bluuebunny/crossref_metadata_2025 huggingface dataset:
A Low-Barrier Hydrogen Bond in the Catalytic Triad of Serine Proteases? Theory Versus Experiment
Authors: Ash Elissa L. , Sudmeier James L. , De Fabo Edward C. , Bachovchin William W.
Published: July 2002

Cleland and Kreevoy recently advanced the idea that a special type of hydrogen bond (H-bond), termed a low-barrier hydrogen bond (LBHB), may account for the “missing” transition state stabilization underlying the catalytic power of many enzymes, and Frey
et al.
have proposed that the H-bond between aspartic acid 102 and histidine 57 in the catalytic triad of serine proteases is an example of a catalytically important LBHB. Experimental facts are here considered regarding the aspartic acid–histidine and
cis–
urocanic H-bonds that are inconsistent with fundamental tenets of the LBHB hypothesis. The inconsistencies between theory and experiment in these paradigm systems cast doubt on the existence of LBHBs, as currently defined, within enzyme active sites.
Combinatorial Chemistry in Insects: A Library of Defensive Macrocyclic Polyamines
Authors: Schröder Frank C. , Farmer Jay J. , Attygalle Athula B. , Smedley Scott R. , Eisner Thomas , Meinwald Jerrold
Published: July 2002

The pupal defensive secretion of the coccinellid beetle
Epilachna borealis
is composed principally of a combinatorial library of macrocyclic polyamines. These compounds constitute a previously unrecognized family of natural products, characterized by extremely large-ring lactonic structures derived from a small set of (2-hydroxyethylamino)alkanoic acids. The combinatorial assembly of these simple building blocks generates a high degree of structural diversity, which is further increased by slow, spontaneous intramolecular rearrangement of the macrocycles.
Helical Polyacetylene Synthesized with a Chiral Nematic Reaction Field
Authors: Akagi K. , Piao G. , Kaneko S. , Sakamaki K. , Shirakawa H. , Kyotani M.
Published: July 2002

Helical polyacetylene was synthesized under an asymmetric reaction field consisting of chiral nematic (N*) liquid crystals (LCs). The chiral nematic LC was prepared by adding a chiroptical binaphthol derivative as a chiral dopant to a mixture of two nematic LCs. Acetylene polymerizations were carried out using the catalyst titanium tetra-
n
-butoxide–triethylaluminum dissolved in the chiral nematic LC solvent. The polyacetylene film was shown by scanning electron microscopy to consist of clockwise or counterclockwise helical structure of fibrils. A Cotton effect was observed in the region of the π → π* transition of the polyacetylene chain in circular dichroism spectra. The high electrical conductivities of ∼1500 to 1800 siemens per centimeter after iodine doping and the chiral helicity of these films may be exploited in electromagnetic and optical applications.
Asymmetric Electrical Structure in the Mantle Beneath the East Pacific Rise at 17°S
Authors: Evans R. L. , Tarits P. , Chave A. D. , White A. , Heinson G. , Filloux J. H. , Toh H. , Seama N. , Utada H. , Booker J. R. , Unsworth M. J.
Published: July 2002

The magnetotelluric component of the Mantle Electromagnetic and Tomography (MELT) Experiment measured the electrical resistivity structure of the mantle beneath the fast-spreading southern East Pacific Rise (EPR). The data reveal an asymmetric resistivity structure, with lower resistivity to the west of the ridge. The uppermost 100 kilometers of mantle immediately to the east of the ridge is consistent with a dry olivine resistivity structure indicating a mantle depleted of melt and volatiles. Mantle resistivities to the west of the ridge are consistent with a low-melt fraction (about 1 to 2 percent interconnected melt) distributed over a broad region and extending to depths of about 150 kilometers. The asymmetry in resistivity structure may be the result of asymmetric spreading rates and a westward migration of the ridge axis and suggests distinct styles of melt formation and delivery in the mantle beneath the two plates.
Melting Dynamics Beneath the Tonga-Kermadec Island Arc Inferred from
231
Pa-
235
U Systematics
Authors: Bourdon Bernard , Turner Simon , Allègre Claude
Published: July 2002

Measurement of
231
Pa-
235
U disequilibrium in Tonga-Kermadec island arc lavas (north of New Zealand) permits discrimination of the time scales and mechanisms of fluid addition and partial melting. For Tonga lavas, the (
231
Pa/
235
U) ratios are less than 1 and can be explained by fluid addition from the subducting plate around 60,000 years ago. In contrast, the effects of partial melting overprinted those of fluid addition in the Kermadec lavas resulting in (
231
Pa/
235
U) ratios greater than 1.
40
K-
40
Ar Constraints on Recycling Continental Crust into the Mantle
Authors: Coltice Nicolas , Albarède Francis , Gillet Philippe
Published: July 2002

Extraction of potassium into magmas and outgassing of argon during melting constrain the relative amounts of potassium in the crust with respect to those of argon in the atmosphere. No more than 30% of the modern mass of the continents was subducted back into the mantle during Earth's history. It is estimated that 50 to 70% of the subducted sediments are reincorporated into the deep continental crust. A consequence of the limited exchange between the continental crust and the upper mantle is that the chemistry of the upper mantle is driven by exchange of material with the deep mantle.
Genetic Diversity of Carbapenem-Hydrolyzing Metallo-β-Lactamases from
Chryseobacterium
(
Flavobacterium
)
indologenes
Authors: Bellais Samuel , Poirel Laurent , Leotard Sophie , Naas Thierry , Nordmann Patrice
Published: July 2002

The class B carbapenem-hydrolyzing β-lactamase IND-1 has been characterized for
Chryseobacterium indologenes
strain 001. With internal primers for the
bla
gene for IND-1 (
bla
IND-1
) and an internal
bla
IND-1
probe, PCR amplifications failed, while hybridization results were positive when DNA from another
C. indologenes
isolate, strain CIP101026, was used as a template. Thus, a
bla
IND
-related gene was cloned from this
C. indologenes
reference strain. Sequencing of the insert of a recombinant plasmid conferring resistance to carbapenems revealed an open reading frame with a G + C content of 39.9% and coding for a 243-amino-acid preprotein named IND-2. IND-2 shared 80% amino acid identity with IND-1 and had a similar broad-spectrum resistance profile, including resistance to carbapenems. It was classified in functional subgroup 3a of class B carbapenem-hydrolyzing β-lactamases. IND-1 and IND-2, despite their genetic diversity, possessed similar kinetic parameters, except that ceftazidime was hydrolyzed less by IND-2. To obtain the entire
bla
IND
-related gene sequences of eight other
C. indologenes
isolates, PCR was performed using internal and external primers, followed by inverse PCR techniques. The likely chromosome-mediated metallo-β-lactamases of the 10
C. indologenes
isolates were divided into several groups and subgroups. IND-1, IND-2, IND-2a, IND-3, and IND-4 shared 77 to 99% amino acid identity.
Interactions between Triazoles and Amphotericin B against
Cryptococcus neoformans
Authors: Barchiesi Francesco , Schimizzi Anna M. , Caselli Francesca , Novelli Andrea , Fallani Stefania , Giannini Daniele , Arzeni Daniela , Di Cesare Simona , Di Francesco Luigi Falconi , Fortuna Moira , Giacometti Andrea , Carle Flavia , Mazzei Teresita , Scalise Giorgio
Published: July 2002

The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates of
Cryptococcus neoformans
. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of
C. neoformans
2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of
C. neoformans
2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against
C. neoformans
in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction.
In Vitro Activities of Linezolid against Multiple
Nocardia
Species
Authors: Brown-Elliott Barbara A. , Ward Shelby C. , Crist Christopher J. , Mann Linda B. , Wilson Rebecca W. , Wallace Richard J.
Published: July 2002

Linezolid was tested by broth microdilution against 140 clinical
Nocardia
isolates belonging to seven species. The MIC at which 50% of the strains are inhibited (MIC
50
) and MIC
90
for all species other than
Nocardia farcinica
were 2 and 4 μg/ml. Linezolid is the first antimicrobial agent demonstrated to be active against all
Nocardia
species.
Emergence of a Pneumococcal Clone with Cephalosporin Resistance and Penicillin Susceptibility
Authors: Smith Anthony M. , Botha Roelof F. , Koornhof Hendrik J. , Klugman Keith P.
Published: July 2002

We report two South African serotype 6B pneumococcal isolates with cephalosporin resistance, yet with susceptibility to penicillin. DNA fingerprinting revealed that they were clonal in origin.
pbp 2X
and
1A
genes showed major alterations typical of cephalosporin-resistant pneumococci. The
pbp 2B
gene was completely unaltered, explaining the penicillin susceptibility of the isolates.
Prediction of Abacavir Resistance from Genotypic Data: Impact of Zidovudine and Lamivudine Resistance In Vitro and In Vivo
Authors: Walter Hauke , Schmidt Barbara , Werwein Marianne , Schwingel Eva , Korn Klaus
Published: July 2002

Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI). Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V. Abacavir resistance has also been observed in NRTI multidrug-resistant samples. Furthermore, abacavir resistance has been described in the context of zidovudine resistance. To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples. Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively. If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible. Therefore, the relative frequencies of other mutations were evaluated. Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples. Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant. Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only. Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase. Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction. In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant. Thus, options for useful sequential combinations of NRTI are limited.
Sterol and Fatty Acid Composition ofCandida lusitaniaeClinical Isolates
Authors: Peyron F. , Favel A. , Calaf R. , Michel-Nguyen A. , Bonaly R. , Coulon J.
Published: July 2002

The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin B-susceptibleCandida lusitaniaeclinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in Δ8→7 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.
Stable Concentrations of Zidovudine, Stavudine, Lamivudine, Abacavir, and Nevirapine in Serum and Cerebrospinal Fluid during 2 Years of Therapy
Authors: van Praag Rieneke M. E. , van Weert Elisabeth C. M. , van Heeswijk Rolf P. G , Zhou Xiao-Jian , Sommadossi Jean-Pierre , Jurriaans Suzanne , Lange Joep M. A. , Hoetelmans Richard M. W. , Prins Jan M.
Published: July 2002

For a number of antiretroviral drugs, prolonged suppression of viral replication is related to drug exposure. Therefore, it is important to maintain stable concentrations during prolonged therapy. While studies suggest that saquinavir concentrations decrease over time, we show that concentrations of zidovudine, stavudine, lamivudine, abacavir, and nevirapine in serum and cerebrospinal fluid are stable during 2 years of therapy.
Intrapulmonary Pharmacokinetics of Telithromycin, a New Ketolide, in Healthy Japanese Volunteers
Authors: Kadota Jun-Ichi , Ishimatsu Yuji , Iwashita Tetsuji , Matsubara Yuichi , Tomono Kazunori , Tateno Masao , Ishihara Roza , Muller-Serieys Claudette , Kohno Shigeru
Published: July 2002

The concentrations of telithromycin, a new ketolide antimicrobial agent, in alveolar macrophages (AMs) and bronchoalveolar epithelial lining fluid (ELF) were determined in order to investigate the transfer of the drug into target tissue, relative to plasma, following multiple oral doses of telithromycin. Twenty-four healthy male Japanese volunteers were randomly allocated to four groups. Each subject was given 600 or 800 mg of telithromycin once daily for 5 days, followed by bronchoalveolar lavage (BAL) 2 or 8 h after the last dose (group A and B: 600 mg, 2 and 8 h BAL time point; group C and D: 800 mg, 2 and 8 h BAL time point). The mean concentrations of the drug in AMs and ELF were 34.54 and 4.92 mg/liter in group A, 50.97 and 2.26 mg/liter in group B, 25.47 and 4.24 mg/liter in group C, and 108.22 and 4.31 mg/liter in group D, respectively, which markedly exceeded concentrations in plasma. These results demonstrated good transfer of telithromycin into AMs and ELF, suggesting good efficacy against common respiratory pathogens, including intracellular pathogens and atypical microorganisms.
Enantiomeric Degradation of 2-(4-Sulfophenyl)Butyrate via 4-Sulfocatechol inDelftia acidovoransSPB1
Authors: Schulz Saskia , Dong Wenbo , Groth Ulrich , Cook Alasdair M.
Published: July 2002

Enrichment cultures with enantiomeric 2-(4-sulfophenyl)butyrate (SPB) as the sole added source(s) of carbon and energy for growth yielded a pure culture of a degradative bacterium, which was identified asDelftia acidovoransSPB1. The organism utilized the enantiomers sequentially.R-SPB was utilized first (specific growth rate [μ] = 0.28 h−1), with transient excretion of an unknown intermediate, which was identified as 4-sulfocatechol (4SC). Utilization ofS-SPB was slower (μ = 0.016 h−1) and was initiated only after the first enantiomer was exhausted. Suspensions of cells grown inS-SPB excreted 4SC, so metabolism of the two enantiomers converged at 4SC. The latter was degraded byorthocleavage via 3-sulfo-cis,cis-muconate. Strain SPB1 grew with 4SC and with 1-(4-sulfophenyl)octane (referred to herein as model LAS) but not with commercial linear alkylbenzenesulfonate (LAS) surfactant, which is subterminally substituted but nontoxic. It would appear that metabolism of the model LAS does not represent metabolism of commercial LAS.
Evaluation of Immunomagnetic Separation for Recovery of
Cryptosporidium parvum
and
Giardia duodenalis
from High-Iron Matrices
Authors: Yakub Gary P. , Stadterman-Knauer Kathleen L.
Published: July 2002

In this study we examined the recovery of
Cryptosporidium parvum
and
Giardia duodenalis
from matrices containing various concentrations of dissolved iron. The organisms were recovered by using the immunomagnetic separation-immunofluorescent assay method, and the levels of recovery were compared to the dissolved iron concentrations. The levels of recovery of
C. parvum
decreased sharply at dissolved iron concentrations greater than 4 mg/liter, while the levels of recovery of
G. duodenalis
decreased sharply at concentrations greater than 40 mg/liter.
A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria
Authors: Grünberg Karen , Wawer Cathrin , Tebo Bradley M. , Schüler Dirk
Published: July 2002

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium
Magnetospirillum gryphiswaldense
that could be assigned to two different genomic regions. Except for
mamA
, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of
M. magnetotacticum
and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including
mamA
and
mamB
) was found to be conserved in all three of the strains investigated. The
mamAB
cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.
Experimental and Field Studies ofEscherichia coliO157:H7 in White-Tailed Deer
Authors: Fischer John R. , Zhao Tong , Doyle Michael P. , Goldberg Martin R. , Brown Cathy A. , Sewell Christopher T. , Kavanaugh Darrell M. , Bauman Christopher D.
Published: July 2002

Studies were conducted to evaluate fecal shedding ofEscherichia coliO157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships betweenE. coliO157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 108CFU ofE. coliO157:H7. Inoculated deer were sheddingE. coliO157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. AlthoughE. coliO157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies,E. coliO157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998,E. coliO157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates ofE. coliO157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence ofE. coliO157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir ofE. coliO157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.
Export of Cytochrome P450 105D1 to the Periplasmic Space of
Escherichia coli
Authors: Kaderbhai Mustak A. , Ugochukwu Cynthia C. , Kelly Steven L. , Lamb David C.
Published: July 2002

CYP105D1, a cytochrome P450 from
Streptomyces griseus,
was appended at its amino terminus to the secretory signal of
Escherichia coli
alkaline phosphatase and placed under the transcriptional control of the native
phoA
promoter. Heterologous expression in
E. coli
phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell extract and whole-cell activity studies showed that the periplasmically located CYP105D1 competently catalyzed NADH-dependent oxidation of the xenobiotic compounds benzo[
a
]pyrene and erythromycin, further revealing the presence in the
E. coli
periplasm of endogenous functional redox partners. This system offers substantial advantages for the application of P450 enzymes to whole-cell biotransformation strategies, where the ability of cells to take up substrates or discard products may be limited.
Adhesion of
Pseudomonas fluorescens
(ATCC 17552) to Nonpolarized and Polarized Thin Films of Gold
Authors: Busalmen J. P. , de Sánchez S. R.
Published: July 2002

The adhesion of
Pseudomonas fluorescens
(ATCC 17552) to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow cell. The influence of the electrochemical potential was evaluated at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under controlled flow. Adhesion to nonpolarized gold surfaces readily increased with the time of exposition at both ionic-strength values. At negative potentials (−0.2 and −0.5 V [Ag/AgCl-KCl saturated {sat.}]), on the other hand, bacterial adhesion was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total at both negative potentials, whereas at 0.1 M NaCl the inhibition was proportional to the magnitude of the potential, being almost total at −0.5 V. The existence of reversible adhesion was investigated by carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high ionic strength (0.1 M NaCl). Theoretical calculations of the Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for the bacteria-gold interaction were in good agreement with experimental results at low ionic strength (0.01 M). At high ionic strength (0.1 M), deviations from DLVO behavior related to the participation of specific interactions were observed, when surfaces were polarized to negative potentials.
Specific Detection of
Bradyrhizobium
and
Rhizobium
Strains Colonizing Rice (
Oryza sativa
) Roots by 16S-23S Ribosomal DNA Intergenic Spacer-Targeted PCR
Authors: Tan Zhiyuan , Hurek Thomas , Vinuesa Pablo , Müller Peter , Ladha Jagdish K. , Reinhold-Hurek Barbara
Published: July 2002

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes
Aeschynomene fluminensis
(IRBG271) and
Sesbania aculeata
(IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from
Bradyrhizobium elkanii
.
Rhizobium
sp. (
Sesbania
) strain IRBG74 was located in the
Rhizobium
-
Agrobacterium
cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with
Agrobacterium tumefaciens;
emended name,
Rhizobium radiobacter
). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus
Bradyrhizobium,
as illustrated here for strains of
Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense,
and
Bradyrhizobium
sp. (
Chamaecytisus
) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.
Characterization of Cyt2Bc Toxin from
Bacillus thuringiensis
subsp.
medellin
Authors: Juárez-Pérez Victor , Guerchicoff Alejandra , Rubinstein Clara , Delécluse Armelle
Published: July 2002

We cloned and sequenced a new cytolysin gene from
Bacillus thuringiensis
subsp.
medellin
. Three IS
240
-like insertion sequence elements and the previously cloned
cyt1Ab
and
p21
genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from
Bacillus thuringiensis
subsp.
israelensis
and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of
B. thuringiensis
. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against
Aedes aegypti
,
Anopheles stephensi
, and
Culex quinquefasciatus
, including strains resistant to the
Bacillus sphaericus
binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant
C. quinquefasciatus
.
Immunoglobulin A (IgA) Deficiency and Alternative Celiac Disease-Associated Antibodies in Sera Submitted to a Reference Laboratory for Endomysial IgA Testing
Authors: Prince Harry E. , Norman Gary L. , Binder Walter L.
Published: July 2002

Immunoglobulin A (IgA) deficiency occurs more frequently in patients with celiac disease (CD) than in the general population and can lead to false-negative results in the best serologic test for CD, endomysial IgA (EMA). To evaluate the impact of IgA deficiency on serologic detection of CD in a reference laboratory setting, IgA levels were measured in 510 consecutive serum specimens submitted for testing for EMA; 510 consecutive serum specimens submitted forHelicobacter pyloriIgG testing served as a gastrointestinal symptom control group. The frequency of IgA deficiency was significantly higher among the specimens submitted for testing for EMA (5.1%) than among the specimens from the symptom control group (1.4%). Three subsets of sera from the group of specimens submitted for testing for EMA were then tested by additional serologic assays for CD; these subsets were EMA-positive sera (n= 25), EMA-negative, IgA-deficient sera (n= 26), and control sera (from EMA-negative, IgA-nondeficient patients age matched to IgA-deficient patients;n= 26). The proportions of EMA-positive sera positive by other assays for CD were 92% for transglutaminase IgA (TG-IgA), 80% for gliadin IgA, 84% for gliadin IgG, 60% for endomysial IgG (EMG), and 32% for transglutaminase IgG (TG-IgG). Very low proportions (0 to 8%) of IgA-deficient sera and control sera were positive for TG-IgA, gliadin IgA, EMG, and TG-IgG. Eight of 26 (31%) IgA-deficient serum samples were positive for gliadin IgG, whereas 3 of 26 (12%) control serum samples were positive for gliadin IgG, but this difference was not statistically significant. Physicians supplied clinical data for 18 of 26 patients with IgA deficiency; only 4 patients had undergone small-bowel biopsy, and 0 of 4 patients showed villous atrophy. These findings show that IgA deficiency is found more frequently among sera submitted for testing for EMA in a reference laboratory setting, but there was no clear-cut serologic or clinical evidence of CD in EMA-negative, IgA-deficient patients.
Prevalence of CagA and VacA Antibodies in Children with
Helicobacter pylori
-Associated Peptic Ulcer Compared to Prevalence in Pediatric Patients with Active or Nonactive Chronic Gastritis
Authors: Alarcón T. , Martínez M. J. , Urruzuno P. , Cilleruelo M. L. , Madruga D. , Sebastian M. , Domingo D. , Sanz J. C. , López-Brea M.
Published: July 2002

VacA and CagA serological responses were detected in pediatric patients: 44 and 56%, respectively, in peptic ulcer (PU) patients, 33.3 and 44.4% in active chronic gastritis (ACG) patients, and 23.2 and 39.2% in non-ACG patients. Higher seroprevalence to CagA+VacA and to CagA+VacA+35-kDa antigen was found among PU patients. However, a low level of sensitivity and specificity was found for indirect detection of PU patients.
Recombinant Glycoprotein Vaccines for Human Immunodeficiency Virus-Infected Children and Their Effects on Viral Quasispecies
Authors: Essajee Shaffiq M. , Yogev Ram , Pollack Henry , Greenhouse Bryan , Krasinski Keith , Borkowsky William
Published: July 2002

In individuals infected with human immunodeficiency virus type 1 (HIV-1), specific immunity is associated with a more diverse viral repertoire and slower disease progression. Attempts to enhance antiviral immunity with therapeutic vaccination have shown that recombinant glycoprotein (RGP) vaccines are safe, well tolerated, and immunogenic, but the effect of RGP vaccines on the viral repertoire is unknown. We evaluated diversification of the viral envelope in 12 HIV-infected children who received placebo or RGP vaccines. At baseline, 11 of 12 patients had multiple viral variants. On follow-up 6 months later, children who had a strong vaccine-associated lymphoproliferative immune response showed less viral diversification than those in whom the immune response was weak or absent. These results suggest that the immune response elicited by RGP vaccines does not exert a significant selection pressure on the viral quasispecies and therefore may not be helpful in changing the course of the disease.
Detection of
Cryptococcus neoformans
DNA in Tissue Samples by Nested and Real-Time PCR Assays
Authors: Bialek Ralf , Weiss Michael , Bekure-Nemariam Kubrom , Najvar Laura K. , Alberdi Maria B. , Graybill John R. , Reischl Udo
Published: July 2002

Two PCR protocols targeting the 18S rRNA gene of
Cryptococcus neoformans
were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 10
1
to 2.9 × 10
4
CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of
C. neoformans
per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast
Malassezia furfur
, which are phylogenetically related to
C. neoformans
. Twenty-four strains, including species of human skin flora like
M. furfur
and
Trichosporon
spp., were PCR negative. Amplification was observed with
Cryptococcus amylolentus
,
Filobasidiella depauperata
,
Cryptococcus laurentii
, and five species unrelated to clinical specimens. LightCycler PCR products from
F. depauperata
and
Trichosporon faecale
could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.
High Immunoglobulin G2 (IgG2) and Low IgG4 Levels Are Associated with Human Resistance toPlasmodium falciparumMalaria
Authors: Aucan Christophe , Traoré Yves , Tall François , Nacro Boubacar , Traoré-Leroux Thérèse , Fumoux Francis , Rihet Pascal
Published: July 2002

There is accumulating evidence for a role of immunoglobulin G (IgG) in protection against malarial infection and disease. Only IgG1 and IgG3 are considered cytophilic and protective againstP. falciparum, whereas IgG2 and IgG4 were thought to be neither and even to block protective mechanisms. However, no clear pattern of association between isotypes and protection has so far emerged. We analyzed the isotypic distribution of the IgG response to conserved epitopes andP. falciparumblood-stage extract in 283 malaria-exposed individuals whose occurrence of infection and malaria attack had been monitored for about 1 year. Logistic regression analyses showed that, at the end of the season of transmission, high levels of IgG2 to RESA and to MSP2 epitopes were associated with low risk of infection. Indeed, IgG2 is able to bind FcγRIIA in individuals possessing the H131 allele, and we showed that 70% of the study subjects had this allele. Also, high specific IgG4 levels were associated with an enhanced risk of infection and with a high risk of malaria attack. Moreover, specific IgG2 and IgG3 levels, as well as the IgG2/IgG4 and IgG3/IgG4 ratios, increased with the age of subjects, in parallel with the protection against infection and disease. IgG4 likely competes with cytophilic antibodies for antigen recognition and may therefore block cytotoxicity mediated by antibody-activated effector cells. In conclusion, these results favor a protective role of IgG3 and IgG2, which may activate effector cells through FcγRIIA, and provide evidence for a blocking role of IgG4 in malarial infection and disease.
Iha: a Novel
Escherichia coli
O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure
Authors: Tarr Phillip I. , Bilge Sima S. , Vary James C. , Jelacic Srdjan , Habeeb Rebecca L. , Ward Teresa R. , Baylor Michael R. , Besser Thomas E.
Published: July 2002

The mechanisms used by Shiga toxin (Stx)-producing
Escherichia coli
to adhere to epithelial cells are incompletely understood. Two cosmids from an
E. coli
O157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) of
Vibrio cholerae
(M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407–2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by
iha
. Iha is 67 kDa in
E. coli
O157:H7 and 78 kDa in laboratory
E. coli
and is structurally unlike other known adhesins. DNA adjacent to
iha
contains tellurite resistance loci and is conserved in structure in distantly related pathogenic
E. coli
, but it is absent from nontoxigenic
E. coli
O55:H7, sorbitol-fermenting Stx-producing
E. coli
O157:H−, and laboratory
E. coli
. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an
E. coli
chromosomal island of conserved structure. Pathogenic
E. coli
O157:H7 has only recently acquired this island.
Xanthine Oxidase Contributes to Host Defense againstBurkholderia cepaciain the p47phox−/−Mouse Model of Chronic Granulomatous Disease
Authors: Segal Brahm H. , Sakamoto Nobuaki , Patel Mayur , Maemura Kosei , Klein Andrew S. , Holland Steven M. , Bulkley Gregory B.
Published: July 2002

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide and downstream microbicidal reactive oxidants, leading to recurrent life-threatening bacterial and fungal infections. Xanthine oxidase (XO) is another enzyme known to produce superoxide in many tissues. Using the p47phox−/−mouse model of CGD, we evaluated the residual antibacterial activity of XO. Clearance ofBurkholderia cepacia, a major pathogen in CGD, was reduced in p47phox−/−mice compared to that in wild-type mice and was further inhibited in p47phox−/−mice by pretreatment with the specific XO inhibitor allopurinol. HepaticB. cepaciaburden was similar in the two genotypes, but allopurinol significantly reduced net hepatic killing and killing efficiency only in p47phox−/−mice. Clearance and killing of intravenousEscherichia coliwas intact in p47phox−/−mice and was unaffected by pretreatment with allopurinol. In CGD, XO may contribute to host defense against a subset of reactive oxidant-sensitive pathogens.
Serological Expression Cloning of Novel Immunoreactive Antigens of
Babesia microti
Authors: Lodes Michael J. , Houghton Raymond L. , Bruinsma Elizabeth S. , Mohamath Raodoh , Reynolds Lisa D. , Benson Darin R. , Krause Peter J. , Reed Steven G. , Persing David H.
Published: July 2002

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent,
Babesia microti
. To develop more sensitive and specific assays for
B. microti
, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique
B. microti
antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to
Plasmodium
sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during
B. microti
infection in humans.
Actinobacillus pleuropneumoniaeIron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response
Authors: Baltes Nina , Tonpitak Walaiporn , Gerlach Gerald-F. , Hennig-Pauka Isabel , Hoffmann-Moujahid Astrid , Ganter Martin , Rothkötter Hermann-J.
Published: July 2002

Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenicA. pleuropneumoniaesingle mutants (ΔexbBand ΔureC) and a newly constructedA. pleuropneumoniaedouble (ΔureCΔexbB) mutant in aerosol infection experiments. Neither theA. pleuropneumoniaeΔexbBmutant nor the double ΔureCΔexbBmutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence ofA. pleuropneumoniaein the porcine respiratory tract. TheA. pleuropneumoniaeΔureCmutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection theA. pleuropneumoniaeΔureCmutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number ofA. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with theA. pleuropneumoniaeΔureCmutant than in the BALF of those infected with the parent strain. These results imply thatA. pleuropneumoniaeurease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positiveA. pleuropneumoniaeparent strain.
Purification of Anthrax Edema Factor from
Escherichia coli
and Identification of Residues Required for Binding to Anthrax Protective Antigen
Authors: Kumar Praveen , Ahuja Nidhi , Bhatnagar Rakesh
Published: July 2002

The structural gene for anthrax edema factor (EF) was expressed in
Escherichia coli
under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from
E. coli
. EF purified from
E. coli
was biologically and functionally as active as its
Bacillus anthracis
counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.
Identification of an Antigenic Epitope in
Helicobacter pylori
Urease That Induces Neutralizing Antibody Production
Authors: Hirota Kaoru , Nagata Kumiko , Norose Yoshihiko , Futagami Seiji , Nakagawa Yohko , Senpuku Hidenobu , Kobayashi Masafumi , Takahashi Hidemi
Published: July 2002

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for
Helicobacter pylori
urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of
H. pylori
, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of
H. pylori
infection.
Chlamydia pneumoniae
Infects and Multiplies in Lymphocytes In Vitro
Authors: Haranaga Shusaku , Yamaguchi Hiroyuki , Friedman Herman , Izumi Shin-ichi , Yamamoto Yoshimasa
Published: July 2002

The obligate intracellular pathogen
Chlamydia (Chlamydophila) pneumoniae
is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between
C. pneumoniae
and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to
C. pneumoniae
infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to
C. pneumoniae
infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with
C. pneumoniae
. Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to
C. pneumoniae
infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for
C. pneumoniae
, i.e., lymphocytes.