
Published: July 2002

A comprehensive investigation of polar stratospheric clouds was performed on 25 January 2000 with instruments onboard a balloon gondola flown from Kiruna, Sweden. Cloud layers were repeatedly encountered at altitudes between 20 and 24 kilometers over a wide range of atmospheric temperatures (185 to 197 kelvin). Particle composition analysis showed that a large fraction of the cloud layers was composed of nitric acid trihydrate (NAT) particles, containing water and nitric acid at a molar ratio of 3:1; this confirmed that these long-sought solid crystals exist well above ice formation temperatures. The presence of NAT particles enhances the potential for chlorine activation with subsequent ozone destruction in polar regions, particularly in early and late winter.
Antimalarial Activities of Dermaseptin S4 Derivatives
Authors: Krugliak Miriam , Feder Rina , Zolotarev Vadim Y. , Gaidukov Leonid , Dagan Arie , Ginsburg Hagai , Mor Amram
Published: July 2002

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC
50
) in the micromolar range. Among these, the substituted S4 analog K
4
K
20
-S4 was the most potent (IC
50
= 0.2 μM), while its shorter version, K
4
-S4(1–13)a, retained a considerable potency (IC
50
= 6 μM). Both K
4
K
20
-S4 and K
4
-S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K
4
K
20
-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K
4
K
20
-S4, the most hemolytic dermaseptin. The same trend was observed with K
4
-S4(1–13)a. The
d
isomers of K
4
K
20
-S4 or K
4
-S4(1–13)a were as active as the
l
counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.
Role of Penicillin-Binding Protein 4 in Expression of Vancomycin Resistance among Clinical Isolates of Oxacillin-Resistant
Staphylococcus aureus
Authors: Finan J. E. , Archer Gordon L. , Pucci Michael J. , Climo Michael W.
Published: July 2002

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible
Staphylococcus aureus
isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible
S. aureus
(VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant
S. aureus
(ORSA) isolates. The nucleotide sequences of the
pbp4
structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of
pbp4
by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded
pbp4
restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.
Cloning and Biochemical Characterization of FOX-5, an AmpC-Type Plasmid-Encoded β-Lactamase from a New York City
Klebsiella pneumoniae
Clinical Isolate
Authors: Queenan Anne Marie , Jenkins Stephen , Bush Karen
Published: July 2002

Klebsiella pneumoniae
5064, isolated in New York, carried plasmid-mediated resistance to multiple β-lactams and was unresponsive to clavulanic acid. The β-lactamase gene responsible for cephalosporin resistance encoded FOX-5, with 96 to 97% amino acid identities to other members of the FOX family of β-lactamases. The
bla
FOX-5
coding region was located next to a transposase gene from the
Aeromonas salmonicida
insertion element IS
AS2
.
In Vivo Monitoring of Intracellular ATP Levels inLeishmania donovaniPromastigotes as a Rapid Method To Screen Drugs Targeting Bioenergetic Metabolism
Authors: Luque-Ortega Juan Román , Rivero-Lezcano Octavio Miguel , Croft Simon L. , Rivas Luis
Published: July 2002

A method for the rapid screening of drugs targeting the bioenergetic metabolism ofLeishmaniaspp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels ofLeishmania donovanipromastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form ofPhothinus pyralisluciferase and incubated with free-membrane permeabled-luciferin analogued-luciferin–[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (ΔΨm) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 μM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.
Safety and Pharmacokinetics of Multiple 750-Milligram Doses of Intravenous Levofloxacin in Healthy Volunteers
Authors: Chow Andrew T. , Fowler Cynthia , Williams R. Rex , Morgan Nancy , Kaminski Susan , Natarajan Jaya
Published: July 2002

The safety and pharmacokinetics of a once-daily high intravenous dose of levofloxacin (750 mg) in 18 healthy volunteers were studied in a double-blind, randomized, placebo-controlled, single-center parallel group study. Levofloxacin was well tolerated, and higher maximum concentration of drug in serum and area under the concentration-time curve values were achieved. For difficult-to-treat infections, high daily doses of levofloxacin may be beneficial, and intravenous administration may be preferred in certain clinical settings, such as when treating patients in intensive care units, warranting further evaluation.
Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among
Enterobacteriaceae
in the People's Republic of China
Authors: Chanawong Aroonwadee , M'Zali Fatima Hannachi , Heritage John , Xiong Jian-Hui , Hawkey Peter Michael
Published: July 2002

Of 15 extended-spectrum β-lactamase (ESBL)-producing isolates of the family
Enterobacteriaceae
collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying
bla
CTX-M
genes revealed that they harbored three different
bla
CTX-M
genes,
bla
CTX-M-9
(5 isolates),
bla
CTX-M-13
(1 isolate), and
bla
CTX-M-14
(6 isolates). These genes have 98% nucleotide homology with
bla
Toho-2
. The
bla
CTX-M
genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the
bla
CTX-M
genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence IS
Ecp1
, found on the upstream region of these genes, may be involved in the translocation of the
bla
CTX-M
genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of
bla
CTX-M
genes and their dissemination among at least three species of the family
Enterobacteriaceae
,
Escherichia coli
,
Klebsiella pneumoniae
, and
Enterobacter cloacae
, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.
Fitness Cost of Chromosomal Drug Resistance-Conferring Mutations
Authors: Sander Peter , Springer Burkhard , Prammananan Therdsak , Sturmfels Antje , Kappler Martin , Pletschette Michel , Böttger Erik C.
Published: July 2002

To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with definedMycobacterium smegmatislaboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.
CTX-M-14, a Plasmid-Mediated CTX-M Type Extended-Spectrum β-Lactamase Isolated from
Escherichia coli
Authors: Ma L. , Ishii Yoshikazu , Chang Feng-Yee , Yamaguchi Keizo , Ho Monto , Siu L. K.
Published: July 2002

Four
Escherichia coli
isolates harboring CTX-M-14, with a single Ala231→Val substitution compared to CTX-M-9, had three different ribotypes. Cefotaxime resistance was plasmid encoded and conjugatively transferable. Three isolates had the same plasmid restriction enzyme digestion profile, suggesting clonal spread of a resistant plasmid. A high
k
cat
/

K
m

value for cefotaxime (20.3 μM
−1
s
−1
) but low values for ceftazidime and aztreonam (<0.02 μM
−1
s
−1
) were observed in hydrolysis assays, indicating resistance to cefotaxime (MIC ⩾ 64 μg/ml) but susceptibility to ceftazidime (MIC ⩽ 2 μg/ml).
Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia
Authors: Ross Ian L. , Alami Younes , Harvey Paul R. , Achouak Wafa , Ryder Maarten H.
Published: July 2002

Rhizobacteria closely related to two recently described species of pseudomonads,
Pseudomonas brassicacearum
and
Pseudomonas thivervalensis
, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against
P. brassicacearum
. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either
Pseudomonas corrugata
or
P. brassicacearum-P. thivervalensis
type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen
Gaeumannomyces graminis
var.
tritici
, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that
P. brassicacearum
and
P. thivervalensis
are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.
Redirection of the Respiro-Fermentative Flux Distribution in
Saccharomyces cerevisiae
by Overexpression of the Transcription Factor Hap4p
Authors: Blom Jolanda , De Mattos M. Joost Teixeira , Grivell Leslie A.
Published: July 2002

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of
Saccharomyces cerevisiae
. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.
Degradation of Xylan to
d
-Xylose by Recombinant
Saccharomyces cerevisiae
Coexpressing the
Aspergillus niger
β-Xylosidase (
xlnD
) and the
Trichoderma reesei
Xylanase II (
xyn2
) Genes
Authors: La Grange D. C. , Pretorius I. S. , Claeyssens M. , van Zyl W. H.
Published: July 2002

The β-xylosidase-encoding
xlnD
gene of
Aspergillus niger
90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the
Saccharomyces cerevisiae
mating factor α1 signal peptide (MFα1
s
) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant β-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The
K

i
(app)

value for
d
-xylose and xylobiose for the recombinant β-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The
XLO2
fusion gene and the
XYN2
β-xylanase gene from
Trichoderma reesei
, located on
URA3
-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast
Saccharomyces cerevisiae
under the control of the alcohol dehydrogenase II gene (
ADH2
) promoter and terminator. These recombinant
S. cerevisiae
strains produced 1,577 nkat/ml of β-xylanase activity when expressing only the β-xylanase and 860 nkat/ml when coexpressing the β-xylanase with the β-xylosidase. The maximum β-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the β-xylanase. Coproduction of the β-xylanase and β-xylosidase enabled
S. cerevisiae
to degrade birchwood xylan to
d
-xylose.
Interaction of Ammonium, Glucose, and Chitin Regulates the Expression of Cell Wall-Degrading Enzymes in
Trichoderma atroviride
Strain P1
Authors: Donzelli Bruno Giuliano Garisto , Harman Gary E.
Published: July 2002

Chitinolytic and glucanolytic fungal cell wall-degrading enzymes have been suggested to be primary determinants of biocontrol by
Trichoderma
spp. We examined the effects of ammonium, glucose, chitin, and chito-oligomers on transcription of specific genes and secretion of fungal cell wall-degrading enzymes. The genes
ech42
,
nag1
, and
gluc78
were examined, as were the enzymes they encode (endochitinase CHIT42,
N
-acetylhexosaminidase CHIT73, and glucan exo-1,3-β-glucanase GLUC78, respectively).
gluc78
could be induced by nitrogen starvation alone, while both
ech42
and
nag1
required nitrogen starvation and the presence of chitin for induction. Starvation for both ammonium and glucose resulted in very early expression and secretion of all cell wall-degrading enzymes examined. In the presence of low levels of ammonium (10 mM), both chito-oligomers and chitin triggered CHIT42 and CHIT40 (chitobiosidase) production. CHIT73 secretion occurred in the presence of
N
-acetylglucosamine and chito-oligomers, while chitin was less effective. The presence of different chito-oligomers resulted in secretion of specific
N
-acetylhexosaminidases, of which CHIT73 is one. Our results indicate that the expression and secretion of cell wall-degrading enzymes is nitrogen repressed, that effects of carbon and nitrogen nutrition are interactive, and that especially for chitinolytic enzymes, the inductive effect of chitin is altered by the level of ammonium or glucose in the medium.
Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR
Authors: Hermansson Anna , Lindgren Per-Eric
Published: July 2002

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the β subdivision of the class
Proteobacteria
in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were ∼6.2 × 10
7
ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.
Plant-Derived Oils Reduce Pathogens and Gaseous Emissions from Stored Cattle Waste
Authors: Varel Vincent H. , Miller Daniel N.
Published: July 2002

Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 × 10
6
to 2.0 × 10
3
cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 × 10
10
to 1.5 × 10
7
cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste.
Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from
Ceriporiopsis subvermispora
and
Phanerochaete chrysosporium
Authors: Larrondo Luis F. , Lobos Sergio , Stewart Phillip , Cullen Dan , Vicuña Rafael
Published: July 2002

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes
Ceriporiopsis subvermispora
(MnP1) and
Phanerochaete chrysosporium
(H4) under control of the α-amylase promoter from
Aspergillus oryzae
in
Aspergillus nidulans
. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from
P. chrysosporium
(pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from
C. subvermispora
grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize
o
-dianisidine and
p
-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from
P. chrysosporium
.
Obligate Sulfide-Dependent Degradation of Methoxylated Aromatic Compounds and Formation of Methanethiol and Dimethyl Sulfide by a Freshwater Sediment Isolate,Parasporobacterium paucivoransgen. nov., sp. nov
Authors: Lomans Bart P. , Leijdekkers Pieter , Wesselink Jan-Jaap , Bakkes Patrick , Pol Arjan , van der Drift Chris , den Camp Huub J. M. Op
Published: July 2002

Methanethiol (MT) and dimethyl sulfide (DMS) have been shown to be the dominant volatile organic sulfur compounds in freshwater sediments. Previous research demonstrated that in these habitats MT and DMS are derived mainly from the methylation of sulfide. In order to identify the microorganisms that are responsible for this type of MT and DMS formation, several sulfide-rich freshwater sediments were amended with two potential methyl group-donating compounds, syringate and 3,4,5-trimethoxybenzoate (0.5 mM). The addition of these methoxylated aromatic compounds resulted in excess accumulation of MT and DMS in all sediment slurries even though methanogenic consumption of MT and DMS occurred. From one of the sediment slurries tested, a novel anaerobic bacterium was isolated with syringate as the sole carbon source. The strain, designatedParasporobacterium paucivorans, produced MT and DMS from the methoxy groups of syringate. The hydroxylated aromatic residue (gallate) was converted to acetate and butyrate. LikeSporobacterium olearium, another methoxylated aromatic compound-degrading bacterium, the isolate is a member of the XIVa cluster of the low-GC-contentClostridialesgroup. However, the new isolate differs from all other known methoxylated aromatic compound-degrading bacteria because it was able to degrade syringate in significant amounts only in the presence of sulfide.
Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20
Authors: Zhong Yan , Chen Feng , Wilhelm Steven W. , Poorvin Leo , Hodson Robert E.
Published: July 2002

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different
Synechococcus
groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.
Purification and Characterization of a Surface Protein from
Lactobacillus fermentum
104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin
Authors: Rojas Maurilia , Ascencio Felipe , Conway Patricia L.
Published: July 2002

An adhesion-promoting protein involved in the binding of
Lactobacillus fermentum
strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of
L. fermentum
104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.
Rapid Detection and Enumeration of
Naegleria fowleri
in Surface Waters by Solid-Phase Cytometry
Authors: Pougnard Claire , Catala Philippe , Drocourt Jean-Louis , Legastelois Stephane , Pernin Pierre , Pringuez Emmanuelle , Lebaron Philippe
Published: July 2002

A new method for the rapid and accurate detection of pathogenic
Naegleria fowleri
amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.
T-Helper-Cell Proliferative Responses to Whole-Killed Human Immunodeficiency Virus Type 1 (HIV-1) and p24 Antigens of Different Clades in HIV-1-Infected Subjects Vaccinated with HIV-1 Immunogen (Remune)
Authors: Moss Ronald B. , Giermakowska Wieslawa , Wallace Mark R. , Savary Jay , Jensen Fred , Carlo Dennis J.
Published: July 2002

The discovery of multiple subtypes of human immunodeficiency virus type 1 (HIV-1) worldwide has created new challenges for the development of both therapeutic and preventive AIDS vaccines. We examined T-helper proliferative responses to HIV-1 clade A, B, C, G, and E whole-killed virus and to HIV-1 clade G and B core (p24) antigens in HIV-1-infected subjects taking potent antiviral drugs who received HIV immunogen (Remune) therapeutic vaccination. Subjects who were immunized mounted strong proliferative responses to both whole virus and core antigens of the different clades. These results suggest that a whole-killed immunogen may have broad applications as a therapeutic as well as a preventive vaccine in the current multiclade HIV-1 pandemic.
Naive Human T Cells Develop into Th1 Effectors after Stimulation withMycobacterium tuberculosis-Infected Macrophages or Recombinant Ag85 Proteins
Authors: Russo Donna M. , Kozlova Natalia , Lakey David L. , Kernodle Douglas
Published: July 2002

Most studies of human T-cell responses in tuberculosis have focused on persons with either active disease or latent infection. Although this work has been critical in defining T-cell correlates of successful versus failed host containment, little is known about the development ofMycobacterium-specific T-cell responses in uninfected persons. To explore this issue, naive T cells from uninfected donors were sensitized in vitro with avirulentMycobacterium tuberculosis-infected autologous macrophages. T-cell lines primed in this manner proliferated and produced cytokines after challenge with mycobacterial antigens. Of 11 such lines, 8 were high Th1 responders, 2 were low Th1 responders, and 1 was a Th2 responder. Furthermore, similar patterns and magnitudes of proliferative and cytokine responses were seen whenMycobacteriuminfection-primed lines were challenged with recombinant antigen 85 (Ag85) proteins. The addition of interleukin 12 (IL-12) during the initial sensitization increased the magnitude of Th1 responses; however, antibody to IL-12 did not eliminate Th1 responses, suggesting that additional factors contributed to the differentiation of these cells. Finally, in the presence of IL-12, recombinant Ag85B was able to prime naive T cells for Th1 responses upon challenge withMycobacterium-infected macrophages or Ag85B. Therefore, under the appropriate conditions, priming with whole bacteria or a subunit antigen can stimulateMycobacterium-specific Th1 effector cell development. Further definition of the antigens and conditions required to drive naive human T cells to differentiate into Th1 effectors should facilitate the development of an improved tuberculosis vaccine.
Human Monocyte Receptors Involved in Tumor Necrosis Factor Responses to Group B Streptococcal Products
Authors: Cuzzola Maria , Mancuso Giuseppe , Beninati Concetta , Biondo Carmelo , von Hunolstein Christina , Orefici Graziella , Espevik Terje , Flo Trude H. , Teti Giuseppe
Published: July 2002

Several group B streptococcal products have been previously found to stimulate human monocytes to produce tumor necrosis factor alpha. In order to identify the receptors involved in these responses, monocytes were stimulated with purified group- or type-specific carbohydrates or lipoteichoic acid in the presence of anti-receptor monoclonal antibodies, soluble CD14, or lipopolysaccharide-binding protein. Results indicate that CD14 plays an important role in tumor necrosis factor alpha responses to all of the stimuli tested. Moreover, both CD14 and complement receptor type 3 may be involved in responses to the group-antigen.
Innate Lung Defenses and CompromisedPseudomonas aeruginosaClearance in the Malnourished Mouse Model of Respiratory Infections in Cystic Fibrosis
Authors: Yu H. , Nasr S. Z. , Deretic V.
Published: July 2002

Cystic fibrosis (CF) is characterized by dysfunction of the digestive and respiratory tracts resulting in generalized malnutrition and chronic respiratory infections. Chronic lung infections withPseudomonas aeruginosa, intense neutrophil-dominated airway inflammation, and progressive lung disease are the major cause of high morbidity and mortality in CF. Here we investigated the effects of malnutrition in CF on innate lung defenses, susceptibility toP. aeruginosacolonization, and associated inflammation, using aerosol models of acute and chronic infections in normal, malnourished, and transgenic mice.CFTRm1Unc−/−knockout mice displayed body weight variations and showed variable pulmonary clearance ofP. aeruginosa. This variability was not detected in bitransgenicCFTRm1Unc−/−(FABP-hCFTR) mice in which the intestinal defect had been corrected. Diet-induced protein calorie malnutrition in C57BL/6J mice resulted in impaired pulmonary clearance ofP. aeruginosa. Tumor necrosis factor alpha (TNF-α) and nitrite levels detected upon exposure toP. aeruginosaaerosols were lower in the lungs of the malnourished C57BL/6J mice relative than in lungs of mice fed a normal diet. The role of TNF-α and reactive nitrogen intermediates inP. aeruginosaclearance was tested in TNF-α and inducible nitric oxide synthase (iNOS) knockout mice.P. aeruginosaclearance was diminished in transgenic TNF-α- and iNOS-deficient mice. In contrast to the effects of TNF-α and iNOS, gamma interferon knockout mice retained a full capacity to eliminateP. aeruginosafrom the lung. Malnutrition also contributed to excessive inflammation in C57BL/6J mice upon chronic challenge withP. aeruginosa. The repeatedly infected malnourished host did not produce interleukin-10, a major anti-inflammatory cytokine absent or diminished in the bronchoalveolar fluids of CF patients. These results are consistent with a model in which defectiveCFTRin the intestinal tract leads to nutritional deficiency which in turn contributes to compromised innate lung defenses, bacterial colonization, and excessive inflammation in the CF respiratory tract.
Transforming Growth Factor-β Inhibits Lipopolysaccharide-Stimulated Expression of Inflammatory Cytokines in Mouse Macrophages through Downregulation of Activation Protein 1 and CD14 Receptor Expression
Authors: Imai Kenichi , Takeshita Akira , Hanazawa Shigemasa
Published: July 2002

The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-β1 (TGF-β1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-β1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-β1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fosand c-junoligonucleotides. Since TGF-β1 pretreatment inhibited LPS-stimulated expression of c-fosand c-jungenes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-β1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1β and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-β1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-β1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-β1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-β1 inhibition of LPS-induced septic shock.
Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from
Fasciola hepatica
Authors: Kim Tong-Soo , Jung Younghun , Na Byoung-Kuk , Kim Ki-Sun , Chung Pyung-Rim
Published: July 2002

The cytosolic superoxide dismutase (SOD) of
Fasciola hepatica
, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An
F. hepatica
cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the
F. hepatica
Cu/Zn-SOD was cloned and expressed in
Escherichia coli
. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.
Mutator Natural
Escherichia coli
Isolates Have an Unusual Virulence Phenotype
Authors: Picard Bertrand , Duriez Patrick , Gouriou Stéphanie , Matic Ivan , Denamur Erick , Taddei François
Published: July 2002

A small percentage of natural
Escherichia coli
isolates (both commensal and pathogenic) have a mutator phenotype related to defects in methyl-directed mismatch repair (MR) genes. We investigated whether there was a direct link between the mutator phenotype and virulence by (i) studying the relationships between mutation rate and virulence in a mouse model of extraintestinal virulence for 88 commensal and extraintestinal pathogenic
E. coli
isolates and (ii) comparing the virulence in mice of MR-deficient and MR-proficient strains that were otherwise isogenic. The results provide no support for the hypothesis that the mutator phenotype has a direct role in virulence or is associated with increased virulence. Most of the natural mutator strains studied displayed an unusual virulence phenotype with (i) a lack of correspondence between the number of virulence determinants and pathogenicity in mice and (ii) an intermediate level of virulence. On a large evolutionary scale, the mutator phenotype may help parasites to achieve an intermediate rate of virulence which mathematical models predict to be selected for during long-term parasite-host interactions.
Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of
Helicobacter pylori
Authors: Fischer Wolfgang , Buhrdorf Renate , Gerland Elke , Haas Rainer
Published: July 2002

Helicobacter pylori
produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in
H. pylori
the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the
H. pylori
outer membrane and that the CtxB domain is exposed on the
H. pylori
surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the
H. pylori
surface and hence acts as a functional autotransporter.
Comparison of Genetic Divergence and Fitness between Two Subclones of
Helicobacter pylori
Authors: Björkholm Britta , Lundin Annelie , Sillén Anna , Guillemin Karen , Salama Nina , Rubio Carlos , Gordon Jeffrey I. , Falk Per , Engstrand Lars
Published: July 2002

Helicobacter pylori
has a very plastic genome, reflecting its high rate of recombination and point mutation. This plasticity promotes divergence of the population by the development of subclones and presumably enhances adaptation to host niches. We have investigated the genotypic and phenotypic characteristics of two such subclones isolated from one patient as well as the genetic evolution of these isolates during experimental infection. Whole-genome genotyping of the isolates using DNA microarrays revealed that they were more similar to each other than to a panel of other genotyped strains recovered from different hosts. Nonetheless, they still showed significant differences. For example, one isolate (67:21) contained the entire Cag pathogenicity island (PAI), whereas the other (67:20) had excised the PAI. Phenotypic studies disclosed that both isolates expressed adhesins that recognized human histo-blood group Lewis
b
glycan receptors produced by gastric pit and surface mucus cells. In addition, both isolates were able to colonize, to equivalent density and with similar efficiency, germ-free transgenic mice genetically engineered to synthesize Lewis
b
glycans in their pit cells (12 to 14 mice/isolate). Remarkably, the Cag PAI-negative isolate was unable to colonize conventionally raised Lewis
b
transgenic mice harboring a normal gastric microflora, whereas the Cag PAI-positive isolate colonized 74% of the animals (39 to 40 mice/isolate). The genomic evolution of both isolates during the infection of conventionally raised and germ-free mice was monitored over the course of 3 months. The Cag PAI-positive isolate was also surveyed after a 10 month colonization of conventionally raised transgenic animals (
n
= 9 mice). Microarray analysis of the Cag PAI and sequence analysis of the
cagA
,
recA
, and 16S rRNA genes disclosed no changes in recovered isolates. Together, these results reveal that the
H. pylori
population infecting one individual can undergo significant divergence, creating stable subclones with substantial genotypic and phenotypic differences.
Mucosal Vaccination with Recombinantly Attenuated Staphylococcal Enterotoxin B and Protection in a Murine Model
Authors: Stiles Bradley G. , Garza Anthony R. , Ulrich Robert G. , Boles James W.
Published: July 2002

Previous work in our laboratory revealed that mice parenterally vaccinated with recombinantly attenuated staphylococcal enterotoxin (SE) or toxic shock syndrome toxin 1 develop protective antibodies against a lethal intraperitoneal (i.p.) toxin challenge. This study investigated the efficacy of nasal and oral immunizations with an SEB vaccine (SEBv) toward an i.p. or mucosal (via an aerosol) toxin challenge. Both vaccination routes, with the immunoadjuvant cholera toxin (CT), elicited comparable SEB-specific immunoglobulin A (IgA) and IgG levels in saliva. Nasal or oral inoculations also generated SEB-specific IgA, IgG, and IgM in the serum, but the nasal route yielded higher specific IgG titers. SEBv alone, when given nasally or orally, did not induce any detectable SEB-specific antibody. Mice vaccinated mucosally were protected against a 50% lethal dose of wild-type SEB given i.p. or mucosally, thus demonstrating that nasal or oral administration of this SEBv, with CT, elicits systemic and mucosal antibodies to SEB that protect against SEB-induced lethal shock.
Staphylococcus aureusFibronectin Binding Proteins Are Essential for Internalization by Osteoblasts but Do Not Account for Differences in Intracellular Levels of Bacteria
Authors: Ahmed Saddif , Meghji Sajeda , Williams Rachel J. , Henderson Brian , Brock Jeremy H. , Nair Sean P.
Published: July 2002

Staphylococcus aureusis a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake ofS. aureusby osteoblasts. AnS. aureusseptic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains ofS. aureusblocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains ofS. aureusare internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.
Priming by DNA Immunization Augments Protective Efficacy ofMycobacterium bovisBacille Calmette-Guerin against Tuberculosis
Authors: Feng Carl G. , Palendira Umaimainthan , Demangel Caroline , Spratt Joanne M. , Malin Adam S. , Britton Warwick J.
Published: July 2002

Sequential immunization with mycobacterial antigen Ag85B-expressing DNA andMycobacterium bovisbacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting againstMycobacterium tuberculosisinfection. Depletion of the CD8+T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8+T cells.