bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive ion stream etching utilizing electron cyclotron resonance plasma
Authors: Ono Toshiro , Oda Masatoshi , Takahashi Chiharu , Matsuo Seitaro
Published: July 2002

Reactive ion stream etching has been developed for highly accurate etching with little bombardment induced damage by utilizing an electron cyclotron resonance (ECR) plasma. The ion energy during etching is controlled by utilizing the interaction between the ECR plasma and a divergent magnetic field, in a low energy range from 20 to 50 eV at low gas pressures of 10−2 Pa. Highly accurate submicron patterns of polysilicon and molybdenum were obtained with high selectivities to SiO2, larger than 30, by using Cl2 gas as the main etching gas.
Can photoemission measure valence-band discontinuities?
Authors: List R. S. , Spicer W. E.
Published: July 2002

The progress in understanding heterojunction band lineups has increasingly become dependent upon the availability of accurate and reliable measurements. The reported values of the band offset for the Si/GaAs system measured by photoemission range from 0.05 to 0.70 eV. Such a large scatter in experimentally determined discontinuities is common for other systems as well and not only makes the evaluation of different theories difficult, but raises the question as to whether such variations are real and representative of technological variations in the offset. Clearly the understanding and control of these variations is essential before any device applications can be realized. We attempted to study the source of these variations in the Si/GaAs(110) heterojunction. We altered the growth conditions to effect modifications in the overlayer crystalline order, strain, and chemical reactivity. While some of these studies have been previously reported, their collective consideration gives us a much more coherent overview of the problems involved in discontinuity measurements. In our studies we found the offset to be 0.23±0.05 eV for both amorphous and crystalline overlayers, either strained or unstrained. We did, however, observe discontinuities as large as 0.7 eV for room temperature growths. We show that such large discontinuities are due to dangling bond type states which extend into the band gap and change the apparent valence band maximum energy. These states can be removed by annealing the sample without crystallization. Offsets as small as 0.05 eV may be inferred from our data if we fail to account for the contributions of rather subtle chemical effects at the interface. Overall, we found the discontinuity to be more dependent on the interpretation of the data than on technological variations. If we discern and properly account for extrinsic effects and unambiguously define the discontinuity, photoemission can yield accurate and single valued measurements of the valence band discontinuity.
Characterization of near-field holography grating masks for optoelectronics fabricated by electron beam lithography
Authors: Tennant D. M. , Koch T. L. , Mulgrew P. P. , Gnall R. P. , Ostermeyer F. , Verdiell J-M.
Published: July 2002

Direct write e-beam lithography and reactive ion etching was used to fabricate square-wave gratings in quartz substrates which serve as pure phase masks in the near-field holographic printing of gratings. This method of fabricating these masks extends the flexibility of the printing technique by allowing both abrupt phase shifts as well as multiple grating pitches to be simultaneously printed from a single contact mask. Grating masks with periods in the 235–250 nm range have been produced and measured to be within 0.15 nm of the design period. Transmitted and diffracted beam powers have also been measured for various duty cycles and etch depths and are shown to be important parameters for ‘‘balancing’’ these interfering beams. Simple scalar diffraction modeling is used to qualitatively examine the dependence of diffraction on grating parameters, but the need for a more comprehensive modeling is illustrated. Prototype masks have been used to produce grating patterns on InP substrates using two different ultraviolet illumination sources: an argon ion laser and a conventional mercury lamp.
Characterization of chemically assisted ion beam etching of InP
Authors: Youtsey C. , Grundbacher R. , Panepucci R. , Adesida I. , Caneau C.
Published: July 2002

Chemically assisted ion beam etching involving an Ar ion beam and a Cl2 ambient was investigated for the etching of high quality facets in InP. Detailed investigations on how the etch anisotropy as well as surface morphology could be optimized under different etch conditions were performed. It was necessary to elevate the substrate temperature above 150 °C to obtain smooth surfaces and above 225 °C to achieve good anisotropy. At these elevated temperatures, very high etch rates in excess of 2 μm/min were obtained. The choice of mask material was found to have a strong influence on the surface quality due to micromasking effects. Hard baked photoresist and Ti masks were used to obtain surfaces free of ‘‘grasslike’’ roughness. Free standing InP wires of various widths were fabricated to estimate the extent of sidewall damage under different etch conditions.
Low-damage specimen preparation technique for transmission electron microscopy using iodine gas-assisted focused ion beam milling
Authors: Yamaguchi Akira , Nishikawa Takeshi
Published: July 2002

A novel method is presented for the preparation of high-resolution electron microscopy specimens with gas-assisted focused ion beam (FIB) milling. An etching rate of InP is enhanced 11–13 times with iodine gas-assisted FIB milling, compared to conventional FIB milling. Specimens with lower radiation damage can be prepared due to lower ion dose during FIB milling, and show no anomalous contrast which comes from the artifacts during FIB milling. Damaged layer introduced during FIB milling was also investigated.
Structure and electronic states on reduced BaTiO3 (100) surface observed by scanning tunneling microscopy and spectroscopy
Authors: Bando Hiroshi , Shimitzu Tetsushi , Aiura Yoshihiro , Haruyama Yuichi , Oka Kunihiko , Nishihara Yoshikazu
Published: July 2002

Structure and electronic states on the (100) surface of reduced BaTiO3 single crystals were observed by scanning tunneling microscopy and spectroscopy. After annealing at 1150 °C, the direction of the edges ordered to run along the equivalents of [011] and [001] directions. All the observed step edges were of unit-cell height. On each terrace atomlike corrugation with the periodicity multiples of the √5×√5 R26.6° superlattice was observed in the filled state images. A straightforward method to deduce the local density of states from the scanning tunneling spectroscopy data was introduced, based on multiplication of an inverted triangular transfer matrix, and was applied to the scanning tunneling spectroscopy data on the terraces. The amplitude of the local density of states at EF−1 eV varied in phase with the atomlike corrugation on the terrace, which was consequently ascribed to electronic states at the spatially ordered Ti 3d–oxygen vacancy complexes on the surface.
Molecular arrangement of copper phthalocyanine on hydrogen-terminated Si(111): Influence of surface roughness
Authors: Nakamura M. , Morita Y. , Mori Y. , Ishitani A. , Tokumoto H.
Published: July 2002

The molecular arrangement of copper phthalocyanine (CuPc) crystals on hydrogen-terminated Si(111) surfaces was investigated by using both scanning probe microscopy and x-ray diffraction in terms of influence of the surface roughness. On a rough surface with a root-mean-square roughness of 0.20 nm, the molecules were stacked so as to form an α crystal where the molecular column was parallel to the surface. On the other hand, a new crystal form with its column exactly perpendicular to the Si(111) plane was grown on a atomically flat surface. In this case, the molecules were stacked perpendicular to the substrate with the underlying molecules situated directly below. These molecular arrangements were independent of the growth temperature in the range of 60–180 °C. On the atomically flat surfaces, the strong interactive force between the surface and the planar CuPc molecule may result in the new growth behavior.
Fabrication of 200 nm period nanomagnet arrays using interference lithography and a negative resist
Authors: Farhoud Maya , Ferrera Juan , Lochtefeld Anthony J. , Murphy T. E. , Schattenburg Mark L. , Carter J. , Ross C. A. , Smith Henry I.
Published: July 2002

Magnetic information storage density has increased at the rate of 60% per year for the past seven years. There is wide agreement that continuation of this trend beyond the physical limits of the continuous thin-film media currently used will likely require a transition to discrete, lithographically defined magnetic pillars. Interference lithography (IL) appears to be the most cost-effective means of producing two-dimensional arrays of such pillars. IL can rapidly expose large areas with relatively simple equipment, without the need for a mask, and with fine control of the ratio of pillar diameter to period. We show that negative-tone imaging yields three times the contrast of positive-tone imaging for the generation of holes in photoresist, suitable for subsequent deposition or electroplating of magnetic material. We use a negative i-line, chemically-amplified resist (OHKA THMR-iN PS1) to form 200 nm period arrays of magnetic dots in Co and Ni. Such arrays, with a variety of well controlled diameters, are used to study the effect of particle size on magnetic behavior.
Investigators Detail HIV's Fatal Handshake
Authors: Cohen Jon
Published: July 2002

A new study published in this issue of Science (p.
602
) describes the elaborate “handshake” the AIDS virus uses to wangle its way into white blood cells, HIV's main target.
Germany Joins the Biotech Race
Authors: Dickman Steven
Published: July 2002

Five years ago, Germany provided perhaps the most inhospitable climate for biotechnology in the Western world. Now the German government is trying to transform the country virtually overnight into a biotech powerhouse. Last week, it picked three regions of the country for special grants and loans for start-ups and industry-academia collaborations—the spearheads in its attempt to make up lost ground.
HIV's Other Immune-System Targets: Macrophages
Authors: Balter Michael
Published: July 2002

Varenna, Italy—Last month, more than 100 AIDS experts gathered here to contemplate a major unsolved riddle of HIV infection: What role do immune-system cells other than T lymphocytes play in the progression of the disease? Researchers reported that macrophages and dendritic cells may be an important site of virus production and may also be HIV's gateway to the brain, an accomplice in the killing of T cells, and the refuge of HIV strains with different susceptibilities to antiviral drugs.
Space Carbon--Neutral Pathways?
Authors: Heath James Richard , Saykally Richard James
Published: July 2002

Complex chemistry takes place in interstellar matter, aided by the energetic photons of ultraviolet radiation that ionize molecules and induce reactions. In their Perspective, Heath and Saykally discuss results published in the same issue by Kaiser
et al
. (
p. 1508
) that reveal the importance of neutral species in interstellar chemistry. Such reactions explain the reaction-product branching ratios in such different objects as the TMC-1 molecular cloud and the carbon star IRC+10216.
Funding Inequality Threatens Novel Bioscience Program
Authors: Williams Nigel
Published: July 2002

The Human Frontier Science Program (HFSP) has this year won plaudits from the leaders of the G7 industrial nations, and an external review of its scientific achievements gave it a glowing endorsement. But Japan, which originally proposed HFSP, is growing tired of the fact that its international partners contribute little money to the program.
Dissecting How Presenilins Function—and Malfunction
Authors: Marx Jean
Published: July 2002

Two recently identified proteins are moving onto center stage in Alzheimer's disease research: the presenilins, which are mutated in about half of all inherited cases. A recent crop of results implies that these proteins play a role in the cell's internal protein-handling systems. If so, faulty presenilins could contribute to the abnormal buildup of other proteins seen in the brains of Alzheimer's patients.
Plateaus Observed in the Field Profile of Thermal Conductivity in the Superconductor Bi
2
Sr
2
CaCu
2
O
8
Authors: Krishana K. , Ong N. P. , Li Q. , Gu G. D. , Koshizuka N.
Published: July 2002

Quasi-particles (QPs) are excitations of the superconducting state. The behavior of QPs in Bi
2
Sr
2
CaCu
2
O
8
in a magnetic field was probed by measurement of the thermal conductivity κ. An anomaly in κ was observed at low temperatures. At a transition field H
k
, κ displayed a sharp break in slope, followed by a plateau region in which it ceased to change with increasing field. The nonanalytic nature of the break at H
k
suggests a phase transition of the condensate to a state in which the QP current is zero (the system remains superconducting). Detailed measurements of the new regime are presented, and implications for the QPs and the superconducting state are discussed.
Synergistic Predation, Density Dependence, and Population Regulation in Marine Fish
Authors: Hixon Mark A. , Carr Mark H.
Published: July 2002

Understanding natural causes of density dependence is essential for identifying possible sources of population regulation. Field experiments on a model system of coral reef fishes showed that small juveniles of
Chromis cyanea
suffer heavy mortality that is spatially density-dependent only in the presence of two suites of predators: transient piscivores attacking from above, and reef-resident piscivores attacking from below. In the absence of either kind of predator, early mortality of
Chromis
is virtually density-independent. Because piscivores may have regulatory roles in this and similar marine systems, overfishing these predators may have ramifications for the remainder of the exploited community.
The Role of Ocean-Atmosphere Interactions in Tropical Cooling During the Last Glacial Maximum
Authors: Bush Andrew B. G. , Philander S. George H.
Published: July 2002

A simulation with a coupled atmosphere-ocean general circulation model configured for the Last Glacial Maximum delivered a tropical climate that is much cooler than that produced by atmosphere-only models. The main reason is a decrease in tropical sea surface temperatures, up to 6°C in the western tropical Pacific, which occurs because of two processes. The trade winds induce equatorial upwelling and zonal advection of cold water that further intensify the trade winds, and an exchange of water occurs between the tropical and extratropical Pacific in which the poleward surface flow is balanced by equatorward flow of cold water in the thermocline. Simulated tropical temperature depressions are of the same magnitude as those that have been proposed from recent proxy data.
Slope Water Current over the Laurentian Fan on Interannual to Millennial Time Scales
Authors: Keigwin L. D. , Pickart R. S.
Published: July 2002

The strength and position of surface and deep currents in the slope water south of Newfoundland are thought to vary as a coupled system in relation to the dipole in atmospheric sea level pressure known as the North Atlantic oscillation (NAO). Paleoceanographic data from the Laurentian Fan, used as a proxy for sea surface temperature, reveal that surface slope waters north of the Gulf Stream experienced warming during the Little Ice Age of the 16th to 19th centuries and support the notion of an NAO-driven coupled system. The NAO may be a useful model for millennial-scale ocean variability during interglacial climate states.
Step-by-Step Engineered Multiparticle Entanglement
Authors: Rauschenbeutel Arno , Nogues Gilles , Osnaghi Stefano , Bertet Patrice , Brune Michel , Raimond Jean-Michel , Haroche Serge
Published: July 2002

After quantum particles have interacted, they generally remain in an entangled state and are correlated at a distance by quantum-mechanical links that can be used to transmit and process information in nonclassical ways. This implies programmable sequences of operations to generate and analyze the entanglement of complex systems. We have demonstrated such a procedure for two atoms and a single-photon cavity mode, engineering and analyzing a three-particle entangled state by a succession of controlled steps that address the particles individually. This entangling procedure can, in principle, operate on larger numbers of particles, opening new perspectives for fundamental tests of quantum theory.
Discovery of a Transient Absorption Edge in the X-ray Spectrum of GRB 990705
Authors: Amati Lorenzo , Frontera Filippo , Vietri Mario , Zand Jean J. M. in 't , Soffitta Paolo , Costa Enrico , Sordo Stefano Del , Pian Elena , Piro Luigi , Antonelli Lucio A. , Fiume D. Dal , Feroci Marco , Gandolfi Giangiacomo , Guidorzi Cristiano , Heise John , Kuulkers Erik , Masetti Nicola , Montanari Enrico , Nicastro Luciano , Orlandini Mauro , Palazzi Eliana
Published: July 2002

We report the discovery of a transient equivalent hydrogen column density with an absorption edge at ∼3.8 kiloelectron volts in the spectrum of the prompt x-ray emission of gamma-ray burst (GRB) 990705. This feature can be satisfactorily modeled with a photoelectric absorption by a medium located at a redshift of ∼0.86 and with an iron abundance of ∼75 times the solar one. The transient behavior is attributed to the strong ionization produced in the circumburst medium by the GRB photons. The high iron abundance points to the existence of a burst environment enriched by a supernova along the line of sight. The supernova explosion is estimated to have occurred about 10 years before the burst. Our results agree with models in which GRBs originate from the collapse of very massive stars and are preceded by a supernova event.
Distribution of Lipid Formulations of Amphotericin B into Bone Marrow and Fat Tissue in Rabbits
Authors: Groll Andreas H. , Mickiene Diana , Piscitelli Stephen C. , Walsh Thomas J.
Published: July 2002

The distribution of the three currently available lipid formulations of amphotericin B (AmB) into bone marrow and fat tissue was evaluated in noninfected rabbits. Groups of four animals each received either 1 mg of AmB deoxycholate (D-AmB) per kg of body weight per day or 5 mg of AmB colloidal dispersion, AmB lipid complex, or liposomal AmB per kg per day for seven doses. Plasma, bone marrow, fat, and liver were collected at autopsy, and AmB concentrations were determined by high-performance liquid chromatography. At the investigated dosages of 5 mg/kg/day, all AmB lipid formulations achieved at least fourfold-higher concentrations in bone marrow than did standard D-AmB at a dosage of 1 mg/kg/day. Concentrations in bone marrow were 62 to 76% of concurrent AmB concentrations in the liver. In contrast, all AmB formulations accumulated comparatively poorly in fat tissue. The results of this study show that high concentrations of AmB can be achieved in the bone marrow after administration of lipid formulations, suggesting their particular usefulness against disseminated fungal infections involving the bone marrow and against visceral leishmaniasis.
Novel α- and β-Amino Acid Inhibitors of Influenza Virus Neuraminidase
Authors: Kati Warren M. , Montgomery Debra , Maring Clarence , Stoll Vincent S. , Giranda Vincent , Chen Xiaoqi , Laver W. Graeme , Kohlbrenner William , Norbeck Daniel W.
Published: July 2002

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 α- and β-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited
K

i

values in the 50 μM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-
N
-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the α-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the β-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the α- or β-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.
Low Sensitivity of
Listeria monocytogenes
to Quaternary Ammonium Compounds
Authors: Mereghetti L. , Quentin R. , Marquet-Van Der Mee N. , Audurier A.
Published: July 2002

Ninety-seven epidemiologically unrelated strains of
Listeria monocytogenes
were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the
mdrL
gene, which encodes a multidrug efflux pump, and the
orfA
gene, a putative transcriptional repressor of
mdrL
. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the
qacA
and
smr
genes, encoding multidrug efflux systems previously described for the genus
Staphylococcus
. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall.
Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “
Bacillus cereus
Group”
Authors: Daffonchio Daniele , Cherif Ameur , Borin Sara
Published: July 2002

Bacillus anthracis
,
Bacillus cereus
,
Bacillus mycoides
,
Bacillus pseudomycoides
,
Bacillus thuringiensis
, and
Bacillus weihenstephanensis
are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “
B. cereus
group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as
B. anthracis
or serotypes of
B. thuringiensis
. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included
B. mycoides
,
B. weihenstephanensis
, and
B. pseudomycoides
. The second included
B. cereus
and
B. thuringiensis
,
B. anthracis
appeared as a lineage of
B. cereus
.
Molecular Mating Type Assay for
Fusarium circinatum
Authors: Wallace M. Margaret , Covert Sarah F.
Published: July 2002

A rapid and reliable mating type assay for
Fusarium circinatum
was created by applying primers specific for the
MAT1-1
and
MAT1-2
mating type alleles to genomic DNA in a single PCR. A similar approach may be applied to fungi not previously shown to reproduce sexually, thus enabling studies of population structure and inheritance.
Molecular Cloning, Sequencing, and Expression of
omp-40
, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium
Thiobacillus ferrooxidans
Authors: Guiliani Nicolas , Jerez Carlos A.
Published: July 2002

Thiobacillus ferrooxidans
is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized
omp40
, the gene coding for the major outer membrane protein from
T. ferrooxidans
. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the
T. ferrooxidans
surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic
T. ferrooxidans
to prevent free movement of protons across its outer membrane.
Temperature Affects Species Distribution in Symbiotic Populations of
Vibrio
spp
Authors: Nishiguchi Michele K.
Published: July 2002

The genus
Sepiola
(Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All
Sepiola
species have a light organ that contains at least one of two species of luminous bacteria,
Vibrio fischeri
and
Vibrio logei
. The two
Vibrio
species coexist in at least four
Sepiola
species (
S. affinis
,
S. intermedia
,
S. ligulata
, and
S. robusta
), and their concentrations in the light organ depend on changes in certain abiotic factors, including temperature. Strains of
V. fischeri
grew faster in vitro and in
Sepiola
juveniles when they were incubated at 26°C. In contrast, strains of
V. logei
grew faster at 18°C in culture and in
Sepiola
juveniles. When aposymbiotic
S. affinis
or
S. ligulata
juveniles were inoculated with one
Vibrio
species, all strains of
V. fischeri
and
V. logei
were capable of infecting both squid species at the optimum growth temperatures, regardless of the squid host from which the bacteria were initially isolated. However, when two different strains of
V. fischeri
and
V. logei
were placed in direct competition with each other at either 18 or 26°C, strains of
V. fischeri
were present in sepiolid light organs in greater concentrations at 26°C, whereas strains of
V. logei
were present in greater concentrations at 18°C. In addition to the competition experiments, the ratios of the two bacterial species in adult
Sepiola
specimens caught throughout the season at various depths differed, and these differences were correlated with the temperature in the surrounding environment. My findings contribute additional data concerning the ecological and environmental factors that affect host-symbiont recognition and may provide insight into the evolution of animal-bacterium specificity.
Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the
ail
Gene of Pathogenic
Yersinia enterocolitica
Authors: Jourdan Alissa D. , Johnson Scott C. , Wesley Irene V.
Published: July 2002

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic
Yersinia enterocolitica
. The assay targets the chromosomally encoded attachment and invasion gene,
ail
. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of
ail
were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified
Y. enterocolitica
DNA. TM1 identified all 26
Y. enterocolitica
strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the
Y. enterocolitica
strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-
Y. enterocolitica
strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4
Y. enterocolitica
CFU/ml in pure culture and 10
Y. enterocolitica
CFU/ml independent of the presence of 10
8
CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of
Y. enterocolitica
per g of ground pork or feces after a 24-h enrichment in a
Yersinia
selective broth.
Inactivation of Viable
Ascaris
Eggs by Reagents during Enumeration
Authors: Nelson Kara L. , Darby Jeannie L.
Published: July 2002

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate
Ascaris
eggs under typical laboratory conditions. Two methods were used to enumerate indigenous
Ascaris
eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H
2
SO
4
) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-μm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of
Ascaris suum
eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO
4
, MgSO
4
, and NaCl) or during incubation (0.1 N H
2
SO
4
and 0.5% formalin) inactivated the
Ascaris
eggs under the conditions studied.
Concentration and Detection of
Cryptosporidium
Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration
Authors: Simmons Otto D. , Sobsey Mark D. , Heaney Christopher D. , Schaefer Frank W. , Francy Donna S.
Published: July 2002

The protozoan parasite
Cryptosporidium parvum
is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of
Cryptosporidium
oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of
C. parvum
oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of
C. parvum
oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although
C. parvum
oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered
C. parvum
oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.
Anti-CagA Immunoglobulin G Responses Correlate with Interleukin-8 Induction in Human Gastric Mucosal Biopsy Culture
Authors: Ando Takafumi , Perez-Perez Guillermo I. , Kusugami Kazuo , Ohsuga Masahiro , Bloch Karen C. , Blaser Martin J.
Published: July 2002

Helicobacter pyloripersists in the human stomach despite eliciting both cellular and humoral immune responses and inducing proinflammatory cytokines. To determine whether local humoral and cytokine responses are related to each other and to histologic responses, we studied 66 Japanese patients who underwent gastroscopy. Using specific enzyme-linked immunosorbent assays, we examined gastric antral mucosal-organ biopsy culture supernatants to assess interleukin-6 (IL-6) and interleukin-8 (IL-8) levels and antibody responses toH. pyloriwhole-cell antigens CagA, HspA, and HspB. Of the patients studied, 11 wereH. pylorinegative and 55 wereH. pyloripositive; by PCR, all strains werecagA+. As expected, compared toH. pylori-negative patients,H. pylori-positive patients had significantly higher humoral responses to allH. pyloriantigens and had higher IL-8 (47.8 ± 3.5 versus 10.1 ± 4.3 ng/mg of biopsy protein;P< 0.001) and IL-6 levels (2.8 ± 0.3 versus 0.26 ± 0.2 ng/mg of protein;P< 0.001). Among theH. pylori-positive patients, supernatant anti-CagA immunoglobulin G (IgG) levels were significantly associated withH. pyloridensity (P< 0.005) and neutrophil infiltration (P< 0.005) scores. Anti-CagA immunoglobulin A levels were correlated with intestinal metaplasia (P< 0.05). Mononuclear cell infiltration scores were significantly associated with supernatant IL-6 levels (P< 0.005) and with IgG responses to whole-cell antigens (P< 0.05). Supernatant IL-8 levels were significantly associated with anti-CagA IgG (r= 0.75,P< 0.001). Anti-CagA responses correlated with neutrophil infiltration, intestinal metaplasia,H. pyloridensity, and IL-8 levels, suggesting that the absolute levels of these antibodies may be markers for gastric inflammation and premalignant changes in individual hosts.
CpG Oligodeoxynucleotides Act as Adjuvants for Pneumococcal Polysaccharide-Protein Conjugate Vaccines and Enhance Antipolysaccharide Immunoglobulin G2a (IgG2a) and IgG3 Antibodies
Authors: Chu Rose S. , McCool Tera , Greenspan Neil S. , Schreiber John R. , Harding Clifford V.
Published: July 2002

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses againstStreptococcus pneumoniaepolysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM197. BALB/c ByJ mice were injected with 19F-CRM197or 6B-CRM197with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM197alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM197alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM197IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.
Altered Gene Expression inStaphylococcus aureusupon Interaction with Human Endothelial Cells
Authors: Vriesema Aldwin J. M. , Beekhuizen Henry , Hamdi Mohamed , Soufan Alexandre , Lammers Aart , Willekens Ben , Bakker Onno , Welten Angelique G. A. , Veltrop Marcel H. A. M. , van de Gevel Joke S. , Dankert Jacob , Zaat Sebastian A. J.
Published: July 2002

Staphylococcus aureusis isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding ofS. aureusto human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction betweenS. aureusand human vascular endothelium. Scanning electron microscopic analyses showed that binding ofS. aureusto human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase ofS. aureushad a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library ofS. aureuswas used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of theS. aureustransposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression inS. aureus.
Inactivation of the
srtA
Gene in
Streptococcus gordonii
Inhibits Cell Wall Anchoring of Surface Proteins and Decreases In Vitro and In Vivo Adhesion
Authors: Bolken Tové C. , Franke Christine A. , Jones Kevin F. , Zeller Gloria O. , Jones C. Hal , Dutton Emma K. , Hruby Dennis E.
Published: July 2002

The
srtA
gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium
Staphylococcus aureus
. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013–5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if
srtA
plays a similar role in other gram-positive bacteria. To that end, the
srtA
gene in the human oral commensal organism
Streptococcus gordonii
was insertionally inactivated. The
srtA
mutant
S. gordonii
exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the
srtA
mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.
Cytokine Networking in Lungs of Immunocompetent Mice in Response to InhaledAspergillus fumigatus
Authors: Brieland Joan K. , Jackson Craig , Menzel Fred , Loebenberg David , Cacciapuoti Anthony , Halpern Judy , Hurst Stephen , Muchamuel Tony , Debets Reno , Kastelein Rob , Churakova Tatyana , Abrams John , Hare Roberta , O'Garra Anne
Published: July 2002

Cytokine networking in the lung in response to inhaledAspergillus fumigatuswas assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulentA. fumigatus(6 × 106CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-α), IL-12, and gamma interferon (IFN-γ) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-α protein, which preceded induction of immunoreactive IL-12 and IFN-γ. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-α, IL-12, and IFN-γ mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-α (anti-TNF-α MAb), IL-12 (anti-IL-12 MAb), and/or IFN-γ (anti-IFN-γ MAb), and effects on intrapulmonary cytokine activity and growth ofA. fumigatuswere assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-α, while neutralization of IL-18, TNF-α, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-γ. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-α alone or in combination with IL-12, IL-18, or IFN-γ also resulted in a significant increase inA. fumigatusCFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-α, through their modulatory effects on both intrapulmonary cytokine activity and growth ofA. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.
Helicobacter pylori
Resists Phagocytosis by Macrophages: Quantitative Assessment by Confocal Microscopy and Fluorescence-Activated Cell Sorting
Authors: Ramarao Nalini , Meyer Thomas F.
Published: July 2002

Helicobacter pylori
infection of the stomach epithelium is characterized by an infiltration of polymorphonuclear and mononuclear cells. These immune cells contribute to mucosal damage which may eventually lead to gastritis, peptic ulcer, gastric cancer, and/or MALT-associated gastric lymphoma. Here we show that
H. pylori
inhibits its own uptake, as well as in
trans
the phagocytosis of
Neisseria gonorrhoeae
, by human and murine macrophages. This antiphagocytic activity is dependent on the presence of the
cag
pathogenicity island in the
H. pylori
genome. We demonstrate that
H. pylori
also expresses its antiphagocytic activity towards the myelomonocytic cell line JOSKM, thus providing a potent model for the study of the interaction between
H. pylori
and phagocytes. Our data were obtained using laser confocal microscopy and flow cytometry after quenching the fluorescence of labeled extracellular bacteria. The antiphagocytic activity of
H. pylori
may explain the persistence of
H. pylori
and its pathological consequences. The use of cell lines and flow cytometry will hopefully facilitate progress in our understanding of the immune escape of these persistent bacteria.
Arg-Gingipain A DNA Vaccine Induces Protective Immunity against Infection by
Porphyromonas gingivalis
in a Murine Model
Authors: Yonezawa Hideo , Ishihara Kazuyuki , Okuda Katsuji
Published: July 2002

Arginine-specific cysteine proteinases (RgpA and RgpB) produced by the periodontal pathogen
Porphyromonas gingivalis
are suspected virulence factors and are involved in interrupting host defense mechanisms as well as in penetrating and destroying periodontal connective tissues. To induce a protective immune response against
P. gingivalis
, we constructed an
rgpA
DNA vaccine. BALB/c mice were immunized intradermally by Gene Gun with plasmid DNA carrying
rgpA
. Antibody responses against
P. gingivalis
were determined by an enzyme-linked immunosorbent assay. The
rgpA
DNA vaccine induced high levels of serum antibodies against
P. gingivalis
. Sera from the
rgpA
DNA vaccine-immunized mice diminished the proteolytic activity of RgpA and RgpB and inhibited the binding of
P. gingivalis
to a type I collagen sponge. Moreover, the sera effectively reduced the hemagglutination of
P. gingivalis
, indicating that the hemagglutinin activity of the organism is associated with RgpA. We found with a murine abscess model that mice immunized with the
rgpA
DNA vaccine were resistant to an invasive
P. gingivalis
W50 challenge. These results suggest that the
rgpA
DNA vaccine induced specific antibodies against the enzyme and that this vaccine could confer protective immunity against
P. gingivalis
infection.
Exacerbation ofAcanthamoebaKeratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies
Authors: Hurt Michael , Apte Sherine , Leher Henry , Howard Kevin , Niederkorn Jerry , Alizadeh Hassan
Published: July 2002

Neutrophils are thought to be involved in many infectious diseases and have been found in high numbers in the corneas of patients withAcanthamoebakeratitis. Using a Chinese hamster model of keratitis, conjunctival neutrophil migration was manipulated to determine the importance of neutrophils in this disease. Inhibition of neutrophil recruitment was achieved by subconjunctival injection with an antibody against macrophage inflammatory protein 2 (MIP-2), a powerful chemotactic factor for neutrophils which is secreted by the cornea. In other experiments, neutrophils were depleted by intraperitoneal injection of anti-Chinese hamster neutrophil antibody. The inhibition of neutrophils to the cornea resulted in an earlier onset and more severe infection compared to controls. Anti-MIP-2 antibody treatment produced an almost 35% reduction of myeloperoxidase activity in the cornea 6 days postinfection, while levels of endogenous MIP-2 secretion increased significantly. Recruitment of neutrophils into the cornea via intrastromal injections of recombinant MIP-2 generated an initially intense inflammation that resulted in the rapid resolution of the corneal infection. The profound exacerbation ofAcanthamoebakeratitis seen when neutrophil migration was inhibited, combined with the rapid clearing of the disease in the presence of increased neutrophils, strongly suggests that neutrophils play an important role in combatingAcanthamoebainfections in the cornea.