bluuebunny/crossref_metadata_2025 huggingface dataset:
DPC 817: a Cytidine Nucleoside Analog with Activity against Zidovudine- and Lamivudine-Resistant Viral Variants
Authors: Schinazi Raymond F. , Mellors John , Bazmi Holly , Diamond Sharon , Garber Sena , Gallagher Karen , Geleziunas Romas , Klabe Ron , Pierce Michael , Rayner Marlene , Wu Jing-Tao , Zhang Hangchun , Hammond Jennifer , Bacheler Lee , Manion Douglas J. , Otto Michael J. , Stuyver Lieven , Trainor George , Liotta Dennis C. , Erickson-Viitanen Susan
Published: July 2002

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents.
Reassessment of
Clostridium difficile
Susceptibility to Metronidazole and Vancomycin
Authors: Peláez T. , Alcalá L. , Alonso R. , Rodríguez-Créixems M. , García-Lechuz J. M. , Bouza E.
Published: July 2002

Clostridium difficile
is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea. The drugs most commonly used to treat diseases associated with
C. difficile
are metronidazole and vancomycin. Most clinical laboratories assume that all
C. difficile
isolates are susceptible to metronidazole and vancomycin. We report on the antimicrobial susceptibilities of 415
C. difficile
isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000). The overall rate of resistance to metronidazole at the critical breakpoint (16 μg/ml) was 6.3%. Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1%. One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin. Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients. Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested.
Reduction of Fe(III), Mn(IV), and Toxic Metals at 100°C by
Pyrobaculum islandicum
Authors: Kashefi Kazem , Lovley Derek R.
Published: July 2002

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When
Pyrobaculum islandicum
was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of
P. islandicum
per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite.
P. islandicum
could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction,
P. islandicum
grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species
P. aerophilum
grew via Fe(III) reduction in a similar complex medium. Cell suspensions of
P. islandicum
reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.
Engineering Desiccation Tolerance in
Escherichia coli
Authors: Billi Daniela , Wright Deborah J. , Helm Richard F. , Prickett Todd , Potts Malcolm , Crowe John H.
Published: July 2002

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in
Escherichia coli
BL21DE3 by using the
spsA
gene of the cyanobacterium
Synechocystis
sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.
An In Vitro Study of the Effect of Fluoridated Milk on Oral Bacterial Biofilms
Authors: Pratten J. , Bedi R. , Wilson M.
Published: July 2002

Microcosmic dental plaques were grown in artificial saliva and supplemented with either milk or fluoridated milk. The presence of fluoride in the milk increased the pH of the biofilms and reduced the proportions of streptococci, demonstrating that in this model, fluoridation of milk produces biofilms with reduced cariogenic potential.
A Few Cosmopolitan Phylotypes Dominate Planktonic Archaeal Assemblages in Widely Different Oceanic Provinces
Authors: Massana Ramon , DeLong Edward F. , Pedrós-Alió Carlos
Published: July 2002

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.
A
Mycobacterium
Strain with Extended Capacities for Degradation of Gasoline Hydrocarbons
Authors: Solano-Serena Floriane , Marchal Rémy , Casarégola Serge , Vasnier Christelle , Lebeault Jean-Michel , Vandecasteele Jean-Paul
Published: July 2002

A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2,4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of
Mycobacterium austroafricanum
was found.
Mycobacterium
sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including
n
-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl
t
-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.
Cloning, Expression, and Sequence Analysis of the Gene Encoding the Alkali-Stable, Thermostable Endoxylanase from Alkalophilic, Mesophilic
Bacillus
sp. Strain NG-27
Authors: Gupta Naveen , Reddy Vanga Shiva , Maiti Sankar , Ghosh Amit
Published: July 2002

Alkalophilic
Bacillus
sp. strain NG-27 produces a 42-kDa endoxylanase active at 70°C and at a pH of 8.4. The gene for this endoxylanase was cloned and sequenced. The gene contained one open reading frame of 1,215 bases. An active site characteristic of the family 10 β-glycanases was recognized between amino acids 303 and 313, with the active glutamate at position 310. Though highly thermostable, the enzyme contains no cysteine residue.
Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged
Pseudomonas fluorescens
A506
Authors: Lowder M. , Unge A. , Maraha N. , Jansson J. K. , Swiggett J. , Oliver J. D.
Published: July 2002

The green fluorescent protein (GFP) gene,
gfp
, of the jellyfish
Aequorea victoria
is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the
gfp
gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows
gfp
-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a
gfp
-tagged strain of
Pseudomonas fluorescens
continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that
gfp
-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.
Anhydrobiotic Engineering of Gram-Negative Bacteria
Authors: García de Castro Arcadio , Bredholt Harald , Strøm Arne R. , Tunnacliffe Alan
Published: July 2002

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for
Escherichia coli
and
Pseudomonas putida
by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.
Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure
Authors: Masschalck Barbara , Van Houdt Rob , Van Haver Ellen G. R. , Michiels Chris W.
Published: July 2002

We have studied the inactivation of six gram-negative bacteria (
Escherichia coli
,
Pseudomonas fluorescens
,
Salmonella enterica
serovar Typhimurium,
Salmonella enteritidis
,
Shigella sonnei
, and
Shigella flexneri
) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both
Salmonella
species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except
E. coli
and
P. fluorescens
were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.
Peptide Synthetase Gene in
Trichoderma virens
Authors: Wilhite S. E. , Lumsden R. D. , Straney D. C.
Published: July 2002

Trichoderma virens
(synonym,
Gliocladium virens
), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of
T. virens
with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (
Psy
1) from
T. virens
using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the
Psy
1 gene as a gene that encodes a peptide synthetase. Disruption of the native
Psy
1 gene through gene replacement was used to identify the function of this gene.
Psy
1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that
Psy
1 plays a role in siderophore production.
Psy
1 disruptants cannot produce the major
T. virens
siderophore dimerum acid, a dipetide of acylated
N
δ
-hydroxyornithine. Biocontrol activity against damping-off diseases caused by
Pythium ultimum
and
Rhizoctonia solani
was not reduced by the
Psy
1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.
Isolation, Characterization, and Polyaromatic Hydrocarbon Degradation Potential of Aerobic Bacteria from Marine Macrofaunal Burrow Sediments and Description ofLutibacterium anuloederansgen. nov., sp. nov., andCycloclasticus spirillensussp. nov
Authors: Chung W. K. , King G. M.
Published: July 2002

Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene. Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth. Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile. Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genusCycloclasticusand represents a new species,Cycloclasticus spirillensussp. nov. Strain LC8 appears to represent a new genus and species,Lutibacterium anuloederansgen. nov., sp. nov., within theSphingomonadaceae. However, when inoculated into sediment slurries with or without exogenous phenanthrene, onlyL. anuloederansappeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation. In addition, onlyL. anuloederansappeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.
Viability of
Giardia intestinalis
Cysts and Viability and Sporulation State of
Cyclospora cayetanensis
Oocysts Determined by Electrorotation
Authors: Dalton C. , Goater A. D. , Pethig R. , Smith H. V.
Published: July 2002

Electrorotation is a noninvasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. Electrorotation studies are reported for two intestinal parasites,
Giardia intestinalis
and
Cyclospora cayetanensis
. It is concluded that viable and nonviable
G. intestinalis
cysts can be differentiated by this technique, and support for this conclusion was obtained using a fluorogenic vital dye assay and morphological indicators. The viability of
C. cayetanensis
oocysts (for which no vital dye assay is currently available) can also be determined by electrorotation, as can their sporulation state. Modeling of the electrorotational response of these organisms was used to determine their dielectric properties and to gain an insight into the changes occurring within them. Electrorotation offers a new, simple, and rapid method for determining the viability of parasites in potable water and food products and as such has important healthcare implications.
Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria
Authors: Metcalf James S. , Bell Steven G. , Codd Geoffrey A.
Published: July 2002

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Yersinia pseudotuberculosis
mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several
Yersinia
virulence proteins (
Yersinia
outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on
Yersinia
virulence determinant expression and secretion, as well as the host immune response to
Yersinia
antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of
Yersinia
proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.
Fate ofEntamoeba histolyticaduring Establishment of Amoebic Liver Abscess Analyzed by Quantitative Radioimaging and Histology
Authors: Rigothier Marie-Christine , Khun Hout , Tavares Paulo , Cardona Ana , Huerre Michel , Guillén Nancy
Published: July 2002

The protozoan parasiteEntamoeba histolyticais the causative agent of amoebiasis, a human disease characterized by dysentery and liver abscess. The physiopathology of hepatic lesions can be satisfactorily reproduced in the hamster animal model by the administration of trophozoites through the portal vein route. Hamsters were infected with radioactively labeled amoebas for analysis of liver abscess establishment and progression. The radioimaging of material from parasite origin and quantification of the number inflammation foci, with or without amoebas, described here provides the first detailed assessment of trophozoite survival and death during liver infection byE. histolytica. The massive death of trophozoites observed in the first hours postinfection correlates with the presence of a majority of inflammatory foci without parasites. A critical point for success of infection is reached after 12 h when the lowest number of trophozoites is observed. The process then enters a commitment phase during which parasites multiply and the size of the infection foci increases fast. The liver shows extensive areas of dead hepatocytes that are surrounded by a peripheral layer of parasites facing inflammatory cells leading to acute inflammation. Our results show that the host response promotes massive parasite death but also suggest also that this is a major contributor to the establishment of inflammation during development of liver abscess.
Restart of Exponential Growth of Cold-Shocked
Yersinia enterocolitica
Occurs after Down-Regulation of
cspA1/A2
mRNA
Authors: Neuhaus Klaus , Rapposch Sonja , Francis Kevin P. , Scherer Siegfried
Published: July 2002

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication
cspA1/A2
and growth of
Yersinia enterocolitica
were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0°C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of
cspA1/A2
mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade
cspA1/A2
mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing
cspA1/A2
mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.
Footprinting Studies of Specific Complexes Formed by RepA, a Replication Initiator of Plasmid pCU1, and Its Binding Site
Authors: Papp Péter Pál , Élö Péter , Semsey Szabolcs , Orosz László
Published: July 2002

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the
oriB
origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions −6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.