bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-term reliability of Pt and Mo diffusion barriers in Ti–Pt–Au and Ti–Mo–Au metallization systems for GaAs digital integrated circuits
Authors: Kitaura Y. , Hashimoto T. , Inoue T. , Ishida K. , Uchitomi N. , Nii R.
Published: July 2002

Reliability of Ti–Pt–Au and Ti–Mo–Au systems has been investigated for GaAs integrated circuit first-level metallizations on semi-insulating GaAs substrates and second-level metallizations on interlayer SiO2 films using Auger depth profile analysis, residual resistance examination and temperature storage step-stress testing. Auger analysis and residual resistance examination showed significant reaction between first-level Ti–Pt–Au and GaAs substrates during metallization processes, while Ti–Mo–Au system with the electron-beam evaporated Mo film showed higher thermal stability because the Mo film acted as a good diffusion barrier between GaAs and Au. The second-level Ti–Pt–Au on SiO2 was found to be free from the reaction with GaAs substrates, and its degradation was ascribed to interdiffusion of composite metals. The resistance increase in step-stress testing for the second Ti–Pt–Au was analyzed on the basis of a new diffusion-controlled model, and long-term reliability was estimated. A mean time to failure value of 3×105 h at 150 °C was obtained for a failure defined as 10% increase in resistance. Much higher reliability was estimated for Ti–Mo–Au, because the resistance continued to decrease as long as 3000 h at 250 °C. The decrease in resistance clearly indicates defect annealing with reduced defect scattering in Au layers. This also shows that foreign metal diffusion into Au, acting as impurity scattering centers, is perfectly eliminated by Mo diffusion barriers.
Energy broadening by Coulomb interactions of an electron beam emitted from a point source
Authors: Elswijk H. B. , van Rooy T. , Schiller C.
Published: July 2002

Recently, it has been proven possible to fabricate tungsten field emitter tips emitting from areas as small as a few atoms down to a single atom. Emission is then limited to a small solid angle and is achieved at low extraction voltages. These emitters may find practical application in electron microscopy. We investigate the broadening of the energy distribution of the electrons in the beam by statistical Coulomb interactions using Monte Carlo simulations and we present an analytical model. We conclude that this broadening increases linearly with the current density and is inversely proportional to the square root of the electron energy. It exceeds the Fowler–Nordheim width of the energy distribution only at currents higher than approximately 1 μA at energies ranging from 100 to 1000 eV.
Practical feasibility of the multistage deflection theory
Authors: Hosokawa Teruo
Published: July 2002

A magnetic five-stage deflection system has been developed to prove the practicality of a design method based on the multistage deflection theory. High-precision printed coils are used for five-stage deflection. Coil manufacturing accuracy is 30 μm. By using this system, beam resolution of less than 0.25 μm has been obtained at 7×7 mm2 field corners with an 8 mrad beam half-angle. This system satisfies the requirement for deflective telecentricity.
Fabrication and characterization of platinum nanocrystalline material grown by electron-beam induced deposition
Authors: Koops H. W. P. , Kaya A. , Weber M.
Published: July 2002

The technique of electron-beam induced deposition allows three-dimensional structures to be generated on the nanometer scale. This is achieved in a scanning electron microscope equipped with a lithography attachment that enables separate position and time control for every pixel. By decomposing adsorbed molecules with the electron beam, structures are created on arbitrarily chosen substrates with nm precision under computer control. Deposits containing metallic nanocrystallites can be produced using organometallic precursor materials. The decomposition of cyclopentadienylplatinum (IV)-trimethyl (CpPtMe3) results in platinum single crystals with a 2 nm diameter embedded in a carbon-containing amorphous matrix. The metal content of deposits can be adjusted by choosing an appropriate acceleration voltage and beam current for deposition. The growth rate of deposits from CpPtMe3 is superior to that of frequently used organogold compounds. Tips can be deposited with growth rates up to 150 nm/s. This property is in favor of a higher throughput for nanofabrication. The deposition cross section for this precursor molecule is estimated at 1×10−16 cm2. The electrical resistivity of material deposited at room temperature is measured by a two-point technique and amounts to 1–100 Ω cm depending on the current employed for deposition. The technique is applied to generate fields of dot marks visible in the optical microscope for metrology purposes. These dot arrays can be fabricated on the surface of finished three-dimensional structures without additional treatments like resist deposition or development.
Focused ion beam insulator deposition
Authors: Young Richard J. , Puretz Joseph
Published: July 2002

Focused ion beam (FIB) induced deposition of insulating films from TEOS, a SiO2 precursor, is reported. Deposition was typically carried out using a 30 kV beam of Ga+ ions with a range of ion beam currents from 40 to 2000 pA. The deposition rate was measured as a function of beam current density into the scanned area and scan period, with a typical rate of 0.3 μm3/nC being noted. An important application of the FIB is to repair integrated circuits enabling faster debugging and shorter design cycles. With the continuing trend toward devices containing more interconnect layers having narrower tracks and spaces, it is becoming increasingly difficult to access lower metal levels when they are completely covered by wide upper tracks. Localized FIB insulator deposition enables contact to lower level metal without first removing large areas of the upper metal layers which would otherwise be required. In such structures isolation resistances of more than 15 GΩ have been measured between the metal layers, corresponding to resistivities greater than 108 Ω cm, with a breakdown voltage for the material in the range of 20 to greater than 100 V/μm.
Deep trench fabrication by Si (110) orientation dependent etching
Authors: Theil Jeremy A.
Published: July 2002

A simple technique for creating trench structures in silicon using readily available wafer processing techniques is discussed. By using orientation dependent etching of (110) Si, it is possible to create trenches in silicon with vertical sidewalls. The etching anisotropy of certain solutions used with this technique is greater than 600:1 [110]:[111] etching, making it possible to fabricate virtually any value of aspect ratio trench. For this technique, which makes use of two etchants, an anisotropy of 50:1 is demonstrated. The equipment, materials, and processing steps required are outlined.
Metal–insulator–metal capacitors by using electron cyclotron resonance plasma-SiO2
Authors: Machida K. , Imai K. , Miura K. , Ozaki Y. , Arai E.
Published: July 2002

This paper describes the fabrication process and the reliable characteristics of a metal–insulator–metal capacitor using electron cyclotron resonance plasma-SiO2 as an insulator film. The metal–insulator–metal capacitors are additionally fabricated during the Al multilevel interconnection process since the deposition temperature of electron cyclotron resonance plasma-SiO2 is below 200 °C. Also, the leakage current characteristics of the film are excellent even without densification at high temperature. The experimental results confirm that a metal–insulator–metal capacitor fabrication process using electron cyclotron resonance plasma-SiO2 can be applied to analog large scale integration implementation.
Improved retarding field optics via image outside field
Authors: Hordon L. S. , Boyer B. B. , Pease R. F. W.
Published: July 2002

The advantages of a retarding field immersion lens for focusing low energy electrons and ions have been well chronicled. One recent development is a retarding field lens in which the sample is outside the retarding field (i.e., a nonimmersion lens). Thus, any sample roughness will not distort the focusing field. We describe here a simple model and analysis that indicates that the performance of such a lens can be almost as good as that of a retarding field immersion lens. For example, it should be possible to focus 1 keV electrons with an energy spread of 1 eV into a beam diameter of 4 nm using realistic values of maximum voltage and retarding field strength; the effective resolution under these conditions is estimated to be 1.4 nm.
Pattern recognition of trench width using a confocal microscope
Authors: Wang Yao-Ting , Schaper Charles D. , Kailath Thomas
Published: July 2002

A pattern recognition algorithm is developed to estimate the top width and bottom width of trenches of semiconductor wafers simultaneously. A confocal microscope is used in this study because of its excellent depth resolution. The signals from the confocal microscope are compared to a set of a priori waveforms with known trench dimensions. An optimal estimate is chosen. Principle component decomposition of the set of waveforms is used to achieve reduced storage and improved computational speed. An interpolation method is used to improve the accuracy of the estimate. Experimental results of deep trenches are provided to demonstrate the approach.
Effects of synchrotron radiation irradiation on selective Si epitaxial growth by disilane molecular beam epitaxy
Authors: Utsumi Yuichi , Akazawa Housei
Published: July 2002

Perfect selectivity in Si epitaxial growth on a Si/SiO2 substrate has been achieved by SR irradiation during disilane molecular-beam epitaxy (MBE). This differs from conventional selective growth by gas-source MBE using disilane in that no polycrystalline Si is deposited on the SiO2 irrespective of growth time. A temperature of over 700 °C is necessary for perfect selective growth. The effects of SR irradiation on the initial stage of polycrystalline Si film formation on SiO2 are also investigated as they are closely related to the appearance of perfect selectivity. It is demonstrated that a photoenhanced reduction completely removes Si from SiO2 and that this process brings about perfect selectivity.
A new theory for silicon oxidation
Authors: Peng Kuang-Yao , Wang Long-Ching , Slattery John C.
Published: July 2002

A new theory for oxidation of silicon is derived which is based upon diffusion of molecular oxygen in stoichiometric silicon dioxide, consistent with the mass balance at the silicon-silicon dioxide phase interface. The results are compared with the experimental data of Lie et al. and Adams et al.
Polycide gate etching using a helical resonator on an applied materials precision 5000 platform
Authors: Chang C. P. , Klemens F. P. , Maynard H. L. , Lee T. C. , Kornblit A. , Ibbotson D. E.
Published: July 2002

We modified a polycide gate etching chamber of an Applied Materials Precision 5000 platform to accept a commercially available Prototech 150-mm-diam helical resonator plasma source to extend the utility of this proven manufacturing platform to 0.25 μm technology and beyond. We successfully demonstrated the use of the helical resonator on the Precision 5000 platform to etch WSix/α-Si gate stacks using a multistep process. At low pressures (<10 mTorr), the helical resonator etches polysilicon and WSix more uniformly than a standard Precision 5000 MERIE chamber. In addition, the WSix-to-α-Si selectivity for the WSix etching step is also higher with the high-density source.
SiC x-ray lithography mask fabricated by electron cyclotron resonance plasma source coupled with divided microwaves
Authors: Shimada Masaru , Ono Toshiro , Okada Ikuo , Matsuo Seitaro
Published: July 2002

A newly developed electron cyclotron resonance plasma source coupled with divided microwaves was investigated for depositing SiC films as an x-ray mask membrane. Silicon carbide (SiC) films were deposited with high reliability and high repeatability using this source. The stress in SiC films has easily been controlled for a self-supported mask membrane by the addition of heat treatment. The SiC membrane has a very smooth surface (less than 1 nm of Rrms), and high stability against the x-ray irradiation, which enables highly accurate fine pattern replication with x-ray lithography.
Paleostress in Cratonic North America: Implications for Deformation of Continental Interiors
Authors: van der Pluijm Ben A. , Craddock John P. , Graham Brita R. , Harris John H.
Published: July 2002

Compressive paleostresses, as recorded by twinned calcite in carbonate rocks that cover cratonic northwestern North America, are perpendicular to the orogenic front of the Late Cretaceous to Early Cenozoic Sevier fold-thrust belt. Inferred differential stresses decrease from ∼100 megapascals (MPa) at the orogenic front to ∼20 MPa up to 2000 kilometers inland. New analyses near the Late Paleozoic Appalachian front refine earlier results from the eastern Midcontinent. The Appalachian and Sevier stress data in North America's continental interior are remarkably similar in spite of distinctly different tectonic properties. This suggests that continental interior stresses are largely insensitive to tectonic characteristics of compressive plate margins and that far-field stress transmission is filtered by deformation styles in mountain belts.
Origin of Multikilometer Earth- and Mars-Crossing Asteroids: A Quantitative Simulation
Authors: Migliorini Fabio , Michel Patrick , Morbidelli Alessandro , Nesvorný David , Zappalà Vincenzo
Published: July 2002

Orbital dynamic simulations show that many asteroids in the main asteroid belt are driven toward Mars-crossing orbits by numerous weak mean motion resonances, which slowly increase the orbital ellipticity of the asteroids. In addition, half of the Mars-crossing asteroids (MCAs) transition to Earth-crossing asteroids (ECAs) in less than 20 million years. This scenario quantitatively explains the observed number of large ECAs and MCAs.
Nanoscale Science of Single Molecules Using Local Probes
Authors: Gimzewski James K. , Joachim Christian
Published: July 2002

Experiments on individual molecules using scanning probe microscopies have demonstrated an exciting diversity of physical, chemical, mechanical, and electronic phenomena. They have permitted deeper insight into the quantum electronics of molecular systems and have provided unique information on their conformational and mechanical properties. Concomitant developments in experimentation and theory have allowed a diverse range of molecules to be studied, varying in complexity from simple diatomics to biomolecular systems. At the level of an individual molecule, the interplays of mechanical and electronical behavior and chemical properties manifest themselves in an unusually clear manner. In revealing the crucial role of thermal, stochastic, and quantum-tunneling processes, they suggest that dynamics is inescapable and may play a decisive role in the evolution of nanotechnology.
Quantum-Critical Conductivity Scaling for a Metal-Insulator Transition
Authors: Lee H.-L. , Carini John P. , Baxter David V. , Henderson W. , Grüner G.
Published: July 2002

Temperature (
T
)- and frequency (ω)-dependent conductivity measurements are reported here in amorphous niobium-silicon alloys with compositions (
x
) near the zero-temperature metal-insulator transition. There is a one-to-one correspondence between the frequency- and temperature-dependent conductivity on both sides of the critical concentration, thus establishing the quantum-critical nature of the transition. The analysis of the conductivity leads to a universal scaling function and establishes the critical exponents. This scaling can be described by an
x
-,
T
-, and ω-dependent characteristic length, the form of which is derived by experiment.
High-Level Expression of Chromosomally Encoded SHV-1 β-Lactamase and an Outer Membrane Protein Change Confer Resistance to Ceftazidime and Piperacillin- Tazobactam in a Clinical Isolate of
Klebsiella pneumoniae
Authors: Rice Louis B. , Carias Lenore L. , Hujer Andrea M. , Bonafede Mary , Hutton Rebecca , Hoyen Claudia , Bonomo Robert A.
Published: July 2002

We describe
Klebsiella pneumoniae
15571, a clinical isolate resistant to ceftazidime MIC = 32 μg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 μg/ml).
K. pneumoniae
15571 expresses a single β-lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector in
Escherichia coli
, this
bla
SHV-1
gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described
bla
SHV-1
genes. Outer membrane protein (OMP) analysis of
K. pneumoniae
15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in
K. pneumoniae
44NR, a low-level ampicillin-resistant strain that also expresses a chromosomally determined
bla
SHV-1
. Crude β-lactamase enzyme extracts from
K. pneumoniae
15571 produced roughly 200-fold more β-lactamase activity than
K. pneumoniae
44NR. Northern hybridization analysis revealed that this difference was explainable by quantifiable differences in transcription of the
bla
SHV-1
gene in the two strains. Primer extension analysis of
bla
SHV-1
mRNA from
K. pneumoniae
15571 and 44NR indicated that the transcriptional start sites were identical in the two strains. DNA sequencing of the promoter regions upstream of the of
bla
SHV-1
open reading frames in the two
K. pneumoniae
strains revealed an A→C change in the second position of the −10 region in
K. pneumoniae
44NR compared to that in 15571. Site-directed mutagenesis of the cloned
K. pneumoniae
15571
bla
SHV-1
, in which the A in the second position of the 15571 −10 region was changed to a C, resulted in a substantial lowering of the MIC of ampicillin. When the levels of β-lactamase enzyme expression in
E. coli
were compared, the
bla
SHV-1
downstream of the altered −10 region produced 17-fold less β-lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a combination of increased enzyme production and minor OMP changes and that levels of chromosomally encoded SHV-1 β-lactamase production can vary substantially with a single-base-pair change in promoter sequence.
Pharmacokinetics and Pharmacodynamics of Intravenous Artesunate in Severe Falciparum Malaria
Authors: Davis Timothy M. E. , Phuong Hoang Lan , Ilett Kenneth F. , Hung Nguyen Canh , Batty Kevin T. , Phuong Vu Duong Bich , Powell Shane M. , Thien Huynh Van , Binh Tran Quang
Published: July 2002

To provide novel data relating to the dispositions, effects, and toxicities of the artemisinin derivatives in severe malaria, we studied 30 Vietnamese adults with slide-positive falciparum malaria treated with intravenous artesunate. Twelve patients with complications (severe; group 1) and 8 patients without complications but requiring parenteral therapy (moderately severe; group 2) received 120 mg of artesunate by injection, and 10 patients with moderately severe complications (group 3) were given 240 mg by infusion. Serial concentrations of artesunate and its active metabolite dihydroartemisinin in plasma were measured by high-performance liquid chromatography. The time to 50% parasite clearance (PCT
50
) was determined from serial parasite densities. Full clinical (including neurological) assessments were performed at least daily. In noncompartmental pharmacokinetic analyses, group mean artesunate half-lives (
t
1/2
) were short (range, 2.3 to 4.3 min). The dihydroartemisinin
t
1/2
(range, 40 to 64 min), clearance (range, 0.73 to 1.01 liters/h/kg), and volume of distribution (range, 0.77 to 1.01 liters/kg) were also similar both across the three patient groups (
P
> 0.1) and to previously reported values for patients with uncomplicated malaria. Parasite clearance was prompt (group median PCT
50
range 6 to 9 h) and clinical recovery was complete under all three regimens. These data indicate that the pharmacokinetics of artesunate and dihydroartemisinin are not influenced by the severity of malaria. Since the pharmacokinetic parameters for both artesunate and dihydroartemisinin were similar regardless of whether injection or infusion was used, artesunate can be considered a prodrug that is converted stoichiometrically to dhydroartemisinin. Conventional doses of artesunate are safe and effective when given to patients with complications of falciparum malaria.
In Vitro Activities of Two Antimitotic Compounds, Pancratistatin and 7-Deoxynarciclasine, against
Encephalitozoon intestinalis
, a Microsporidium Causing Infections in Humans
Authors: Ouarzane-Amara Meryem , Franetich Jean-François , Mazier Dominique , Pettit George R. , Meijer Laurent , Doerig Christian , Desportes-Livage Isabelle
Published: July 2002

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with
Encephalitozoon intestinalis
, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC
50
s) of pancratistatin and 7-deoxynarciclasine for
E. intestinalis
were 0.18 μM and 0.2 μM, respectively, approximately eightfold lower than the IC
50
s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.
Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of
Mycoplasma gallisepticum
Mutants Obtained In Vitro
Authors: Reinhardt A. K. , Bébéar C. M. , Kobisch M. , Kempf I. , Gautier-Bouchardon A. V.
Published: July 2002

Mycoplasma gallisepticum
enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.
Molecular Characterization of a Novel Class 1 Integron Containing
bla
GES-1
and a Fused Product of
aac(3)-Ib/aac(6
"
)-Ib
" Gene Cassettes in
Pseudomonas aeruginosa
Authors: Dubois Véronique , Poirel Laurent , Marie Caroline , Arpin Corinne , Nordmann Patrice , Quentin Claudine
Published: July 2002

As seen by the disk diffusion method, the clinical strain of
Pseudomonas aeruginosa
Pa695, resistant to all extended-spectrum cephalosporins and aminoglycosides, exhibited an unusual synergistic effect between ceftazidime and imipenem. This isolate produced an extended-spectrum β-lactamase (ESBL) with a pI of 5.8 that appeared to be chromosomally encoded. Cloning experiments revealed that this ESBL was encoded by
bla
GES-1
, previously described in an integron from
Klebsiella pneumoniae
. In
P. aeruginosa
Pa695, a higher level of resistance to ceftazidime than to ticarcillin was observed, and no synergy between the β-lactamase inhibitors and extended-spectrum cephalosporins was detected, in contrast to the resistance pattern observed in
K. pneumoniae
. Further sequence analysis demonstrated that the
bla
GES-1
gene cassette was located in a class 1 integron, which contained another sequence corresponding to the fused
aac(3)-Ib
and
aac(6
"
)-Ib
" gene cassettes. The fusion product was functional, as was the product of each gene cloned separately: AAC(3)-I, despite the deletion of the four last amino acids, and AAC(6"), which carried three amino acid changes compared with the most homologous sequence. The AAC(3)-I protein conferred an expected gentamicin and fortimicin resistance, and the AAC(6"), despite the Leu-119→Ser substitution, yielded resistance to kanamycin, tobramycin, and dibekacin, but slightly affected netilmicin and amikacin, and had no apparent effect on gentamicin. The fusion product conveyed a large profile of resistance, combining the AAC(6") activity with a higher level of gentamicin resistance without accompanying fortimicin resistance.
Impact of
N
-Chlorotaurine on Viability and Production of Secreted Aspartyl Proteinases of
Candida
spp
Authors: Nagl Markus , Gruber Andreas , Fuchs Anita , Lell Claudia P. , Lemberger Eva-Maria , Borg-von Zepelin Margarete , Würzner Reinhard
Published: July 2002

N
-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against
Candida
spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of
N
-chlorotaurine as an antimicrobial agent in yeast infections.
A Strain of
Enterococcus faecium
(18C23) Inhibits Adhesion of Enterotoxigenic
Escherichia coli
K88 to Porcine Small Intestine Mucus
Authors: Jin L. Z. , Marquardt R. R. , Zhao X.
Published: July 2002

Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if
Enterococcus faecium
18C23 has the ability to inhibit the adhesion of
Escherichia coli
K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% of
E. faecium
18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living
E. faecium
18C23 culture efficiently inhibited the adhesion of
E. coli
K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of
E. coli
K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 10
9
CFU or more of living
E. faecium
18C23 culture per ml was added simultaneously with
E. coli
to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of
E. coli
K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of
E. coli
K88ac by
E. faecium
18C23 or its supernatant might occur through steric hindrance.
Influence of Catalase and Superoxide Dismutase on Ozone Inactivation of
Listeria monocytogenes
Authors: Fisher Christopher W. , Lee Dongha , Dodge Beth-Anne , Hamman Kristen M. , Robbins Justin B. , Martin Scott E.
Published: July 2002

The effects of ozone at 0.25, 0.40, and 1.00 ppm on
Listeria monocytogenes
were evaluated in distilled water and phosphate-buffered saline. Differences in sensitivity to ozone were found to exist among the six strains examined. Greater cell death was found following exposure at lower temperatures. Early stationary-phase cells were less sensitive to ozone than mid-exponential- and late stationary-phase cells. Ozonation at 1.00 ppm of cabbage inoculated with
L. monocytogenes
effectively inactivated all cells after 5 min. The abilities of in vivo catalase and superoxide dismutase to protect the cells from ozone were also examined. Three listerial test strains were inactivated rapidly upon exposure to ozone. Both catalase and superoxide dismutase were found to protect listerial cells from ozone attack, with superoxide dismutase being more important than catalase in this protection.
Chlorine-Susceptible and Chlorine-Resistant Type 021N Bacteria Occurring in Bulking Activated Sludges
Authors: Séka M. A. , Kalogo Y. , Hammes F. , Kielemoes J. , Verstraete W.
Published: July 2002

Two filamentous bacteria causing bulking in two activated sludges were examined. Investigations using morphological features, staining techniques, and fluorescent in situ hybridization identified both filaments as type 021N. However, an examination of the effect of chlorine on the sludges revealed a chlorine-susceptible type 021N in one sludge and a chlorine-resistant type 021N in the other.
Chromosomal Gene Inactivation in the Green Sulfur Bacterium
Chlorobium tepidum
by Natural Transformation
Authors: Frigaard Niels-Ulrik , Bryant Donald A.
Published: July 2002

Conditions for inactivating chromosomal genes of
Chlorobium tepidum
by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting
nifD
with various antibiotic resistance markers. Growth of wild-type
C. tepidum
at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml
−1
, 2 μg of erythromycin ml
−1
, 30 μg of chloramphenicol ml
−1
, or 1 μg of tetracycline ml
−1
or a combination of 300 μg of streptomycin ml
−1
and 150 μg of spectinomycin ml
−1
. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10
−7
were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10
−3
were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in
C. tepidum
, whose 2.15-Mb genome has recently been completely sequenced.
Antacid Increases Survival of
Vibrio vulnificus
and
Vibrio vulnificus
Phage in a Gastrointestinal Model
Authors: Koo Jaheon , Marshall Douglas L. , DePaola Angelo
Published: July 2002

Viable counts of three strains of
Vibrio vulnificus
and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37°C.
V. vulnificus
was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable
V. vulnificus
cells delivered from the gastric compartment during the first 30 min of exposure reached 10
6
to 10
8
CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid
V. vulnificus
counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10
7
to 10
9
CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both
V. vulnificus
and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.
Sinorhizobium meliloti
Cells Require Biotin and either Cobalt or Methionine for Growth
Authors: Watson Robert J. , Heys Roselyn , Martin Teresa , Savard Marc
Published: July 2002

Sinorhizobium meliloti
is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B
12
-dependent homocysteine methyltransferase for methionine biosynthesis.
Isomaltulose Synthase from
Klebsiella
sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability
Authors: Zhang Daohai , Li Xianzhen , Zhang Lian-Hui
Published: July 2002

The gene (
palI
) encoding isomaltulose synthase (PalI) from a soil bacterial isolate,
Klebsiella
sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)
8
-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp
241
, Glu
295
, Asp
369
, His
145
, and His
368
) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a

K
m

of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe
3+
and Hg
2+
and was enhanced by Mn
2+
and Mg
2+
. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu
498
and Arg
310
with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.
Immune Function in Healthy Inner-City Children
Authors: Bartlett Jacqueline A. , Goldklang Arielle R. , Schleifer Steven J. , Keller Steven E.
Published: July 2002

The importance of investigating immunity in healthy children has been underscored in the last few years by studies of the immune pathology of childhood illnesses, including human immunodeficiency virus. This study reports both ennumerative and functional immune measures in healthy inner city children. A total of 152 of 207 children studied were completely heathy at the time of venipuncture and were included in this study. Laboratory immune batteries were completed (or begun) the same day as venipuncture. Relationships between age, gender, ethnicity, and immunity were then analyzed. We found that gender predicted both the absolute number and the percentage of T cells and helper cells and the percentage of natural killer cells. Total leukocyte counts and percentages of lymphocytes and granulocytes were related to ethnicity, as was the response to mitogen stimulation (concanavalin A and pokeweed mitogen) and phagocytic ability. In conclusion, age, gender, and ethnicity factors were found to contribute to differences in various immune measures in children and require further investigation.
Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays
Authors: Schmitz John L. , Denny Thomas N. , Garcia Ambrosia , Lathey Janet L.
Published: July 2002

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (
gag
) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm
3
, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.
Plasmid-Encoded Toxin of Enteroaggregative
Escherichia coli
is Internalized by Epithelial Cells
Authors: Navarro-Garcı́a Fernando , Canizalez-Roman Adrián , Luna José , Sears Cynthia , Nataro James P.
Published: July 2002

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative
Escherichia coli
(EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet (J. M. Villaseca, F. Navarro-Garcı́a, G. Mendoza-Hernández, J. P. Nataro, A. Cravioto, and C. Eslava, Infect. Immun. 68:5920–5927, 2000).
Escherichia coliCdtB Mediates Cytolethal Distending Toxin Cell Cycle Arrest
Authors: Elwell Cherilyn , Chao Kinlin , Patel Kamlesh , Dreyfus Lawrence
Published: July 2002

We previously reported that the CdtB polypeptide ofEscherichia colicytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with anE. coliextract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtBH154Alacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.
vspGene Expression byGiardia lambliaClone GS/M-83-H7 during Antigenic Variation In Vivo and In Vitro
Authors: Bienz M. , Siles-Lucas M. , Wittwer P. , Müller N.
Published: July 2002

Giardia lambliainfections are associated with antigenic variation of the parasite, which is generated by a continuous change of the variant-specific surface proteins (VSPs). Many investigations on the process of antigenic variation were based on the use ofG. lambliaclone GS/M-83-H7, which expresses VSP H7 as its major surface antigen. In the present study, mice were infected with the aforementioned clonal line to investigatevspgene expression during the complex process of antigenic variation of the parasite. Trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for theirvspgene expression patterns, i.e., without cultivating the recovered parasites in vitro. Because few trophozoites were recovered at late time points p.i., a combined 5′ rapid amplification of cDNA ends–reverse transcription-PCR approach was utilized. This allowed detection and subsequent sequence analysis ofvspgene transcripts upon generation of amplified cDNA analogues. The same PCR approach was applied for analysis ofvspgene expression in variants obtained after negative selection of axenic GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view of the entire panel of in vivo- and in vitro-derived parasite populations, expression of 29 differentvspgene sequences could be demonstrated. In vivo antigenic variation ofG. lambliaclone GS/M-83-H7 was shown to be a continuous process involving the consecutive appearance of relatively distinct sets ofvsptranscripts. During the 42-day infection period investigated, this process activated at least 22 differentvspgenes. Comparative molecular analyses of the amino acid level demonstrated that all cDNA segments identified encode structural elements typical of the terminal segment ofGiardiaVSP. The similarity of most of the GS/M-83-H7 VSP sequences identified in the present study supports previous suggestions thatvspgene diversification inG. lambliais the result of ancestral gene duplication, mutation, and/or recombination events.
Identification and Characterization of Mycobacterial Proteins Differentially Expressed under Standing and Shaking Culture Conditions, Including Rv2623 from a Novel Class of Putative ATP-Binding Proteins
Authors: Florczyk Matthew A. , McCue Lee Ann , Stack Robert F. , Hauer Charles R. , McDonough Kathleen A.
Published: July 2002

The environmental signals that affect gene regulation in
Mycobacterium tuberculosis
remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including
acr
(also known as
hspX
) (α-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling of
M. bovis
BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified
M. tuberculosis
Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in
M. tuberculosis
's response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.
Transfer of the Core Region Genes of the
Yersinia enterocolitica
WA-C Serotype O:8 High-Pathogenicity Island to
Y. enterocolitica
MRS40, a Strain with Low Levels of Pathogenicity, Confers a Yersiniabactin Biosynthesis Phenotype and Enhanced Mouse Virulence
Authors: Pelludat Cosima , Hogardt Michael , Heesemann Jürgen
Published: July 2002

The high-pathogenicity island (HPI) of yersiniae encodes an iron uptake system represented by its siderophore yersiniabactin (Ybt). The HPI is present in yersiniae with high levels of pathogenicity—i.e.,
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
biogroup (BG) 1B—but absent in
Y. enterocolitica
strains with low (BG 2 to 5) and no (BG 1A) levels of pathogenicity and has been shown to be an important virulence factor. Comparison of the HPI in
Y. enterocolitica
(Yen-HPI) and that in
Y. pestis
and
Y. pseudotuberculosis
revealed that, in contrast to genes of the variable region, genes of the core region (genes
irp9
to
fyuA
) are highly homologous. In the present work the Yen-HPI core genes were rescued from the chromosome of
Y. enterocolitica
WA-C (BG 1B, serotype O:8) using the
FRT
-FLP recombinase system. Transfer of the resulting plasmid pCP1 into the siderophore-deficient strain
Y. enterocolitica
NF-O (BG 1A) led to no halo on siderophore indicator chrome azurol S (CAS) agar. Transfer of pCP1 into the
Y. enterocolitica
strain MRS40 (serotype O:9, BG 2; phenotype, CAS negative) led to a CAS halo larger than that of parental strain WA-C, indicating high Ybt production. pCP1 was highly unstable in iron-deficient medium, and no enhanced mouse virulence conferred by MRS40 carrying pCP1 could be detected. To overcome the problem of instability, pCP1 was integrated into the chromosome of MRS40, leading to the formation of a CAS halo comparable to that seen with WA-C and correspondingly to increased mouse virulence. Thus, the core genes of Yen-HPI are sufficient to confer a positive CAS phenotype and mouse virulence to
Y. enterocolitica
MRS40, BG 2, but are insufficient to confer this phenotype to
Y. enterocolitica
NF-O, BG 1A.