bluuebunny/crossref_metadata_2025 huggingface dataset:
Coadsorption of sodium and water on MgO(100)/Mo(100) studied by ultraviolet photoelectron and metastable impact electron spectroscopies
Authors: Günster J. , Liu G. , Kempter V. , Goodman D. W.
Published: July 2002

The coadsorption of D2O and Na on the MgO(100)/Mo(100) surface has been studied by metastable impact electron spectroscopy and ultraviolet photoelectron spectroscopy (He I). The initial layer of D2O adsorbed on the MgO(100) surface at 100 K “wets” the surface. For multilayer adsorption, the outermost water molecules exhibit an electronic structure which is very similar to gas phase water. Na dosed onto a D2O- precovered MgO surface leads to the formation of a hydroxide species, most likely NaOH. This hydroxide species is stable to 530 K.
Chemical and photochemical processes in sulfide passivation of GaAs(100): In situ optical study and photoemission analysis
Authors: Berkovits V. L. , Ulin V. P. , Paget D. , Bonnet J. E. , L’vova T. V. , Chiaradia P. , Lantratov V. M.
Published: July 2002

We demonstrate a new approach to study semiconductor surface passivation. This approach, which we have applied to the case of GaAs(100) passivation by sodium sulfide water solutions, consists of using both reflectance anisotropy spectroscopy during passivation and ultraviolet photoemission spectroscopy after removing the sample from the solution. We find that (i) complete chemical passivation requires a treatment duration significantly longer than what was used in previous works; in our case, the oxygen contamination is strongly reduced; (ii) photochemical processes play an important role for improving the surface electronic properties. As a result, photoemission spectroscopy, which uses powerful light excitation, may strongly alter the chemical bonds. From experimental evidence and using a chemical model based on first principles estimates, we propose a detailed description both of the surface chemical reactions that occur during passivation and of the chemical bonds that are formed at the semiconductor surface and in the passivating overlayer.
Molecular imaging of ordered and disordered naphthalene on Pt(111)
Authors: Hallmark V. M. , Chiang S. , Wöll Ch.
Published: July 2002

Scanning tunneling microscopy (STM) has been used to examine the adsorption system of naphthalene on Pt(111). The images show molecules in three discrete rotational orientations, 120° apart. Images of an ordered naphthalene monolayer show molecules on a (3×3) Pt lattice, with internal domain structure which exhibits pseudo-(6×3) symmetry. Low coverage naphthalene forms a disordered layer uniformly distributed over the surface, with evidence for molecular rotations and translations between subsequent STM images. Images of cleaned Pt(111) without molecules show the interaction of carbon islands on the surface with Pt step edges.
Surface photovoltage and band bending at metal/GaAs interfaces: A contact potential difference and photoemission spectroscopy study
Authors: Mao D. , Kahn A. , Le Lay G. , Marsi M. , Hwu Y. , Margaritondo G. , Santos M. , Shayegan M. , Florez L. T. , Harbison J. P.
Published: July 2002

Contact potential difference (CPD) measurements using a Kelvin probe coupled with synchrotron radiation are used to investigate various aspects of the problem of surface photovoltage (SPV) induced by the synchrotron radiation at (110) and (100) GaAs surfaces. A large and quasipermanent SPV is found at surfaces of low doped and low temperature (110) samples. SPV discharge mechanisms are investigated. Finally, the CPD technique is used to define conditions which minimize SPV and allow accurate measurements of band bending at low temperature. Band bending measurements are reported for interfaces between metals and (110) and (100) GaAs surfaces.
Very sharp gold and platinum tips to modify gold surfaces in scanning tunneling microscopy
Authors: Libioulle L. , Houbion Y. , Gilles J.-M.
Published: July 2002

To induce modifications of gold (111) surface using a scanning tunneling microscope, we have developed new electrochemical procedures to obtain very sharp gold and platinum tips. The resulting geometry is very reproducible and the mean curvature radius is 10 and 5 nm for the two materials, respectively. We have studied the formation of mounds and holes obtained by applying voltage pulses using these prepared tips. The resulting modifications of sample and tip geometry are observed. The threshold pulse amplitude is related to the tip to sample distance and the material transfer is described as a metal–metal bonding.
Deep level formation at ZnSe/GaAs(100) interfaces
Authors: Raisanen A. D. , Brillson L. J. , Vanzetti L. , Bonanni A. , Franciosi A.
Published: July 2002

We have used low energy cathodo- and photoluminescence spectroscopies to characterize the formation of charge states deep within the ZnSe band gap and localized near the ZnSe/GaAs interface. These deep levels are a common feature of the ZnSe/GaAs heterostructure and depend sensitively on growth conditions and post-growth annealing. Here we report in situ measurements of deep level formation near the ZnSe/GaAs interface during post-growth annealing and their dramatic dependence on the initial epitaxical growth conditions. We used 1–2 keV electron beam energies in tandem with HeCd and HeNe excitation to obtain emission spectra characteristic of the free ZnSe surface, the ZnSe bulk, and the ZnSe/GaAs(100) interface. Annealing in ultrahigh vacuum in the 300–500 °C range produces a new emission feature at 1.9–2.0 eV whose intensity increases exponentially with temperature, exhibiting an activation energy of 1.10±0.08 eV. This feature appears enhanced (suppressed) in structures fabricated in Se(Zn)-rich growth, and its intensity may therefore be related to the Zn vacancy concentration or the Ga indiffusion. These results emphasize the importance of local composition in the formation of interface deep level defects and their subsequent effects on luminescence efficiency and heterojunction stability.
Effects of potassium and lithium metal deposition on the emission characteristics of Spindt-type thin-film field emission microcathode arrays
Authors: Talin A. A. , Felter T. E. , Devine D. J.
Published: July 2002

The effects of K and Li deposition on the emission characteristics of Spindt-type thin-film field emission microcathode arrays [Spindt et al. J. Appl. Phys. 47, 5248 (1976)] (TFFEMC) with Mo tips are discussed. The amount by which each alkali metal lowered the effective work function of a TFFEMC is reported and compared to results of similar measurements performed with a clean Mo(100) surface. In addition, it is shown that the emission current is enhanced during exposure to K flux, even after a saturation coverage has been established. These results are interpreted with a model based on an excess K coverage produced during dosing, which enhances the overall emission of the array.
Fabrication of a variable diffraction efficiency phase mask by multiple dose ion implantation
Authors: Erickson L. E. , Champion H. G. , Albert J. , Hill K. O. , Malo B. , Thériault S. , Bilodeau F. , Johnson D. C.
Published: July 2002

Apodized fiber Bragg gratings show reductions in the unwanted sidebands from those of uniform Bragg grating. A phase mask whose diffraction efficiency varied from the center to the ends was fabricated by implanting a grating pattern in a SiO2 substrate with Si++ and wet etching in diluted HF. The phase mask diffraction efficiency vs ion dose was measured. Using this phase mask, apodized Bragg gratings were photoimprinted into fibers. The sidebands of the apodized fiber gratings were 26 dB below the peak of the central resonance compared to 12 dB for the uniform Bragg grating. The modeled values were 29 and 13.2 dB, respectively.
Active control of the emission current of field emitter arrays
Authors: Yokoo Kuniyoshi , Arai Manabu , Mori Masahiro , Bae Jongsuck , Ono Shoichi
Published: July 2002

The current control and the stabilization in field emission of gated field emitter arrays (FEAs) are the highest demand for applications to a flat panel display and other beam devices. The concept of the field-effect-controlled field emission cathode is very useful for these purposes. We carried out preliminary experiments of the idea and showed that the controllability and the stability of emission current of FEA were significantly improved by an actively controlled FEA. Additionally, we discussed beam focusing of FEA for a flat panel display application.
Analytical and seminumerical models for gated field emitter arrays. II. Comparison of theory to experiment
Authors: Jensen K. L. , Zaidman E. G. , Kodis M. A. , Goplen B. , Smithe D. N.
Published: July 2002

The utility of analytic and seminumerical models for a gated field emitter array (FEA) unit cell is judged by comparing the predictions of the Saturn and seminumerical models against the numerically simulated behavior for FEA parameters deemed most important from the standpoint of rf amplifier applications. The numerical simulation algorithm based on the Boundary Element method, used to validate the seminumerical model and to test the qualitative predictions of the Saturn model, is briefly described. Further, by varying the distribution of emitters, work function, and tip radius, we match experimental data given in the literature for molybdenum FEAs: the manner in which the relevant geometrical and material parameters are chosen is explained and shown to be compatible with physically based expectations about emitter geometry and materials.
Simulation of field emission and electrodynamic characteristics for triode near-cathode modulators with edge field emitter arrays
Authors: Zakharchenko Yu. F. , Sinitsyn N. L. , Gulyaev Yu. V.
Published: July 2002

On the basis of the theory of conformal transformations of complex functions, a method was developed to rigorously solve the electrostatic problem for two-dimensional diode and triode systems having edge arrays whose faces are formed by a periodic nano-dimensional array of field emitting centers. The method proposed is used to simulate the emission and electrodynamic characteristics of near-cathode megawatt triodelike modulators.
Computer-controlled fabrication of ultra-sharp tungsten tips
Authors: Kim Young-Guon , Choi Eun-Ha , Kang Seung-Oun , Cho Guangsup
Published: July 2002

In order to monitor the end-point current during electrochemical etching, an analog to digital converter circuit aided by a personal computer has been setup. At the moment the lower part of the needle drops off during the etching process, a maximum current change across the reference resistor is detected by the PC interface card and the applied voltage is then cut off within a few milliseconds. Using these circuits to control the etching time, our experiment has been able to fabricate an ultra-sharp tip of ∼200 Å radius with a higher reproduction rate and reliability than the conventional method.
Electron scattering and transmission through SCALPEL masks
Authors: Mkrtchyan M. M. , Liddle J. A. , Novembre A. E. , Waskiewicz W. K. , Watson G. P. , Harriott L. R. , Muller D. A.
Published: July 2002

Electron scattering in thin solid films used for the fabrication of masks for electron projection lithography, e.g., SCALPEL®, is investigated. We have developed an analytical model to calculate electron transmission through the mask membrane and image contrast due to different scattering properties of the patterned area and the membrane. The model utilizes cross sections for electron elastic and inelastic scattering on an atom with exponentially screened Coulomb potential of the nucleus derived in the first Born approximation. The variety and controversy of theoretical and empirical adjustments of the screening parameter are briefly analyzed and attributed to the misinterpretation of experimental data ignoring the effects mostly due to plural scattering of electrons and dense packing of atoms in thin solid films. This model frees us from the computational limitations of Monte Carlo simulations and proves to be effective for the straightforward characterization of various alternative materials for SCALPEL mask membrane and scatterer. The model includes the appropriate effects of the projection optics and back-focal plane aperture.
A Link Between Virulence and Ecological Abundance in Natural Populations of
Staphylococcus aureus
Authors: Day Nicholas P. J. , Moore Catrin E. , Enright Mark C. , Berendt Anthony R. , Smith John Maynard , Murphy Michael F. , Peacock Sharon J. , Spratt Brian G. , Feil Edward J.
Published: July 2002

Staphylococcus aureus
is a major cause of severe infection in humans and yet is carried without symptoms by a large proportion of the population. We used multilocus sequence typing to characterize isolates of
S. aureus
recovered from asymptomatic nasal carriage and from episodes of severe disease within a defined population. We identified a number of frequently carried genotypes that were disproportionately common as causes of disease, even taking into account their relative abundance among carriage isolates. The existence of these ecologically abundant hypervirulent clones suggests that factors promoting the ecological fitness of this important pathogen also increase its virulence.
Endosomal Targeting by the Cytoplasmic Tail of Membrane Immunoglobulin
Authors: Weiser Peter , Müller Ralph , Braun Uschi , Reth Michael
Published: July 2002

Membrane-bound immunoglobulin (mIg) of the IgG, IgA, and IgE classes have conserved cytoplasmic tails. To investigate the function of these tails, a B cell line was transfected with truncated or mutated γ2a heavy chains. Transport to the endosomal compartment of antigen bound by the B cell antigen receptor did not occur in the absence of the cytoplasmic tail; and one or two mutations, respectively, in the Tyr-X-X-Met motif of the tail partially or completely interrupted the process. Experiments with chimeric antigen receptors confirmed these findings. Thus, a role for the cytoplasmic tail of mIg heavy chains in endosomal targeting of antigen is revealed.
Atomic Selectivity in the Chemisorption of ICl (Iodochloride) on Silicon Surfaces
Authors: Liu Yong , Masson Denis P. , Kummel Andrew C.
Published: July 2002

When ICl adsorbs on a clean silicon (111)-(7×7) surface, the reaction is chemically selective. This process has been studied with the use of scanning tunneling microscopy and Auger spectroscopy. The dominant mechanism is the formation of silicon monoiodide by abstraction and the ejection of the chlorine atoms back into the gas phase. This pathway is not only chemically selective but also the least exothermic of all the possible reaction mechanisms. Although atomic chemical selectivity in gas-phase reactions is quite common, atomic chemical selectivity is unexpected in gas-surface reactions on clean surfaces because of the high density of reactive sites.
Accurate Experimental Electronic Properties of DL-Proline Monohydrate Obtained Within 1 Day
Authors: Koritsánszky T. , Flaig R. , Zobel D. , Krane H.-G. , Morgenroth W. , Luger P.
Published: July 2002

A 1-day x-ray diffraction experiment on
dl
-proline monohydrate was performed at 100 kelvin with synchrotron radiation and a charge-coupled device area detection technique. The accuracy of the charge density distribution and of the related electronic properties extracted from these data is comparable or even superior to the accuracy obtained from a 6-week experiment on
dl
-aspartic acid with conventional x-ray diffraction methods. A data acquisition time of 1 day is comparable to the time needed for an ab initio calculation on the isolated molecules. This technique renders larger molecular systems of biological importance accessible to charge density experiments.
Requirement for MAPK Activation for Normal Mitotic Progression in
Xenopus
Egg Extracts
Authors: Guadagno Thomas M. , Ferrell James E.
Published: July 2002

The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M phase in
Xenopus
oocytes. This report examines whether it also plays a role in normal mitotic progression. MAPK was transiently activated during mitosis in cycling
Xenopus
egg extracts after activation of the cyclin-dependent kinase Cdc2–cyclin B. Interference with MAPK activation by immunodepletion of its activator MEK, or by addition of the MEK inhibitor PD98059, caused precocious termination of mitosis and interfered with production of normal mitotic microtubules. Sustained activation of MAPK arrested extracts in mitosis in the absence of active Cdc2–cyclin B. These findings identify a role for MEK and MAPK in maintaining the mitotic state.
Evolution of Complexity in Paleozoic Ammonoid Sutures
Authors: Saunders W. B. , Work D. M. , Nikolaeva S. V.
Published: July 2002

The septal sutures of 588 genera of Paleozoic ammonoids showed a 1600 percent increase in mean complexity over 140 million years. Within 475 ancestor/descendant pairs, descendants were more than twice as likely to be more complex than their ancestors. Twelve subclades (373 genera) averaged 34 percent increased complexity. These patterns are compatible with an active or driven system of long-term bias for increased complexity. Mass extinctions acted in opposition to this long-term trend, tending to eliminate more-complex forms and resetting the trend with each extinction event.
A Light-Emitting Field-Effect Transistor
Authors: Schön J. H. , Dodabalapur A. , Kloc Ch. , Batlogg B.
Published: July 2002

We report here on the structure and operating characteristics of an ambipolar light-emitting field-effect transistor based on single crystals of the organic semiconductor α-sexithiophene. Electrons and holes are injected from the source and drain electrodes, respectively. Their concentrations are controlled by the applied gate and drain-source voltages. Excitons are generated, leading to radiative recombination. Moreover, above a remarkably low threshold current, coherent light is emitted through amplified spontaneous emission. Hence, this three-terminal device is the basis of a very promising architecture for electrically driven laser action in organic semiconductors.
In Vitro Efficacy of Nikkomycin Z against the Human Isolate of the Microsporidian Species
Encephalitozoon hellem
Authors: Bigliardi Elisa , Bernuzzi Anna Maria , Corona Silvia , Gatti Simonetta , Scaglia Massimo , Sacchi Luciano
Published: July 2002

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of
Encephalitozoon hellem
to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 μg/ml, reduced the number of parasitic foci by about 35% ± standard deviation after 7 days of culture (
P
< 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.
Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in
Streptomyces clavuligerus
Authors: Jensen Susan E. , Elder Kenneth J. , Aidoo Kwamena A. , Paradkar Ashish S.
Published: July 2002

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a “supercluster” in
Streptomyces clavuligerus
. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.
Roles of
gyrA
Mutations in Resistance of Clinical Isolates and In Vitro Mutants of
Bacteroides fragilis
to the New Fluoroquinolone Trovafloxacin
Authors: Bachoual Rafik , Dubreuil Luc , Soussy Claude-James , Tankovic Jacques
Published: July 2002

We determined whether
gyrA
mutations were present in fluoroquinolone-resistant laboratory mutants derived from the
Bacteroides fragilis
reference strain ATCC 25285 and in clinical isolates of
B. fragilis
. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of
gyrA
mutations, whereas two of the three CIP-selected second-step mutants studied presented the same
gyrA
mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 μg/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of
B. fragilis
were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO.
Pharmacodynamics of Trovafloxacin and Levofloxacin against
Bacteroides fragilis
in an In Vitro Pharmacodynamic Model
Authors: Peterson M. L. , Hovde L. B. , Wright D. H. , Brown G. H. , Hoang A. D. , Rotschafer J. C.
Published: July 2002

An in vitro pharmacodynamic investigation was conducted to explore whether the area under the concentration time curve from 0 to 24 h (AUC
0–24
)/MIC ratio could predict fluoroquinolone performance against
Bacteroides fragilis
. An in vitro model was used to generate kill curves for trovafloxacin (TVA) and levofloxacin (LVX) at AUC
0–24
/MIC ratios of 1 to 406 against three strains of
B. fragilis
(ATCC 25285, ATCC 23745, and clinical isolate M97-117). TVA and LVX were bolused prior to the start of experiments to achieve the corresponding AUC
0–24
/MIC ratio. Experiments were performed in duplicate over 24 h and in an anaerobic environment. Analyses of antimicrobial performance were conducted by comparing the rates of bacterial kill (K) using nonlinear regression analysis with 95% confidence intervals. Statistical significance was defined as a lack of overlap in the 95% confidence limits generated from the slope of each kill curve. For both TVA and LVX, K was maximized once an AUC
0–24
/MIC ratio of ≥40 was achieved and was not further increased despite a 10-fold increase in AUC
0–24
/MIC from approximately 40 to 400 against all three strains of
B. fragilis
. No significant differences were found in K between AUC
0–24
/MIC ratios of approximately 40 to 200. In experiments where AUC
0–24
/MIC ratios that were ≥ 5 and ≤ 44 were conducted, 64% demonstrated regrowth at 24 h. Resistant strains were selected in 50% of those experiments, demonstrating regrowth, which resulted in increased MICs of two- to 16-fold for both TVA and LVX. Regrowth did not occur, nor were resistant strains selected in any studies with an AUC/MIC that was > 44. Our findings suggest that fluoroquinolones provide antibacterial effects against
B. fragilis
in a concentration-independent manner associated with an AUC
0–24
/MIC ratio of ≥40. Also, the potential for the selection of resistant strains of
B. fragilis
may increase with an AUC
0–24
/MIC ratio of ≤44.
Plasmidic Extended-Spectrum β-Lactamases in
Vibrio cholerae
O1 El Tor Isolates in Argentina
Authors: Petroni Alejandro , Corso Alejandra , Melano Roberto , Cacace María Luisa , Bru Ana María , Rossi Alicia , Galas Marcelo
Published: July 2002

Since 1992 there have been seven major outbreaks of cholera in Argentina. Susceptibility analysis of 1,947 isolates (40% of reported cases) of
Vibrio cholerae
O1 biotype El Tor suggested the presence of extended-spectrum β-lactamases (ESBLs) in 28 isolates. Because of their different susceptibility profiles,
V. cholerae
isolates M1502, M1516, M1573, and M3030 (all of which are of the Ogawa serotype) were selected for the present study. By susceptibility analysis, isoelectric focusing, and PCR-based restriction fragment length polymorphism analysis, CTX-M-type enzymes were identified in three isolates, whereas a PER-2-type enzyme, in addition to a TEM-1-like enzyme, was identified in the other isolate. The presence of these ESBLs in
V. cholerae
isolates resulted in MICs well below those commonly observed for members of the family
Enterobacteriaceae
. Genes that encode both ESBLs were transferred to
Escherichia coli
by conjugation, together with all determinants of resistance to non-β-lactam antibiotics (gentamicin, kanamycin, and sulfamethoxazole for all isolates; amikacin and streptomycin for three isolates; trimethoprim, tetracycline, and chloramphenicol for two isolates). Plasmid profile analysis and Southern blotting revealed the presence of single plasmids of about 150 kb in the four
V. cholerae
isolates and their respective transconjugants and revealed that the plasmids harbored genes encoding CTX-M-type or PER-2-type ESBLs. These results strongly suggest the broad spread of these ESBLs among genera belong to families other than the
Enterobacteriaceae
.
Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria
Authors: Kuai Linping , Nair Arjun A. , Polz Martin F.
Published: July 2002

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10
1
and 10
5
cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.
Secretion of RTX Leukotoxin by
Actinobacillus actinomycetemcomitans
Authors: Kachlany Scott C. , Fine Daniel H. , Figurski David H.
Published: July 2002

Actinobacillus actinomycetemcomitans
, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of
A. actinomycetemcomitans
is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of
A. actinomycetemcomitans
. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of
A. actinomycetemcomitans
, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the
tad
genes, which are required for tight nonspecific adherence of
A. actinomycetemcomitans
to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that
A. actinomycetemcomitans
has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of
A. actinomycetemcomitans
.
The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and Enterohemorrhagic
Escherichia coli
Authors: Elliott Simon J. , Sperandio Vanessa , Girón Jorge A. , Shin Sooan , Mellies Jay L. , Wainwright Leslie , Hutcheson Steven W. , McDaniel Timothy K. , Kaper James B.
Published: July 2002

Regulation of virulence gene expression in enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the
LEE2
,
LEE3
, and
LEE4
promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including
rorf2
,
orf10
,
rorf10
,
orf19
, and
espF
, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. Δ
ler
mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.
Genetic Dissection of Primary and Secondary Responses to a Widespread Natural Pathogen of the Gut,Eimeria vermiformis
Authors: Smith Adrian L. , Hayday Adrian C.
Published: July 2002

Because most pathogens initially challenge the body at epithelial surfaces, it is important to dissect the mechanisms that underlie T-cell responses to infected epithelial cells in vivo. The coccidian parasites of the genusEimeriaare protozoan gut pathogens that elicit a potent, protective immune response in a wide range of host species. CD4+ αβ T cells and gamma interferon (IFN-γ) are centrally implicated in the primary immunoprotective response. To define any additional requirements for the primary response and to develop a comparison between the primary and the secondary response, we have studiedEimeriainfections of a broad range of genetically altered mice. We find that a full-strength primary response depends on β2-microglobulin (class I major histocompatibility complex [MHC] and class II MHC and on IFN-γ and interleukin-6 (IL-6) but not on TAP1, perforin, IL-4, Fas ligand, or inducible nitric oxide synthetase. Indeed, MHC class II-deficient and IFN-γ-deficient mice are as susceptible to primary infection as mice deficient in all αβ T cells. Strikingly, the requirements for a highly effective αβ-T-cell-driven memory response are less stringent, requiring neither IFN-γ nor IL-6 nor class I MHC. The class II MHC dependence was also reduced, with adoptively transferable immunity developing in MHC class II−/−mice. Besides the improved depiction of an immune response to a natural gut pathogen, the finding that effective memory can be elicited in the absence of primary effector responses appears to create latitude in the design of vaccine strategies.
Necrosis of Lung Epithelial Cells during Infection withMycobacterium tuberculosisIs Preceded by Cell Permeation
Authors: Dobos Karen M. , Spotts Ellen A. , Quinn Frederick D. , King C. Harold
Published: July 2002

Mycobacterium tuberculosisestablishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells withM. tuberculosisstrains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with eitherMycobacterium bovisBCG orMycobacterium smegmatisLR222 did not. Clearing ofM. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment ofM. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated thatM. tuberculosisinduced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific toM. tuberculosisinfection and correlated to A549 cellular necrosis. InactivatedM. tuberculosisor its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.
Role of Streptolysin O in a Mouse Model of Invasive Group A Streptococcal Disease
Authors: Limbago Brandi , Penumalli Vikram , Weinrick Brian , Scott June R.
Published: July 2002

Many of the virulence factors that have been characterized for group A streptococci (GAS) are not expressed in all clinical isolates. One putative virulence factor that is present among most is streptolysin O (Slo), a protein with well-characterized cytolytic activity for many eukaryotic cells types. In other bacterial pathogens, proteins homologous to Slo have been shown to be essential for virulence, but the role of Slo in GAS had not been previously examined. To investigate the role of Slo in GAS virulence, we examined both revertible and stableslomutants in a mouse model of invasive disease. When the revertibleslomutant was used to infect mice, the reversion frequency of bacteria isolated from the wounds and spleens of infected animals was more than 100 times that of the inoculum, indicating that there was selective pressure in the animal for Slo+GAS. Experiments with the stableslomutant demonstrated that Slo was not necessary for the formation of necrotic lesions, nor was it necessary for escape from the lesion to cause disseminated infection. Bacteria were present in the spleens of 50% of the mice that survived infection with the stableslomutant, indicating that dissemination of Slo−GAS does not always cause disease. Finally, mice infected with the stableslomutant exhibited a significant decrease in mortality rates compared to mice infected with wild-type GAS (P< 0.05), indicating that Slo plays an important role in GAS virulence.
Construction of a
Vibrio cholerae
Vaccine Candidate Using Transposon Delivery and FLP Recombinase-Mediated Excision
Authors: Chiang Su L. , Mekalanos John J.
Published: July 2002

Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism,
Vibrio cholerae
. The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients. We therefore converted a motile Inaba
+
vaccine candidate, Peru-2, to a nonmotile Ogawa
+
phenotype using a
mariner
-based transposon carrying
rfbT
, the gene required for expression of the Ogawa phenotype. Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa
+
, and both of these strains had insertions in the
flgE
gene. It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon. The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes.
Involvement of Focal Adhesion Kinase in
Escherichia coli
Invasion of Human Brain Microvascular Endothelial Cells
Authors: Reddy Marpadga A. , Wass Carol A. , Kim Kwang Sik , Schlaepfer David D. , Prasadarao Nemani V.
Published: July 2002

Escherichia coli
K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis. We have previously shown that invasive
E. coli
promotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion. However, signal transduction mechanisms involved in
E. coli
invasion are not defined. In this report we show that tyrosine kinases play a major role in
E. coli
invasion of human BMEC (HBMEC).
E. coli
induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK. Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited
E. coli
invasion of HBMEC. Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in
E. coli
invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required. Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry. Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site. The overexpression of FAK mutants in HBMEC also blocked the
E. coli
-induced tyrosine phosphorylation of FAK and its association with paxillin. These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in
E. coli
invasion of HBMEC.
Contribution of Plasmid-Encoded Fimbriae and Intimin to Capacity of Rabbit-Specific Enteropathogenic
Escherichia coli
To Attach to and Colonize Rabbit Intestine
Authors: Krejany Efrosinia O. , Grant Travis H. , Bennett-Wood Vicki , Adams Louise M. , Robins-Browne Roy M.
Published: July 2002

Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic
Escherichia coli
(EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal
eae
gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H−), in which the
ralE
and
eae
genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a Δ
eae
Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its
eae
-positive parent and far more extensively than an

eae
+
Δral

strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a Δ
eae ΔralE
double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin.
The Nonfunctional Allele TCRBV6S1B Is Strongly Associated withHelicobacter pyloriInfection
Authors: Kunstmann Erdmute , Hardt Cornelia , Elitok Ercan , Harder Marianne , Suerbaum Sebastian , Peitz Ulrich , Schmiegel Wolff , Epplen Jörg T.
Published: July 2002

To determine genetic susceptibility factors forHelicobacter pyloriinfection, polymorphic T-cell receptor gene elements were investigated in 203H. pylori-infected individuals and 180 uninfected individuals (controls).H. pyloriinfection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; χ2= 13;P= 0.00032;Pvalue corrected for multiple comparisons [Bonferroni correction] = 0.00063).
Characterization of the Domain of Fibronectin-Binding Protein I ofStreptococcus pyogenesResponsible for Elicitation of a Protective Immune Response
Authors: Schulze Kai , Medina Eva , Talay Susanne R. , Towers Rebecca J. , Chhatwal Gursharan S. , Guzmán Carlos A.
Published: July 2002

Fibronectin-binding protein I (SfbI) represents a major adhesin ofStreptococcus pyogenes. Mice were intranasally immunized with recombinant proteins spanning different portions of SfbI to identify the minimal fragment able to elicit a protective response against a lethal challenge withS. pyogenes. The strongest cellular responses and the highest levels of antigen-specific secretory immunoglobulin A (IgA) were detected in mice immunized with the fibronectin-binding region of SfbI. In contrast, animals vaccinated with a polypeptide spanning the aromatic and proline-rich regions showed the highest titers and fastest IgG response in serum. Vaccination with either SfbI without a membrane anchor and signal peptide or a polypeptide encompassing its fibronectin-binding regions resulted in efficient protection against heterologous challenge (60% and 80%, respectively), whereas the use of a polypeptide lacking this region conferred marginal protection (10%) with respect to the control group (0%). These results demonstrate that the fibronectin-binding region of SfbI is a promising candidate antigen for developing anti-S. pyogenesvaccines.
Cyclic Guanosine-3′,5′-Monophosphate and Biopteridine Biosynthesis in
Nocardia
sp
Authors: Son Jong-Keun , Rosazza John P. N.
Published: July 2002

Nocardia
sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H
4
B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3′,5′-monophosphate (cGMP) at 9.56 pmol of cGMP h
−1
mg of protein
−1
. Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter
−1
to a maximum of 315 pmol liter
−1
, in response to additions of GTP,
l
-arginine, H
4
B, and sodium nitroprusside to growing
Nocardia
cultures. On the other hand, the NOS inhibitor
N
G
-nitro-
l
-arginine and the GC inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H
4
B biosynthesis, were present in
Nocardia
culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h
−1
mg of protein
−1
, respectively. In
Nocardia
spp., as in mammals, GTP is a key intermediate in H
4
B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.
A Genetic Mechanism for Deletion of the
ser2
Gene Cluster and Formation of Rough Morphological Variants of
Mycobacterium avium
Authors: Eckstein Torsten M. , Inamine Julia M. , Lambert Markus L. , Belisle John T.
Published: July 2002

A major phenotypic trait of the
Mycobacterium avium
complex is the ability to produce rough and smooth colony variants. The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants. Using
M. avium
serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis. One such deletion encompasses the gene cluster (
ser2
) responsible for production of the serovar 2 GPL haptenic oligosaccharide. In this study, nucleotide sequencing revealed that both ends of the
ser2
gene cluster are flanked by a novel insertion sequence (IS
1601
) oriented as direct repeats. Detailed analyses of the site of deletion in the genome of
M. avium
2151 Rg-1 demonstrated that a single copy of IS
1601
remained and that the
ser2
gene cluster was deleted by homologous recombination. This same deletion pattern was observed for 10 out of 15 rough colony variants tested. Additionally, these studies revealed that IS
1601
contains portions of three independent insertion sequences. This report is the first to define the precise genetic basis of colony variation in
Mycobacterium
spp. and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.