bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of air exposures and thermal treatments on the secondary electron yield of copper
Authors: Bojko Iouri , Hilleret Noël , Scheuerlein Christian
Published: July 2002

The variation of the secondary electron yield (SEY) of sputter-cleaned oxygen-free high-conductivity copper has been studied as a function of air exposure duration at room temperature. After short air exposures of some seconds the maximum SEY (δmax) of clean copper is reduced from 1.3 to less than 1.2, due to the oxidation of the copper surface. Prolonged air exposure increases the SEY steadily until, after about 8 days of atmospheric exposure, δmax is higher than 2. Air exposures at higher temperatures have been found to be effective in reducing the SEY of technical copper surfaces. A 5 min air exposure of copper at 350 °C followed by a 350 °C bakeout under vacuum reduces δmax to about 1.05, which is lower than the value of pure copper and that of Cu2O.
Oxidation of sub-50 nm Si columns for light emission study
Authors: Liu Harvey I. , Maluf Nadim I. , Pease R. F. W. , Biegelsen David K. , Johnson Noble M. , Ponce Fernando A.
Published: July 2002

Understanding the optical and electrical properties of Si nanostructures is essential for exploring the potential of using structural quantum confinement to induce light emission from crystalline Si. To this end, sub-50 nm Si columns were fabricated with high resolution electron beam lithography and anisotropic reactive ion etching. The dimensions of the Si nanostructures were further reduced by thermal oxidation. A novel transmission electron microscopy technique was developed to monitor the oxidation progress without removing the oxide structural support. Images of sub-5 nm crystalline Si cores were obtained. The oxidation rates of the Si nanostructures were characterized. Among the various interesting oxidation phenomena are the nonmonotonic oxidation rate with respect to the column size and an unexpectedly slow change of the outer diameters of the oxidized columns. Several likely mechanisms, including the stress retardation of oxidation for a small radius of curvature, the stress induced generation and diffusion of Si interstitials, the stress induced radial strain in the Si core, and SiO sublimation during oxidation, were proposed to explain the unusual oxidation phenomena. Although not thoroughly understood, a photoluminescence spectrum was also obtained for a sample with a large patterned area.
Vapor-deposited gold film formation on highly oriented pyrolitic graphite. A transition from pseudo-two-dimensional branched island growth to continuous film formation
Authors: Blum B. , Salvarezza R. C. , Arvia A. J.
Published: July 2002

Growth of vapor-deposited gold islands on highly oriented pyrolitic graphite (HOPG) using evaporation rates in the range 0.01–0.1 nm/s has been investigated with ex situ scanning tunneling microscopy. Equivalent mean gold thickness and substrate temperature were varied from 1 to 20 ML and 333 to 358 K, respectively. Gold ad-islands several layers high grow both atop large HOPG terraces and along HOPG steps. Images show split and stable tip branched islands, depending on individual ad-island height and equivalent mean gold thickness. Initially, the diffusion of gold atoms towards island edges occurs via the HOPG surface, whereas soon thereafter it includes diffusion via gold terraces and steps. These two very dissimilar possibilities are due to both misfit strain variation with local gold-deposit thickness, and large differences in sticking probabilities of gold on HOPG and gold. Addition of gold to islands more than 20 layers high produces island-top flattening, branch coarsening with pronounced facetting, and tip stabilization, as a result of diffusion of gold from island tops to valleys and interbranch spaces. Extended flat areas present monoatomic-high triangular gold terraces. The transition from split to stable-tip branched islands is associated with a surface diffusion barrier which decreases with island height as a result of reduced lattice mismatch. Seemingly, early stages of branched gold island formation can be assimilated to a deposition, diffusion, and aggregation-type model that nucleates surface atom at island or terrace edges. Once flat gold terraces set in, the growth process can be described by an Edwards–Wilkinson-type model. Coalescence of gold ad-islands more than 20 layers high explains the well-known continuous films obtained in metal coaters under comparable growth conditions.
Steric Effects and Solvent Effects in Ionic Reactions
Authors: Regan Colleen K. , Craig Stephen L. , Brauman John I.
Published: July 2002

Rates of S
N
2 reactions of chloride ion with methyl- and
tert
-butyl–substituted chloroacetonitrile were measured by using Fourier transform–ion cyclotron resonance spectrometry to follow the isotopic exchange reaction. Barrier heights for these reactions indicate that steric effects in the gas phase are diminished relative to apparent steric effects in solution. We attribute the increased barrier in solution to a solvation effect. Monte Carlo simulations done using statistical perturbation theory confirm that steric hindrance to solvation contributes to S
N
2 barriers in solution.
DOAS Measurements of Tropospheric Bromine Oxide in Mid-Latitudes
Authors: Hebestreit Kai , Stutz Jochen , Rosen David , Matveiv Valery , Peleg Mordechai , Luria Menachem , Platt Ulrich
Published: July 2002

Episodes of elevated bromine oxide (BrO) concentration are known to occur at high latitudes in the Arctic boundary layer and to lead to catalytic destruction of ozone at those latitudes; these events have not been observed at lower latitudes. With the use of differential optical absorption spectroscopy (DOAS), locally high BrO concentrations were observed at mid-latitudes at the Dead Sea, Israel, during spring 1997. Mixing ratios peaked daily at around 80 parts per trillion around noon and were correlated with low boundary-layer ozone mixing ratios.
Hanbury Brown and Twiss-Type Experiment with Electrons
Authors: Oliver William D. , Kim Jungsang , Liu Robert C. , Yamamoto Yoshihisa
Published: July 2002

Fermion anti-bunching was directly observed by measuring the cross-covariance of the current fluctuations of partitioned electrons. A quantum point contact was used to inject single-mode electrons into a mesoscopic electron beam splitter device. The beam splitter output currents showed negative cross-covariance, indicating that the electrons arrived individually at the beam splitter and were randomly partitioned into two output channels. As the relative time delay between the outputs was changed, the observed ringing in the cross-covariance was consistent with the bandwidths used to monitor the fluctuations. The result demonstrates a fermion complement to the Hanbury Brown and Twiss experiment for photons.
Porphyrin and Phthalocyanine Antiscrapie Compounds
Authors: Priola Suzette A. , Raines Anne , Caughey Winslow S.
Published: July 2002

The transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases for which no effective treatments are available. The likelihood that a bovine form of TSE has crossed species barriers and infected humans underscores the urgent need to identify anti-TSE drugs. Certain cyclic tetrapyrroles (porphyrins and phthalocyanines) have recently been shown to inhibit the in vitro formation of PrP-res, a protease-resistant protein critical for TSE pathogenesis. We now report that treatment of TSE-infected animals with three such compounds increased survival time from 50 to 300%. The significant inhibition of TSE disease by structurally dissimilar tetrapyrroles identifies these compounds as anti-TSE drugs.
A Dose Ranging Study of the Pharmacokinetics, Safety, and Preliminary Efficacy of Lamivudine in Children and Adolescents with Chronic Hepatitis B
Authors: Sokal Etienne M. , Roberts Eve A. , Mieli-Vergani Giorgina , McPhillips Penny , Johnson Mark , Barber Judy , Dallow Nigel , Boxall Elizabeth , Kelly Deirdre
Published: July 2002

Fifty-three patients with chronic hepatitis B and active viral replication were studied for 4 weeks while on treatment and for 12 weeks after treatment with the oral nucleoside analogue lamivudine. Children aged 2 to 12 years were randomized to receive twice-daily doses of 0.35, 1.5, or 4 mg of lamivudine solution per kg of body weight or once-daily doses of 3 mg of lamivudine solution per kg. Adolescents aged 13 to 17 years received lamivudine at 100 mg (as tablets). Blood samples for pharmacokinetic assay were taken on days 1 and 28. Lamivudine was rapidly absorbed following oral administration, with the maximum concentration in serum being reached 0.5 to 1 h postdosing. Apparent oral clearance (CL/F) was higher in younger children and decreased with age, with CL/F values for adolescents reaching those seen for adults by the age of 12. All doses produced a dramatic fall in serum hepatitis B virus (HBV) DNA levels, with a median reduction of ≥99.5% after 4 weeks of treatment and with the levels returning to the baseline levels posttreatment. The correlation of dose, area under the concentration-time curve (AUC), and changes in HBV DNA levels, as measured by the Chiron Quantiplex assay, showed maximal antiviral effects (99.9% inhibition and a reduction of the amount of HBV DNA of approximately 3 log
10
) at 3 mg/kg/day, with no discernible increase in effect seen whether the drug was given at 4 mg/kg twice daily or whether it was given once daily or twice daily. The limit of detection of the assay (2.5 pg/ml) was reached for some but not all patients across the dose ranges, with the smallest number (
n
= 2) of those having values negative by the Chiron Quantiplex assay being in the lowest-dose group. The 13- to 17-year-olds showed a similar overall response in terms of the HBV DNA level reduction compared to that for patients younger than age 13. Analysis of the same samples by PCR, which has a lower limit of sensitivity than the Chiron Quantiplex assay, also showed average drops in HBV DNA levels of about 3 log
10
at 4 weeks for patients for which the AUC was ≥4,000 ng · h/ml, confirming the conclusions given above. Lamivudine treatment was well tolerated at all doses, with no significant adverse events or laboratory data changes. On the basis of pharmacokinetic and pharmacodynamic data, a 3-mg/kg/day dose in children (ages 2 to 12 years) with chronic hepatitis B provides levels of exposure and trough concentrations similar to those seen in adults following the receipt of doses of 100 mg. The 100-mg dose is being evaluated in a large phase III study with HBV-infected pediatric patients.
Concentrations of Gatifloxacin in Plasma and Urine and Penetration into Prostatic and Seminal Fluid, Ejaculate, and Sperm Cells after Single Oral Administrations of 400 Milligrams to Volunteers
Authors: Naber Christoph K. , Steghafner Michaela , Kinzig-Schippers Martina , Sauber Christian , Sörgel Fritz , Stahlberg Hans-Jürgen , Naber Kurt G.
Published: July 2002

Gatifloxacin (GTX), a new fluoroquinolone with extended antibacterial activity, is an interesting candidate for the treatment of chronic bacterial prostatitis (CBP). Besides the antibacterial spectrum, the concentrations in the target tissues and fluids are crucial for the treatment of CBP. Thus, it was of interest to investigate its penetration into prostatic and seminal fluid. GTX concentrations in plasma, urine, ejaculate, prostatic and seminal fluid, and sperm cells were determined by a high-performance liquid chromatography method after oral intake of a single 400-mg dose in 10 male Caucasian volunteers in the fasting state. Simultaneous application of the renal contrast agent iohexol was used to estimate the maximal possible contamination of ejaculate and prostatic and seminal fluid by urine. GTX was well tolerated. The means (standard deviations) for the following parameters were as indicated: time to maximum concentration of drug in serum, 1.66 (0.91) h; maximum concentration of drug in serum, 2.90 (0.39) μg/ml; area under the concentration-time curve from 0 to 24 h, 25.65 μg · h/ml; and half life, 7.2 (0.90) h. Within 12 h about 50% of the drug was excreted unchanged into the urine. The mean renal clearance was 169 ml/min. The gatifloxacin concentrations in ejaculate, seminal fluid, and prostatic fluid were in the range of the corresponding plasma concentrations which were 1.92 (0.27) μg/ml at approximately the same time point (4 h after drug intake). The concentrations in sperm cells (0.195, 0.076, and 0.011 μg/ml) could be determined in three subjects. The good penetration into prostatic and seminal fluid, the good tolerance, and the previously reported broad antibacterial spectrum suggest that GTX may be a good alternative for the treatment of chronic bacterial prostatitis. Clinical studies should be performed to confirm this assumption.
In Vitro Activity of ABT-773 against
Legionella pneumophila
, Its Pharmacokinetics in Guinea Pigs, and Its Use to Treat Guinea Pigs with
L. pneumophila
Pneumonia
Authors: Edelstein Paul H. , Higa F. , Edelstein Martha A. C.
Published: July 2002

The activity of ABT-773 was studied against extracellular and intracellular
Legionella pneumophila
and for the treatment of guinea pigs with
L. pneumophila
pneumonia. The ABT-773 MIC at which 50% of isolates are inhibited (MIC
50
) for 20 different
Legionella
sp. strains was 0.016 μg/ml, whereas the MIC
50
s of clarithromycin and erythromycin were 0.032 and 0.125 μg/ml, respectively. ABT-773 (1 μg/ml) was bactericidal for two
L. pneumophila
strains grown in guinea pig alveolar macrophages. In contrast, erythromycin and clarithromycin had easily reversible static activity only. Therapy studies of ABT-773 and erythromycin were performed with guinea pigs with
L. pneumophila
pneumonia. When ABT-773 was given to infected guinea pigs by the intraperitoneal route (10 mg/kg of body weight), mean peak levels in plasma were 0.49 μg/ml at 0.5 h and 0.30 μg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 0.55 h, and the area under the concentration-time curve from 0 to 24 h (AUC
0–24
) was 0.65 μg · h/ml. For the same drug dose, mean levels in the lung were 15.9 and 13.2 μg/g at 0.5 and 1 h, respectively, with a half-life of 0.68 h and an AUC
0–24
of 37.0 μg · h/ml. Ten of 15
L. pneumophila
-infected guinea pigs treated with ABT-773 (15 mg/kg/dose given intraperitoneally once daily) for 5 days survived for 9 days post-antimicrobial therapy, as did 14 of 15 guinea pigs treated with erythromycin (30 mg/kg given intraperitoneally twice daily) for 5 days. All of the ABT-773-treated animals that died appeared to do so because of drug-induced peritonitis rather than overwhelming pneumonia. None of 12 animals treated with saline survived. ABT-773 is as effective as erythromycin against
L. pneumophila
in infected macrophages and in a guinea pig model of Legionnaires' disease. These data support studies of the clinical effectiveness of ABT-773 for the treatment of Legionnaires' disease.
Activation by 9-(R)-[2-(Phosphonomethoxy)Propyl]Adenine of Chemokine (RANTES, Macrophage Inflammatory Protein 1α) and Cytokine (Tumor Necrosis Factor Alpha, Interleukin-10 [IL-10], IL-1β) Production
Authors: Zı́dek Zdenĕk , Franková Daniela , Holý Antonı́n
Published: July 2002

Development of a novel group of antiviral agents, acyclic nucleoside phosphonates, has provided a new perspective for treating human immunodeficiency virus (HIV) infection. One of the compounds, 9-(R)-[2-(phosphonomethoxy)propyl]adenine (PMPA) (tenofovir), has been shown to confer complete protection against AIDS in a simian model of the infection. The aim of our study was to investigate whether the antiviral efficacy of PMPA, which depends mainly on inhibition of virus-induced DNA polymerase or of reverse transcriptase, could be contributed by immunomodulatory potential of this drug. We screened for its ability to activate production of cytokines and chemokines that are known to interfere with the replication and/or the entry of HIV in cells. Using the in vitro test system of mouse macrophages and lymphocytes, it has been found that PMPA stimulates macrophage secretion of interleukin-1β (IL-1β), IL-10, and tumor necrosis factor alpha. Production of the chemokines RANTES and macrophage inflammatory protein 1α was activated in both macrophages and lymphocytes, and also in human cell line U937. Other cytokines—i.e., IL-2, IL-12, IL-13, and gamma interferon—remained uninfluenced by PMPA. The cytokines were stimulated in a dose-dependent fashion, with rapid onset, and peak concentrations were achieved within 5 to 24 h. The findings contribute to a more complex understanding of mechanisms of antiviral effectiveness of PMPA and support the view that this drug could become a promising candidate for therapeutic exploitation in anti-HIV preventive medicine.
Heat Treatment of Amphotericin B Modifies Its Serum Pharmacokinetics, Tissue Distribution, and Renal Toxicity following Administration of a Single Intravenous Dose to Rabbits
Authors: Kwong Evan H. , Ramaswamy Manisha , Bauer Emily A. , Hartsel Scott C. , Wasan Kishor M.
Published: July 2002

The purpose of this investigation was to determine the serum pharmacokinetics, tissue distribution, and renal toxicity of amphotericin B (AmpB) following administration of a single intravenous dose (1 mg/kg of body weight) of Fungizone (FZ) and a heat-treated form of FZ (HFZ) to New Zealand White female rabbits. FZ solutions were heated at 70°C for 20 min to produce HFZ. Blood samples were obtained before drug administration and serially thereafter. After collection of the 48-h blood sample, each rabbit was humanely sacrificed and the right kidney, spleen, lungs, liver, and heart were harvested for AmpB analysis. Serum creatinine levels were measured before and 10 h after drug administration. AmpB concentrations in the serum and tissues were analyzed using high-performance liquid chromatography. FZ administration to rabbits resulted in a greater-than-50% increase in serum creatinine concentrations compared to baseline. However, HFZ administration resulted in no difference in serum creatinine concentrations compared to baseline. The AmpB area under the concentration-time curve (AUC) after HFZ administration was significantly lower than the AmpB AUC in rabbits administered FZ. However, AmpB systemic total body clearance was significantly greater in rabbits administered HFZ than in rabbits administered FZ without any differences in volume of distribution at steady state. Kidney tissue AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. Likewise, lung and spleen AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. However, liver AmpB concentrations were significantly lower in rabbits administered FZ than in rabbits administered HFZ. No significant differences in heart AmpB concentration between rabbits administered FZ and those given HFZ were found. These findings suggest that the pharmacokinetics, tissue distribution, and renal toxicity of AmpB are modified following administration of HFZ. HFZ could be an improved low-cost AmpB drug delivery system that has a potentially higher therapeutic index than FZ.
Sequence Differences in the Diagnostic Target Region of the Oocyst Wall Protein Gene of
Cryptosporidium
Parasites
Authors: Xiao Lihua , Limor Josef , Morgan Una M. , Sulaiman Irshad M. , Thompson R. C. Andrew , Lal Altaf A.
Published: July 2002

Nucleotide sequences of the
Cryptosporidium
oocyst wall protein (COWP) gene were obtained from various
Cryptosporidium
spp. (
C. wrairi
,
C. felis
,
C. meleagridis
,
C. baileyi
,
C. andersoni
,
C. muris
, and
C. serpentis
) and
C. parvum
genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of
Cryptosporidium
spp. and genotypes.
Rapid Screening Method for Mycobactericidal Activity of Chemical Germicides That Uses
Mycobacterium terrae
Expressing a Green Fluorescent Protein Gene
Authors: Zafer Ahmed A. , Taylor Yvonne E. , Sattar Syed A.
Published: July 2002

The slow growth of mycobacteria in conventional culture methods impedes the testing of chemicals for mycobactericidal activity. An assay based on expression of the green fluorescent protein (GFP) by mycobacteria was developed as a rapid alternative. Plasmid pBEN, containing the gene encoding a red-shifted, high-intensity GFP mutant, was incorporated into
Mycobacterium terrae
(ATCC 15755), and GFP expression was observed by epifluorescence microscopy. Mycobactericidal activity was assessed by separately exposing a suspension of
M. terrae
(pBEN) to several dilutions of test germicides based on 7.5% hydrogen peroxide, 2.4% alkaline glutaraldehyde, 10% acid glutaraldehyde, and 15.5% of a phenolic agent for contact times ranging from 10 to 20 min (22°C), followed by culture of the exposed cells in broth (Middlebrook 7H9) and measurement of fluorescence every 24 h. When the fluorescence was to be compared with CFU, the samples were plated on Middlebrook 7H11 agar and incubated for 4 weeks. No increase in fluorescence or CFU occurred in cultures in which the cells had been inactivated by the germicide concentrations tested. Where the test bacterium was exposed to ineffective levels of the germicides, fluorescence increased after a lag period of 1 to 7 days, corresponding to the level of bacterial inactivation. In untreated controls, fluorescence increased rapidly to reach a peak in 2 to 4 days. A good Pearson correlation coefficient (
r
≥0.85) was observed between the intensity of fluorescence and the number of CFU. The GFP-based fluorescence assay reduced the turnaround time in the screening of chemical germicides for mycobactericidal activity to ≤7 days.
Use of a Dry-Plasma Collection Device to Overcome Problems with Storage and Transportation of Blood Samples for Epidemiology Studies in Developing Countries
Authors: Nurgalieva Zhannat Z. , Almuchambetova R. , Machmudova A. , Kapsultanova D. , Osato Michael S. , Peacock Jeffrey , Zoltek Richard P. , Marchildon Patrice A. , Graham David Y. , Zhangabylov Abai
Published: July 2002

Studies are difficult in areas lacking modern facilities due to the inability to reliably collect, store, and ship samples. Thus, we sought to evaluate the use of a dry plasma collection device for seroepidemiology studies. Plasma was obtained by fingerstick using a commercial dry plasma collection device (Chemcard Plasma Collection Device) and serum (venipuncture) from individuals in Kazakhstan. Plasma samples were air dried for 15 min and then stored desiccated in foil zip-lock pouches at 4 to 6°C and subsequently shipped to the United States by air at ambient temperature. Serum samples remained frozen at −20°C until assayed.
Helicobacter pylori
status was determined by enzyme-linked immunosorbent assay (HM-CAP EIA) for the dry plasma and the serum samples. The results were concordant in 250 of the 289 cases (86.5%). In 25 cases (8.6%), the dry plasma samples gave indeterminate results and could not be retested because only one sample was collected. Five serum samples were positive, and the corresponding dry plasma samples were negative; one serum sample was negative, and the corresponding plasma sample was positive. The relative sensitivity and specificity of the Chemcard samples to serum were 97.6 and 97.9%, respectively, excluding those with indeterminate results. Repeated freeze-thawing had no adverse effect on the accuracy of the test. We found the dry plasma collection device to provide an accurate and practical alternative to serum when venipuncture may be difficult or inconvenient and sample storage and handling present difficulties, especially for seroepidemiologic studies in rural areas or developing countries and where freeze-thawing may be unavoidable.
Serodiagnosis of Human Cysticercosis by Using Antigens from Vesicular Fluid of
Taenia crassiceps
Cysticerci
Authors: Bueno Ednéia C. , Snege Miriam , Vaz Adelaide J. , Leser Paulo G.
Published: July 2002

Neurocysticercosis (NC), caused by the presence of
Taenia solium
metacestodes in tissues, is a severe parasitic infection of the central nervous system with universal distribution. To determine the efficiency of enzyme-linked immunosorbent assay (ELISA) and immunoblot with antigens of
T. crassiceps
vesicular fluid (Tcra) compared to standard techniques (indirect immunofluorescence test [IFT] and complement fixation test [CFT]) using
T. solium
cysticerci (Tso) for the serodiagnosis of NC, we studied serum samples from 24 patients with NC, 30 supposedly healthy individuals, 76 blood bank donors, 45 individuals with other non-NC parasitoses, and 97 samples from individuals screened for cysticercosis serology (SC). The sensitivity observed was 100% for ELISA-Tso and ELISA-Tcra, 91.7% for the IFT, and 87.5% for the CFT. The specificity was 90% for ELISA-Tso, 96.7% for ELISA-Tcra, 50% for IFT, and 63.3% for CFT. The efficiency was highest for ELISA-Tcra, followed by ELISA-Tso, IFT, and CFT. Of the 23 samples from SC group, which were reactive to ELISA-Tso and/or ELISA-Tcra, only 3 were positive to immunblot-Tcra (specific peptides of 14- and 18-kDa) and to glycoprotein peptides purified from Tcra antigen (gp-Tcra), showing the low predictive value of ELISA for screening. None of the samples from the remaining groups showed specific reactivity in immunoblot-Tcra. These results demonstrate that ELISA-Tcra can be used as a screening method for the serodiagnosis of NC and support the need for specific tests for confirmation of the results. The immunoblot can be used as a confirmatory test both with Tcra and gp-Tcra, with the latter having an advantage in terms of visualization of the results.
Viral Replication and Host Gene Expression in Alveolar Macrophages Infected with Ebola Virus (Zaire Strain)
Authors: Gibb Tammy R. , Norwood David A. , Woollen Neal , Henchal Erik A.
Published: July 2002

In order to characterize the cellular response to and identify potential diagnostic markers for the early detection of Ebola virus, an in vitro culture system involving nonhuman primate alveolar macrophages was developed. Ebola virus replication in the alveolar macrophages was characterized by plaque assay, immunohistochemical analysis, and in situ hybridization. Fluorogenic 5′ -nuclease assays specific for nonhuman primate proinflammatory cytokines and chemokines were designed and used to evaluate mRNA transcription in macrophages infected with Ebola virus. Transient increases in cytokine and chemokine mRNA levels were observed immediately following exposure to Ebola virus. At 2 h postexposure, levels of cytokine and chemokine mRNAs were markedly reduced. Although Ebola virus infection of alveolar macrophages failed to induce a sustained increase in proinflammatory cytokine and chemokine mRNA transcription (potentially reducing the use of these markers as diagnostic tools), the fluorogenic 5′-nuclease assays developed may have prognostic value for individuals infected with Ebola virus. Recently published data have indicated that persons who remain asymptomatic after exposure to Ebola virus are capable of mounting an early proinflammatory cytokine response and that those who become clinically ill are not. If implemented immediately after exposure, these assays could be used to predict which individuals will be more likely to remain asymptomatic as opposed to those who will be more likely to develop clinical signs and eventually succumb to the virus.
Production and Application of New Monoclonal Antibodies Specific for a FecalHelicobacter pyloriAntigen
Authors: Suzuki Nobuyuki , Wakasugi Masahiko , Nakaya Seigo , Okada Keiko , Mochida Ritsuko , Sato Masami , Kajiyama Hirofumi , Takahashi Ryoki , Hirata Haruhisa , Ezure Yohji , Koga Yasuhiro , Fukuda Yoshihiro , Shimoyama Takashi
Published: July 2002

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecalHelicobacter pyloriantigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity forH. pyloriantigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form ofH. pylori, and fecal samples fromH. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41H. pyloriclinical isolates and in fecal samples from sevenH. pylori-positive subjects. Several kinds ofHelicobacterspecies (Helicobacter felis,Helicobacter hepaticus,Helicobacter mustelae, andHelicobacter cinaedi) exceptH. pylori, major bacteria in feces (Campylobacter jejuni,Bacteroides vulgatus,Bifidobacterium breve,Bifidobacterium infantis, andEscherichia coli), and fecal samples from sixH. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis ofH. pyloriinfection.
Immunization with Alpha-Toxin Toxoid Protects the Cornea against Tissue Damage during Experimental
Staphylococcus aureus
Keratitis
Authors: Hume Emma B. H. , Dajcs Joseph J. , Moreau Judy M. , O'Callaghan Richard J.
Published: July 2002

Alpha-toxin is a major virulence factor in
Staphylococcus aureus
keratitis. Active or passive immunization with alpha-toxin toxoid could protect against corneal damage. Results show that either form of immunization did not kill bacteria but did significantly protect against corneal pathology, especially epithelial erosion.
Cloning and DNA Sequence Analysis of an Immunogenic Glucose-Galactose MglB Lipoprotein Homologue fromBrachyspira pilosicoli, the Agent of Colonic Spirochetosis
Authors: Zhang P. , Cheng X. , Duhamel G. E.
Published: July 2002

Colonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirocheteBrachyspira(formerlySerpulina)pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of humanB. pilosicolistrain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the clonedB. pilosicoligene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putativemglBgene was a 3′-truncated open reading frame with 73.8% similarity and 66.3% identity tomglAofEscherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present inB. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetesSerpulina intermedia,Brachyspira innocens,Brachyspira murdochii, and the avianBrachyspira alvinipulli, but not in porcineBrachyspira hyodysenteriae, humanBrachyspira aalborgi, and porcineTreponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [3H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis ofB. pilosicoliin vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis.
Attenuated
Shigella flexneri
2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic
Escherichia coli
Authors: Koprowski Hilary , Levine Myron M. , Anderson Richard J. , Losonsky Genevieve , Pizza Mariagrazia , Barry Eileen M.
Published: July 2002

A multivalent live oral vaccine against both
Shigella
spp. and enterotoxigenic
Escherichia coli
(ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into
S. flexneri
2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM
1
binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-
S. flexneri
2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type
S. flexneri
2a. This prototype
Shigella
-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated
Shigella
vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.
Analysis of the Cellular Localization of Bdr Paralogs in
Borrelia burgdorferi
, a Causative Agent of Lyme Disease: Evidence for Functional Diversity
Authors: Roberts David M. , Theisen Michael , Marconi Richard T.
Published: July 2002

The
bdr
(
Borrelia
direct repeat) gene family of the genus
Borrelia
encodes a polymorphic group of proteins that carry a central repeat motif region containing putative phosphorylation sites and a hydrophobic carboxyl-terminal domain. It has been postulated that the Bdr proteins may anchor to the inner membrane via the C-terminal domain. In this study, we used cellular fractionation methodologies, salt and detergent treatments, and immunoblot analyses to assess the association of the Bdr proteins with the cellular infrastructure in both
Borrelia burgdorferi
(a Lyme disease spirochete) and
B. turicatae
(a relapsing fever spirochete). Triton X-114 extraction and partitioning experiments demonstrated that most Bdr paralogs are associated with the inner membrane-peptidoglycan complex. Analyses of cells treated with the highly chaotropic bile salt detergent deoxycholic acid demonstrated that some Bdr paralogs may also interact with the peptidoglycan, as evidenced by their tight association with the insoluble cellular matrix. In addition, immunoprecipitation (IP) experiments revealed an enhanced IP of all Bdr paralogs when the cell lysates were boiled prior to addition of the precipitating antibody. Furthermore, some Bdr paralogs were accessible to antibody in the IP experiments only in the boiled cell lysates. These observations suggest that different Bdr paralogs may carry out different structural-functional roles. Demonstration of the inner membrane localization of the Bdr proteins and of the differences in nature of the interaction of individual Bdr paralogs with the cell infrastructure is an important step toward defining the functional role of this unique protein family in the genus
Borrelia
.
The Molecular Weight Distribution of Succinoglycan Produced by
Sinorhizobium meliloti
Is Influenced by Specific Tyrosine Phosphorylation and ATPase Activity of the Cytoplasmic Domain of the ExoP Protein
Authors: Niemeyer Dagmar , Becker Anke
Published: July 2002

It is thought that in the gram-negative soil bacterium
Sinorhizobium meliloti
the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.
Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein in
Bartonella
Species
Authors: Park Sarah Y. , Kelminson Karen L. , Lee Anthea K. , Zhang Peng , Warner Rachel E. , Rehkopf David H. , Calderwood Stephen B. , Koehler Jane E.
Published: July 2002

Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (
fur
) system. We identified and sequenced the
fur
gene and flanking regions of three
Bartonella
species. The most notable difference between
Bartonella
Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a
gfp
reporter gene fused to the 204 nucleotides immediately upstream of the
Bartonella fur
gene.
Bartonella henselae fur
gene expression complemented a
Vibrio cholerae fur
mutant.
Bacillus subtilisfunctional genomics: global characterization of the stringent response by proteome and transcriptome analysis
Authors: Eymann Christine , Homuth Georg , Scharf Christian , Hecker Michael
Published: July 2002

The stringent response inBacillus subtiliswas characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type andrelAmutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response todl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in therelAmutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknowny-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.
Infection of Central Nervous System by Motile
Enterococcus
: First Case Report
Authors: Kurup Asok , Tee Wen Sim Nancy , Loo Liat Hui , Lin Raymond
Published: July 2002

A 66-year-old man with four indwelling ventriculoperitoneal shunts for multiloculated hydrocephalus from a complicated case of meningitis a year before developed shunt infection based on a syndrome of fever, drowsiness, and cerebrospinal fluid neutrophil pleocytosis in the background of repeated surgical manipulation to relieve successive shunt blockages. The cerebrospinal fluid culture, which yielded a motile
Enterococcus
species, was believed to originate from the gut. This isolate was lost in storage and could not be characterized further. The patient improved with vancomycin and high-dose ampicillin therapy. He relapsed a month later with
Enterococcus gallinarum
shunt infection, which responded to high-dose ampicillin and gentamicin therapy. This is probably the first case report of motile
Enterococcus
infection of the central nervous system.
Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease
Authors: Liu Xin , Yang Jenny , Ghazi A. Mohamad , Frey Teryl K.
Published: July 2002

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463–4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn
65
binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV–CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.
Nitric Oxide Synthesis Enhances Human Immunodeficiency Virus Replication in Primary Human Macrophages
Authors: Blond Donatienne , Raoul Hervé , Le Grand Roger , Dormont Dominique
Published: July 2002

Macrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) andl-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction ofl-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed toNG-monomethyll-arginine andNG-nitro-l-arginine methyl ester (l-NAME), two inhibitors of the iNOS. Moreover, an excess ofl-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.
Low Plasma Human Immunodeficiency Virus Type 2 Viral Load Is Independent of Proviral Load: Low Virus Production In Vivo
Authors: Popper Stephen J. , Sarr Abdoulaye Dieng , Guèye-Ndiaye Aïssatou , Mboup Souleymane , Essex Myron E. , Kanki Phyllis J.
Published: July 2002

Levels of virus in the plasma are closely related to the pathogenicity of human immunodeficiency virus type 1 (HIV-1). HIV-2 is much less pathogenic than HIV-1, and infection with HIV-2 leads to significantly lower plasma viral load. To identify the source of this difference, we measured both viral RNA and proviral DNA in matched samples from 34 HIV-2-infected individuals. Nearly half had undetectable viral RNA loads (<100 copies/ml), but levels of proviral DNA were relatively high and confirmed that quantities of provirus in HIV-1 and HIV-2 infection were similar. Overall, HIV-2 proviral DNA load did not correlate with viral RNA load, and higher viral RNA load was associated with increased production of plasma virus from the proviral template. These results suggest that low viral load in HIV-2 infection is due to decreased rates of viral production, rather than differences in target cell infectivity.