bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Measurement of the Reaction Front in Chemically Amplified Photoresists
Authors: Lin Eric K. , Soles Christopher L. , Goldfarb Dario L. , Trinque Brian C. , Burns Sean D. , Jones Ronald L. , Lenhart Joseph L. , Angelopoulos Marie , Willson C. Grant , Satija Sushil K. , Wu Wen-li
Published: July 2002

The continuing drive by the semiconductor industry to fabricate smaller structures using photolithography will soon require dimensional control at length scales comparable to the size of the polymeric molecules in the materials used to pattern them. The current technology, chemically amplified photoresists, uses a complex reaction-diffusion process to delineate patterned areas with high spatial resolution. However, nanometer-level control of this critical process is limited by the lack of direct measurements of the reaction front. We demonstrate the use of x-ray and neutron reflectometry as a general method to measure the spatial evolution of the reaction-diffusion process with nanometer resolution. Measuring compositional profiles, provided by deuterium-labeled reactant groups for neutron scattering contrast, we show that the reaction front within the material is broad rather than sharply defined and the compositional profile is altered during development. Measuring the density profile, we directly correlate the developed film structure with that of the reaction front.
Nonresonant Multiple Spin Echoes
Authors: Brill Thilo M. , Ryu Seungoh , Gaylor Richard , Jundt Jacques , Griffin Douglas D. , Song Yi-Qiao , Sen Pabitra N. , Hürlimann Martin D.
Published: July 2002

Nonresonant manipulation of nuclear spins can probe large volumes of sample situated in inhomogeneous fields outside a magnet, a geometry suitable for mobile sensors for the inspection of roads, buildings, and geological formations. However, the interference by Earth's magnetic field causes rapid decay of the signal within a few milliseconds for protons and is detrimental to this method. Here we describe a technique to suppress the effects of Earth's field by using adiabatic rotations and sudden switching of the applied fields. We observed hundreds of spin echo signals lasting for more than 600 milliseconds and accurately measured the relaxation times of a liquid sample.
Inhibition of Nanobacteria by Antimicrobial Drugs as Measured by a Modified Microdilution Method
Authors: Çíftçíoglu N. , Miller-Hjelle M. A. , Hjelle J. T. , Kajander E. O.
Published: July 2002

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.
Molecular Characterization of Recombinant
Pneumocystis carinii
Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine
Authors: Van Dross Rukiyah T. , Sanders Marilyn M.
Published: July 2002

The
Pneumocystis carinii
topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The
P. carinii
topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg
2+
requirement. Cleavage assays reveal that both the human and
P. carinii
enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4′,6′-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that
P. carinii
topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme,
P. carinii
topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of
P. carinii
and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with
P. carinii
topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.
Experimental Acute Otitis Media Due to NontypeableHaemophilus influenzae: Comparison of High and Low Azithromycin Doses with Placebo
Authors: Babl Franz E. , Pelton Stephen I. , Li Zhong
Published: July 2002

Treatment of acute otitis media (AOM) with azithromycin results in apparent clinical success, but tympanocentesis performed 4 to 6 days after initiation of therapy in children with nontypeableHaemophilus influenzae(NTHI) recovered from initial middle ear cultures demonstrates persistence of infection in more than 50% of episodes. We sought to determine the effect of azithromycin at different doses on the density of middle ear infection due to NTHI to provide additional understanding of this dichotomy between clinical and microbiologic outcome measures in AOM. In a chinchilla model of experimental otitis media (EOM), animals treated with placebo were compared to animals receiving a single daily dose 30 or 120 mg of azithromycin per kg of body weight per day for 5 days. Microbiologic outcome was assessed by obtaining quantitative cultures from the middle ear during a 5-day course and for 1 week following therapy. Azithromycin concentrations were measured to ascertain whether a concentration-dependent effect was present. Azithromycin at 30 and 120 mg/kg/day demonstrated a dose-dependent effect on the quantitative assessment of middle ear infection due to NTHI. A 30-mg/kg dose of azithromycin daily resulted in levels in serum and areas under the serum concentration-time curve at 24 h comparable to published data obtained with children given azithromycin at 5 to 10 mg/kg in multiday regimens. Increased doses of azithromycin (120 mg/kg) achieved 2.5- to 4-fold-higher levels in serum and 3- to 6-fold-higher total levels and levels in extracellular middle ear fluid as well as more rapid reduction in bacterial density and a greater proportion of middle ears with complete sterilization than either placebo or the 30-mg/kg/day regimen.
Comparative Stability Studies of Antipseudomonal β-Lactams for Potential Administration through Portable Elastomeric Pumps (Home Therapy for Cystic Fibrosis Patients) and Motor-Operated Syringes (Intensive Care Units)
Authors: Viaene Eric , Chanteux Hugues , Servais Hélène , Mingeot-Leclercq Marie-Paule , Tulkens Paul M.
Published: July 2002

The stability of antipseudomonal β-lactams in concentrated solutions was examined in view of their potential administration by continuous infusion with external pumps (for intensive care patients) or with portable pumps carried under clothing (for cystic fibrosis patients). Aztreonam (100 g/liter), piperacillin (128 g/liter, with tazobactam), and azlocillin (128 g/liter) remained 90% stable for up to more than 24 h at 37°C (mezlocillin [128 g/liter] was stable at 25°C but not at 37°C). Ceftazidime (120 g/liter), cefpirome (32 g/liter), and cefepime (50 g/liter) remained 90% stable for up to 24, 23.7, and 20.5 h at 25°C but only for 8, 7.25, and 13 h at 37°C, respectively. The control of temperature therefore appears to be critical for all three cephalosporins that cannot be recommended for use in portable pumps carried under clothes for prolonged periods for reasons of stability. Cefpirome and cefepime solutions developed an important color change (from light yellow to dark red) upon exposure when stored at 30°C or higher. Degradation of ceftazidime was accompanied by the liberation of pyridine which, at 37°C, was in excess of what is allowed by the U.S. Pharmacopeia, i.e., 1.1 mg/liter, after 8 and 12 h for drug concentrations of 12 and 8.3%, respectively. Imipenem and meropenem are too unstable (10% degradation at 25°C after 3.5 and 5.15 h, respectively) to be recommended for use by continuous infusion. Faropenem, examined in comparison with imipenem and meropenem, proved as stable as aztreonam or piperacillin.
Stress-Based Identification and Classification of Antibacterial Agents: Second-Generation
Escherichia coli
Reporter Strains and Optimization of Detection
Authors: Shapiro Elyse , Baneyx François
Published: July 2002

Escherichia coli
strains bearing single-copy fusions between the
lacZ
reporter gene and the
cspA
,
ibp
, or P3
rpoH
stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A. A. Bianchi and F. Baneyx, Appl. Environ. Microbiol.
65:
5023-5027, 1999). Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible
sulA
promoter and
lacZ
is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the
tolC
gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.
PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for
Streptococcus pneumoniae
Authors: Belanger Aimee E. , Lai Angel , Brackman Marcia A. , LeBlanc Donald J.
Published: July 2002

Described here are the development and validation of a novel approach to identify genes encoding drug targets in
Streptococcus pneumoniae
. The method relies on the use of an ordered genomic library composed of PCR amplicons that were generated under error-prone conditions so as to introduce random mutations into the DNA. Since some of the mutations occur in drug target-encoding genes and subsequently affect the binding of the drug to its respective cellular target, amplicons containing drug targets can be identified as those producing drug-resistant colonies when transformed into
S. pneumoniae
. Examination of the genetic content of the amplicon giving resistance coupled with bioinformatics and additional genetic approaches could be used to rapidly identify candidate drug target genes. The utility of this approach was verified by using a number of known antibiotics. For drugs with single protein targets, amplicons were identified that rendered
S. pneumoniae
drug resistant. Assessment of amplicon composition revealed that each of the relevant amplicons contained the gene encoding the known target for the particular drug tested. Fusidic acid-resistant mutants that resulted from the transformation of
S. pneumoniae
with amplicons containing
fusA
were further characterized by sequence analysis. A single mutation was found to occur in a region of the
S. pneumoniae
elongation factor G protein that is analogous to that already implicated in other bacteria as being associated with fusidic acid resistance. Thus, in addition to facilitating the identification of genes encoding drug targets, this method could provide strains that aid future mechanistic studies.
In Vitro Activities of Position 2 Substitution-Bearing 6-Nitro- and 6-Amino-Benzothiazoles and Their Corresponding Anthranilic Acid Derivatives againstLeishmania infantumandTrichomonas vaginalis
Authors: Delmas Florence , Di Giorgio Carole , Robin Maxime , Azas Nadine , Gasquet Monique , Detang Claire , Costa Muriel , Timon-David Pierre , Galy Jean-Pierre
Published: July 2002

6-Nitro- and 6-amino-benzothiazoles bearing different chains in position 2 and their corresponding anthranilic acid derivatives were investigated for their in vitro antiparasitic properties against parasites of the speciesLeishmania infantumandTrichomonas vaginaliscompared to their toxicity towards human monocytes. Biological investigations established that the antiprotozoal properties depended greatly on the chemical structure of the position 2 substitution-bearing group. Compound C1, 2-[(2-chloro-benzothiazol-6-yl) amino] benzoic acid, demonstrated an interesting antiproliferative activity towards parasites of the speciesT. vaginalis, while compound C11, 2-({2-[(2-hydroxyethyl) amino]-benzothiazol-6-yl} amino) benzoic acid, exhibited a promising activity against parasites of the speciesL. infantumin their intracellular amastigote form. Additional experiments established that compound C11, which was poorly toxic against the promastigote and the extracellular amastigote forms of the parasite, could improve host-protective mechanisms againstLeishmaniaby preventing parasite internalization by macrophages and stimulating NO production, by means of a mechanism synergistically enhanced by the presence of gamma interferon.
Activities of Six Different Quinolones against Clinical Respiratory Isolates of
Streptococcus pneumoniae
with Reduced Susceptibility to Ciprofloxacin in Spain
Authors: Pérez-Trallero E. , García-Rey C. , Martín-Sánchez A. M. , Aguilar L. , García-de-Lomas J. , Ruiz J.
Published: July 2002

Six quinolones were tested on 817 consecutive pneumococcal isolates for which ciprofloxacin MICs were high (≥2 μg/ml); the isolates had been collected during two recent Spanish surveillance studies. For strains for which the ciprofloxacin MIC was ≥4 μg/ml, the MICs at which 90% of the isolates tested against gemifloxacin, moxifloxacin, gatifloxacin, sparfloxacin, levofloxacin, and ofloxacin were inhibited were 0.25, 1, 1, 1, 4 and 16 μg/ml, respectively, and the corresponding prevalences of resistance were 0, 1, 4.5, 9.5, 8.4 and 23%. The proportion of isolates for which the ciprofloxacin MIC is high has increased over time.
Rapid Detection of a Molecular Marker for Chloroquine-Resistant Falciparum Malaria
Authors: Durand Rémy , Huart Virginie , Jafari Sayeh , Le Bras Jacques
Published: July 2002

A PCR-based technique using molecular beacons was developed to detect the chloroquine resistance-associated
pfcrt
K76T point mutation in
Plasmodium falciparum
. One hundred thirty African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This rapid and inexpensive genomic assay could expand the possibilities for monitoring chloroquine resistance.
Seasonal Fluctuations and Long-Term Persistence of Pathogenic Populations of
Agrobacterium
spp. in Soils
Authors: Krimi Z. , Petit A. , Mougel C. , Dessaux Y. , Nesme X.
Published: July 2002

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic
Agrobacterium
strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 × 10
5
pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a
vir
sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.
Modeling the Interactions of
Lactobacillus curvatus
Colonies in Solid Medium: Consequences for Food Quality and Safety
Authors: Malakar P. K. , Martens D. E. , van Breukelen W. , Boom R. M. , Zwietering M. H. , van 't Riet K.
Published: July 2002

The growth process of
Lactobacillus curvatus
colonies was quantified by a coupled growth and diffusion equation incorporating a volumetric rate of lactic acid production. Analytical solutions were compared to numerical ones, and both were able to predict the onset of interaction well. The derived analytical solution modeled the lactic acid concentration profile as a function of the diffusion coefficient, colony radius, and volumetric production rate. Interaction was assumed to occur when the volume-averaged specific growth rate of the cells in a colony was 90% of the initial maximum rate. Growth of
L. curvatus
in solid medium is dependent on the number of cells in a colony. In colonies with populations of fewer than 10
5
cells, mass transfer limitation is not significant for the growth process. When the initial inoculation density is relatively high, colonies are not able to grow to these sizes and growth approaches that of broth cultures (negligible mass transfer limitation). In foods, which resemble the model solid system and in which the initial inoculation density is high, it will be appropriate to use predictive models of broth cultures to estimate growth. For a very low initial inoculation density, large colonies can develop that will start to deviate from growth in broth cultures, but only after large outgrowth.
Cardiovascular Effects of Fermented Milk Containing Angiotensin-Converting Enzyme Inhibitors Evaluated in Permanently Catheterized, Spontaneously Hypertensive Rats
Authors: Fuglsang Anders , Nilsson Dan , Nyborg Niels C. B.
Published: July 2002

In this study, two strains of
Lactobacillus helveticus
were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of
L. helveticus
gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.
Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake as Revealed by 5S rRNA Analysis
Authors: Dominik Katja , Höfle Manfred G.
Published: July 2002

The community structure of bacterioplankton was studied at different depths (0 to 25 m) of a temperate eutrophic lake (Lake Plußsee in northern Germany) by using comparative 5S rRNA analysis. The relative amounts of taxonomic groups were estimated from 5S rRNA bands separated by high-resolution electrophoresis. Comparison of partial 5S rRNA sequences enabled detection of changes in single taxa over space and during seasons. Overall, the bacterioplankton community was dominated by 3 to 14 abundant (>4% of the total 5S rRNA) taxa. In general, the number of 5S rRNA bands (i.e., the number of bacterial taxa) decreased with depth. In the fall, when thermal stratification and chemical stratification were much more pronounced than they were in the spring, the correlation between the depth layers and the community structure was more pronounced. Therefore, in the fall each layer had its own community structure; i.e., there were different community structures in the epilimnion, the metalimnion, and the hypolimnion. Only three 5S rRNA bands were detected in the hypolimnion during the fall, and one band accounted for about 70% of the total 5S rRNA. The sequences of individual 5S rRNA bands from the spring and fall were different for all size classes analyzed except two bands, one of which was identified as
Comamonas acidivorans.
In the overall analysis of the depth profiles, the diversity in the epilimnion contrasted with the reduced diversity of the bacterioplankton communities in the hypolimnion, and large differences occurred in the composition of the epilimnion at different seasons except for generalists like
C. acidivorans.
Use of 16S Ribosomal DNA for Delineation of Marine Bacterioplankton Species
Authors: Hagström Åke , Pommier Thomas , Rohwer Forest , Simu Karin , Stolte Willem , Svensson Dominika , Zweifel Ulla Li
Published: July 2002

All of the marine bacterioplankton-derived 16S ribosomal DNA sequences previously deposited in GenBank were reanalyzed to determine the number of bacterial species in the oceanic surface waters. These sequences have been entered into the database since 1990. The rate of new additions reached a peak in 1999 and subsequently leveled off, suggesting that much of the marine microbial species richness has been sampled. When the GenBank sequences were dereplicated by using 97% similarity as a cutoff, 1,117 unique ribotypes were found. Of the unique sequences, 609 came from uncultured environmental clones and 508 came from cultured bacteria. We conclude that the apparent bacterioplankton species richness is relatively low.
An Aspartyl Protease Inhibitor Orthologue Expressed byParelaphostrongylus tenuisIs Immunogenic in an Atypical Host
Authors: Duffy Michael S. , MacAfee Nancy , Burt Michael D. B. , Appleton Judith A.
Published: July 2002

Parelaphostrongylus tenuisis a neurotropic nematode common in white-tailed deer (Odocoileus virginianus) of eastern North America. This parasite is the causative agent of a debilitating neurologic disease in atypical hosts, including domestic livestock. In order to identify proteins of potential significance in the host-parasite relationship, a cDNA library was produced from adultP. tenuismRNA. Screening the library with antisera from infected red deer (Cervus elaphus elaphus) and immunized AO strain rats, we identified clones with sequence similarities to aspartyl protease inhibitors from several parasitic nematodes. Antibody that was generated against this recombinant protein ofP. tenuis(Pt-API-1) detected the native protein in E/S products, in muscle and gonad, and on the surface of the cuticle of adult male and femaleP. tenuis. The native protein was detected in internal structures of first-stage (L1) and third-stage (L3) larvae. Reverse transcription-PCR confirmed expression ofPt-api-1in L1, L3, and adult male and female worms. Expression ofPt-API-1 throughout the life cycle ofP. tenuissuggests an essential function. Antibodies specific for recombinantPt-API-1 were detected by enzyme-linked immunosorbent assay in sera from 12 red deer experimentally infected withP. tenuis. Antibodies were detected within 28 to 56 days postinfection. Responses were sustained or biphasic in animals with patent infections, consistent with expression ofPt-API-1 by L1. Our results are compatible with findings in other parasitic nematodes showing that aspartyl protease inhibitors are highly immunogenic.
Sputum Cytokine Levels in Patients with Pulmonary Tuberculosis as Early Markers of Mycobacterial Clearance
Authors: Ribeiro-Rodrigues Rodrigo , Resende Co Tatiana , Johnson John L. , Ribeiro Fabiola , Palaci Moises , Sá Ricardo T. , Maciel Ethel L. , Pereira Lima Fausto E. , Dettoni Valderio , Toossi Zahra , Boom W. Henry , Dietze Reynaldo , Ellner Jerrold J. , Hirsch Christina S.
Published: July 2002

Sputum and serum from patients with active pulmonary tuberculosis (TB), healthy purified protein derivative-positive adults, and patients with bacterial pneumonia were collected to simultaneously assess local immunity in the lungs and peripheral blood. To determine whether cytokine profiles in sputum from TB patients and control subjects were a reflection of its cellular composition, cytospin slides were prepared in parallel and assessed for the presence of relative proportions of epithelial cells, neutrophils, macrophages, and T cells. Gamma interferon (IFN-γ) in sputum from TB patients was markedly elevated over levels for both control groups. With anti-TB therapy, IFN-γ levels in sputum from TB patients decreased rapidly and by week 4 of treatment were comparable to those in sputum from controls. Further, IFN-γ levels in sputum closely followed mycobacterial clearance. Although detected at fourfold-lower levels, IFN-γ immunoreactivities in serum followed kinetics in sputum. TNF-α, interleukin 8 (IL-8) and IL-6 also were readily detected in sputum from TB patients at baseline and responded to anti-TB therapy. In contrast to IFN-γ, however, TNF-α and IL-8 levels also were elevated in sputum from pneumonia controls. These data indicate that sputum cytokines correlate with disease activity during active TB of the lung and may serve as potential early markers for sputum conversion and response to anti-TB therapy.
Validation of an In-House Assay for Cytomegalovirus Immunoglobulin G (CMV IgG) Avidity and Relationship of Avidity to CMV IgM Levels
Authors: Prince Harry E. , Leber Amy L.
Published: July 2002

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of >60%. Thus, low avidity in the in-house ELISA was defined as an AI of ⩽50%, whereas high avidity was defined as an AI of ⩾60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of ⩾3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (
n
= 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of ⩾3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.
Evaluation of Urine as a Clinical Specimen for Diagnosis of Hepatitis A
Authors: Joshi Madhuri S. , Chitambar Shobha D. , Arankalle Vidya A. , Chadha Mandeep S.
Published: July 2002

The present study pertains to the evaluation of urine as a specimen for detection of anti-hepatitis A virus (anti-HAV) antibodies. Immunoglobulin M (IgM), IgG, and IgA capture enzyme-linked immunosorbent assays for hepatitis A were performed on paired serum and urine specimens collected from hepatitis A patients (
n
= 92), healthy individuals (
n
= 100), non-A hepatitis patients (
n
= 70), and patients with nonhepatic diseases (
n
= 64, including 37 renal disease patients). Hepatitis A patients seropositive for anti-HAV IgM showed 95.65% uropositivity. No false-positive reactions were observed in control groups. The uropositivity of anti-HAV IgM persisted during the convalescent phase of the disease. Anti-HAV IgG uropositivity correlated well with corresponding seropositivity in all groups (
P
> 0.05 for each). No significant difference between the proportions of serum and urine positivity for anti-HAV IgA was noted (
P
> 0.05 for each). Using seroreactivity as a “gold standard,” the sensitivity and specificity for anti-HAV IgM, anti-HAV IgG, and anti-HAV IgA tests with urine as a specimen were found to be 95.65 and 100%, 97.76 and 76.47%, and 92.23 and 88.18%, respectively. Urine appears to be comparable to serum for diagnosis of recent and past infection with hepatitis A.
Preadult Stage Parasites and Multiple Timed Exposure to Infective Larvae Are Involved in Development of Limb Edema in
Brugia malayi
-Infected Indian Leaf Monkeys (
Presbytis entellus
)
Authors: Murthy P. K. , Khan M. A. , Rajani H. B. , Srivastava V. M. L.
Published: July 2002

The pathogenesis of filarial limb edema is not known. The role of parasitological variables and parasite-mediated phenomena in the development of limb edema was investigated in the
Presbytis entellus
-
Brugia malayi
model. Infection was initiated with subcutaneous inoculation of infective third-stage larvae (L
3
), and the animals were reexposed to different doses of L
3
at the prepatent, patent, and diminishing microfilaremia (0 to 5% of peak microfilaremia count) stages of infection. A large L
3
inoculum size and repeated inoculation in the ankle region during the prepatent, patent, and diminishing microfilaremia stages of infection were found to be necessary for reproducible induction of limb edema. The preadult stage of the parasite was found to be the most potent inducer of limb edema, followed by L
5
and L
4
. The presence of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β, and interleukin-6 in edema fluid in the leg receiving the parasite challenge indicated that the limb edema development was due to parasite-mediated cytokine responses. The absence of bacterial infection or anti-streptolysin O titer in the edema fluid and blood indicated that bacterial infection is not necessary for the development of limb edema.
Cultivation ofBabesiaandBabesia-Like Blood Parasites: Agents of an Emerging Zoonotic Disease
Authors: Schuster Frederick L.
Published: July 2002

Babesiaand its close relatives are members of a group of organisms called piroplasms, a name which comes from their pear-shaped outlines. Long associated with blood diseases of cattle and other mammals, members of the genusBabesiahave been recognized since the 1950s as infectious agents in humans. Species of this protozoan blood parasite that have routinely been isolated from mice (B. microti) or cattle (B. divergens) have also been isolated from humans. In addition to these familiar species, new isolates that resist being placed in existing taxonomic categories are the basis for rethinking their phylogenetic relationships based on sequencing data. The parasite represents a threat to the safety of the blood supply in that blood from asymptomatic humans can transmitBabesiato blood recipients. Such transmissions have occurred. The development of methods for cultivation of these organisms represents a significant opportunity to study their biology and disease potential. In addition, in vitro cultivation has provided a basis for studying immune responses of mammals to these infectious agents, with the hope of ultimately producing attenuated strains that could be used for immunizing of cattle and, perhaps, humans who live in areas of endemicity. The microaerophilous stationary phase culture technique, which uses a tissue culture medium base supplemented with appropriate serum and erythrocytes, has made it possible to obtain large numbers of parasitized erythrocytes for studying the biology of this parasite.
Microarray-Based Identification of
htrA
, a
Streptococcus pneumoniae
Gene That Is Regulated by the CiaRH Two-Component System and Contributes to Nasopharyngeal Colonization
Authors: Sebert M. E. , Palmer L. M. , Rosenberg M. , Weiser J. N.
Published: July 2002

Nasopharyngeal carriage is the reservoir from which most disease with
Streptococcus pneumoniae
arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes,
htrA
and
spoJ
, that was specifically and strongly downregulated in a Δ
ciaRH
-null mutant. A
S. pneumoniae
strain lacking the
htrA
gene encoding a putative serine protease, but not one lacking
spoJ
, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the Δ
ciaRH
strain.
Phenotypes of a Naturally Defective
recB
Allele in
Neisseria meningitidis
Clinical Isolates
Authors: Salvatore Paola , Bucci Cecilia , Pagliarulo Caterina , Tredici Maurizio , Colicchio Roberta , Cantalupo Giuseppina , Bardaro Marcellino , Del Giudice Luigi , Massardo Domenica R. , Lavitola Alfredo , Bruni Carmelo B. , Alifano Pietro
Published: July 2002

Neisseria meningitidis
strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. The phenotype is consequent to the presence of a nonfunctional
recB
ET-37
allele carrying multiple missense mutations. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type
recB
allele or the
recB
ET-37
allele. Congenic
recB
ET-37
meningococci, in addition to being sensitive to UV, were defective both in repair of DNA lesions induced by UV treatment and, partially, in recombination-mediated transformation. Consistently, the wild-type, but not the
recB
ET-37
, allele was able to complement the
Escherichia coli recB21
mutation to UV resistance and proficiency in recombination.
recB
ET-37
meningococci did not exhibit higher frequencies of spontaneous mutation to rifampin resistance than
recB
-proficient strains. However, mutation rates were enhanced following UV treatment, a phenomenon not observed in the
recB
-proficient counterpart. Interestingly, the results of PCR-based assays demonstrated that the presence of the
recB
ET-37
allele considerably increased the frequency of recombination at the pilin loci. The main conclusion that can be drawn is that the presence of the defective
recB
ET-37
allele in
N. meningitidis
isolates causes an increase in genetic diversity, due to an ineffective RecBCD-dependent DNA repair and recombination pathway, and an increase in pilin antigenic variation.
Induction of a Potential Paracrine Angiogenic Loop between Human THP-1 Macrophages and Human Microvascular Endothelial Cells duringBartonella henselaeInfection
Authors: Resto-Ruiz Sandra I. , Schmiederer Michael , Sweger Debra , Newton Catherine , Klein Thomas W. , Friedman Herman , Anderson Burt E.
Published: July 2002

Bartonella henselaeis responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual,B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma. We hypothesize thatB. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1β (IL-1β), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to liveB. henselae.Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1β induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest thatBartonellamodulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis.B. henselaeinduction of VEGF, IL-1β, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.
High Expression of a C Protein β Antigen Gene among Invasive Strains from Certain Clonally Related Groups of Type Ia and Ib Group B Streptococci
Authors: Nagano Noriyuki , Nagano Yukiko , Taguchi Fumiaki
Published: July 2002

Serotyped strains of group B streptococci can be divided into subtypes based on restriction endonuclease digestion patterns (RDP). Profiles of cell-bound proteins were compared among RDP types. Proteins that showed a remarkable difference in the degree of expression were found among strains of RDP Ia-3, which has been considered potentially virulent, as well as of RDP Ib-1. For RDP Ia-3 strains, the protein was predominant in strains from cerebrospinal fluid (CSF) but was mostly a minor component in other strains. For RDP Ib-1 strains, the protein was predominant in strains from CSF, showed diversity in strains from blood, and was mostly a minor component in other strains. By N-terminal sequencing analysis, the protein was identified as a C protein β antigen. The level of bound immunoglobulin A (IgA) or anti-β antigen monoclonal antibody correlated with the level of expressed β antigen, and invasive strains showed remarkably high levels of binding; the exception was a CSF-derived strain of RDP Ib-1 which produced a large amount of β antigen and showed a high level of binding of anti-β antigen monoclonal antibody but no IgA binding. PCR-based amplification revealed that the β antigen gene was detected in all RDP Ia-3 and Ib-1 strains but was not found in any strains of other RDP types. Competitive reverse transcriptase PCR demonstrated that the difference in the amount of protein produced was due to the difference in the level of expression of the β antigen mRNA. Our findings imply that differences in gene expression for a protein may contribute to the invasiveness of RDP Ia-3 and Ib-1 strains for the host.
DNA Polymerases of Low-GC Gram-Positive Eubacteria: Identification of the Replication-Specific Enzyme Encoded by
dnaE
Authors: Barnes Marjorie H. , Miller Shelley D. , Brown Neal C.
Published: July 2002

dna
E, the gene encoding one of the two replication-specific DNA polymerases (Pols) of low-GC-content gram-positive bacteria (E. Dervyn et al., Science 294:1716-1719, 2001; R. Inoue et al., Mol. Genet. Genomics 266:564-571, 2001), was cloned from
Bacillus subtilis
, a model low-GC gram-positive organism. The gene was overexpressed in
Escherichia coli
. The purified recombinant product displayed inhibitor responses and physical, catalytic, and antigenic properties indistinguishable from those of the low-GC gram-positive-organism-specific enzyme previously named DNA Pol II after the
polB
-encoded DNA Pol II of
E. coli
. Whereas a
polB
-like gene is absent from low-GC gram-positive genomes and whereas the low-GC gram-positive DNA Pol II strongly conserves a
dnaE
-like, Pol III primary structure, it is proposed that it be renamed DNA polymerase III E (Pol III E) to accurately reflect its replicative function and its origin from
dnaE
. It is also proposed that DNA Pol III, the other replication-specific Pol of low-GC gram-positive organisms, be renamed DNA polymerase III C (Pol III C) to denote its origin from
polC
. By this revised nomenclature, the DNA Pols that are expressed constitutively in low-GC gram-positive bacteria would include DNA Pol I, the dispensable repair enzyme encoded by
polA
, and the two essential, replication-specific enzymes Pol III C and Pol III E, encoded, respectively, by
polC
and
dnaE
.
Cleavage of
Treponema denticola
PrcA Polypeptide To Yield Protease Complex-Associated Proteins Prca1 and Prca2 Is Dependent on PrtP
Authors: Lee Si Young , Bian Xue-Lin , Wong Grace W. K. , Hannam Pauline M. , McBride Barry C. , Fenno J. Christopher
Published: July 2002

Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins. The
Treponema denticola
chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion. The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the
T. denticola
major surface protein (Msp). The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of
prtP
. The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins. No proteins with significant homology are known, nor was information available on the third protein of the complex. DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence. The complete gene, designated
prcA
, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa. Isogenic mutants with mutations in
prtP
,
prcA
, and
prcA-prtP
all lacked CTLP protease activity. The
prcA
mutant lacked all three CTLP proteins. The
prcA
-
prtP
mutant produced only a C-terminally-truncated 62-kDa PrcA protein. The
prtP
mutant produced a full-length 70-kDa PrcA. Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2. These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes.
Aquaporin Z of
Escherichia coli
: Reassessment of Its Regulation and Physiological Role
Authors: Soupene Eric , King Natalie , Lee Haidy , Kustu Sydney
Published: July 2002

Transcription of an
aqpZ-lac
fusion in a single copy on the
Escherichia coli
chromosome increased as cells entered the stationary growth phase. This was true in a variety of media, and increased transcription in enriched medium required the RpoS sigma factor. Expression of the
aqpZ
-
lac
fusion was not affected by up- or downshifts in osmolality. Disruption of
aqpZ
had no detectable adverse effects.
Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA
Authors: Giachetti C. , Linnen J. M. , Kolk D. P. , Dockter J. , Gillotte-Taylor K. , Park M. , Ho-Sing-Loy M. , McCormick M. K. , Mimms L. T. , McDonough S. H.
Published: July 2002

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.
Evaluation of 15 Motility Media and a Direct Microscopic Method for Detection of Motility in Enterococci
Authors: Van Horn Kenneth , Tóth Clara , Kariyama Reiko , Mitsuhata Ritsuko , Kumon Hiromi
Published: July 2002

Isolation of motile,
vanC
enterococci has yet to be a major infection control concern; however, rapid detection still is important. We evaluated 15 motility media from three manufacturers and a 2-h direct microscopic method for accurate detection of 89 enterococcal strains, including 72
vanC
enterococcal strains. Resistance genes were confirmed by a multiplex PCR method with the
vanC
gene detected in all motile enterococci. Motility in the 72
vanC
enterococci was detected at 30°C within 72 h in BD Biosciences motility nitrate medium and in Remel motility B medium, motility B medium supplemented with methyl-α-
d
-glucopyranoside (investigational), motility S medium, motility test medium, and motility test medium with tetrazolium indicator. Motility was also observed for all
vanC
enterococci with the 2-h direct (30°C incubation) microscopic detection method. All
Enterococcus faecalis
and
Enterococcus faecium
isolates were observed to be nonmotile in all media and by the direct microscopic method. Since differences between the various motility media tested were observed, the medium used for detection of enterococcal motility must be selected carefully.