bluuebunny/crossref_metadata_2025 huggingface dataset:
Monolayer-level controlled incorporation of nitrogen in ultrathin gate dielectrics using remote plasma processing: Formation of stacked “N–O–N” gate dielectrics
Authors: Niimi H. , Lucovsky G.
Published: July 2002

A low thermal budget approach to monolayer-level controlled incorporation of nitrogen in ultrathin gate dielectrics using 300 °C, remote plasma processing is discussed. Incorporation of approximately 1 ML of nitrogen at the Si–SiO2 interface in an “N–O” structure has been achieved by remote plasma-assisted oxidation of the Si surface followed by N2/He remote plasma nitridation, each at a process pressure of 0.3 Torr. The interface nitridation reduces direct and Fowler–Nordheim tunneling by at least one order of magnitude, independent of film thickness. Incorporation of nitrogen at the top surface of the oxide in a concentration equivalent to about 1–2 molecular layers of silicon nitride in an “O–N” structure has been accomplished by N2/He remote plasma nitridation at 300 °C, but at a reduced process pressure of 0.1 Torr. Top surface nitridation has been shown to prevent boron diffusion out of p+ poly-Si gate electrodes during high-temperature activation anneals, e.g., at 1000 °C. Combining interfacial and top surface nitridation processes resulted in a “N–O–N” structure that was effective in reducing tunneling leakage currents and suppressing boron out-diffusion from p+ poly-Si gate electrodes.
Sub-100 nm metrology using interferometrically produced fiducials
Authors: Schattenburg M. L. , Chen C. , Everett P. N. , Ferrera J. , Konkola P. , Smith Henry I.
Published: July 2002

Pattern-placement metrology plays a critical role in nanofabrication. Not far in the future, metrology standards approaching 0.2 nm in accuracy will be required to facilitate the production of 25 nm semiconductor devices. They will also be needed to support the manufacturing of high-density wavelength-division-multiplexed integrated optoelectronic devices. We are developing a new approach to metrology in the sub-100 nm domain that is based on using phase-coherent fiducial gratings and grids patterned by interference lithography. This approach is complementary to the traditional mark-detection, or “market plot” pattern-placement metrology. In this article we explore the limitations of laser-interferometer-based mark-detection metrology, and contrast this with ways that fiducial grids could be used to solve a variety of metrology problems. These include measuring process-induced distortions in substrates; measuring patterning distortions in pattern-mastering systems, such as laser and e-beam writers; and measuring field distortions and alignment errors in steppers and scanners. We describe a proposed standard for pattern-placement metrology, which includes both a fiducial grid and market-type marks. Finally, a number of methods through which phase-coherent periodic structures can be patterned are shown, including “traditional” interference lithography, achromatic interference lithography, near-field interference lithography, and scanning-beam interference lithography.
High resolution sampling electrostatic force microscopy using pulse width modulation technique
Authors: Said R. A. , Cheung S. P. , Bridges G. E.
Published: July 2002

This article presents a sampling electrostatic force microscopy technique for high frequency integrated circuit internal measurement that utilizes a pulse width modulation method. The electrostatic force microscope is a class of scanning probe microscopes that is capable of providing noninvasive measurements of the electrical signals inside operating integrated circuits with very high spatial resolution. With this technique, the internal circuit voltage is determined by sensing the local electrostatic force on a miniature probe cantilever that is closely positioned above the circuit test point. The ability to extract repetitive voltage wave forms using direct force measurement is typically limited by the mechanical response of the probe. To overcome the measurement bandwidth limitation of the electrostatic force microscope previous methods have employed a modulated high bandwidth sampling pulse approach. The temporal resolution of these pulse sampling methods is limited by the pulse width. This article presents a pulse width modulation method. The resolution of the pulse width modulation technique is not dependent on the pulse width, but rather on the modulation depth. This enhances measurement resolution several times more than previous methods. The current instrument has a voltage sensitivity of 10 mVrms/Hz1/2.
Role of the substrate in the C49–C54 transformation of TiSi2
Authors: La Via F. , Raineri V. , Grimaldi M. G. , Miglio Leo , Iannuzzi M. , Marabelli F. , Bocelli S. , Santucci S. , Phani A. R.
Published: July 2002

In this work we report the results of the crystallographic and morphological characterization of TiSi2 films grown on a patterned and on a blanket Si substrate. We show that the C49 films grown on crystalline silicon exhibit a rougher surface with respect to that grown on polycrystalline substrate. The different surface morphology is maintained after transformation to the C54 phase. This latter is always (040) textured in the case of patterned films, whereas in blanket films texturing occurs only on a polysilicon substrate. The C49 phase displays (200) texturing when the film is grown on single crystalline Si(100), while random orientation of the grains is detected in the other cases. The experimental indications are associated with semiempirical total energy estimations of the surface energies for the two competing phases. From this comparison it turns out that, in the case of texturing, the preferential surfaces facing the substrate are the most energetic.
Field-emission triodes with integrated anodes
Authors: Garner D. M. , Long G. M. , Herbison D. , Amaratunga G. A. J.
Published: July 2002

A silicon microtriode where the gate and anode are fully integrated along with the cathode tip on the silicon substrate is proposed. The device can operate in two modes: a normally on mode and a normally off mode. In the normally on mode, reported here, the anode is a suspended bimetallic layer of chromium and gold above a sharp silicon tip, formed by the anisotropic etching of silicon in potassium hydroxide solution. The gate, in the same plane as the anode, is formed of the same bimetallic layer and approaches from the sides. The process requires only one mask and is extremely simple. Field emission was found between the anode and the cathode tip and, in three-terminal mode, the device was found to have gate control. The extrapolated gate pinch-off voltages for the two devices measured were −230 and −162 V at an anode voltage of 120 V.
Modification of electron field emission properties from surface treated amorphous carbon thin films
Authors: Carey J. D. , Poa C. H. , Forrest R. D. , Burden A. P. , Silva S. R. P.
Published: July 2002

The field emission properties of amorphous hydrogenated carbon (a-C:H) films and nitrogenated a-C:H (a-C:H:N) films subjected to 10 keV Co60 ion implantation are investigated as a function of ion dose. The average threshold electric field for conditioned nitrogenated and non-nitrogenated unimplanted films is found to be 27 and 29 V/μm. Implantation of C60 ions to a dose of 7.5×1013 cm−2 results in an increase in the threshold field of both the nitrogenated and non-nitrogenated films. However, implantation to a dose of 1.25×1014 cm−2 results in a reduction in the threshold fields to values close to those found in the unimplanted samples. At larger doses of 2.65×1014 cm−2, the threshold field for the non-nitrogenated samples remain largely unchanged though there is an increase in the refractive index consistent with an increase in the optical density of the film. In the nitrogenated film, the average threshold field at the highest dose again increases to values comparable to those found for C60 implantation into virgin single crystal Si. The microstructural changes that take place during implantation and the role of sp2 rich conductive layers are discussed.
Field emission from 4.5 in. single-walled and multiwalled carbon nanotube films
Authors: Chung D. S. , Choi W. B. , Kang J. H. , Kim H. Y. , Han I. T. , Park Y. S. , Lee Y. H. , Lee N. S. , Jung J. E. , Kim J. M.
Published: July 2002

Field emission properties of 4.5 in. flat panel displays in a diode type panel using single-walled (SWNTs) and multiwalled carbon nanotube tips (MWNTs) were characterized and compared. The panel, fabricated by a slurry squeezing and surface rubbing technique, enables the generation of more emission sites by removing materials on the surface. The turn-on field of MWNTs decreased from 6.4 to 3 V/μm by treatment of the surface, and that of SWNTs also decreased, from 4.5 to 2 V/μm. The density of aligned MWNTs is approximately 2/μm2, whereas the aligned SWNTs were uniformly distributed, with densities of 5–10/μm2. As a result, SWNT films show higher emission uniformity than MWNT films. A gradual degradation over time was observed in both MWNTs and SWNTs. The current stability curve of the SWNTs decreased about 20%, while that of the MWNTs decreased less than 10%.
Crustal Deformation from 1992 to 1995 at the Mid-Atlantic Ridge, Southwest Iceland, Mapped by Satellite Radar Interferometry
Authors: Vadon Hélène , Sigmundsson Freysteinn
Published: July 2002

Satellite radar interferometry observations of the Reykjanes Peninsula oblique rift in southwest Iceland show that the Reykjanes central volcano subsided at an average rate of up to 13 millimeters per year from 1992 to 1995 in response to use of its geothermal field. Interferograms spanning up to 3.12 years also include signatures of plate spreading and indicate that the plate boundary is locked at a depth of about 5 kilometers. Below that depth, the plate movements are accommodated by continuous ductile deformation, which is not fully balanced by inflow of magma from depth, causing subsidence of the plate boundary of about 6.5 millimeters per year.
The Activity and Size of the Nucleus of Comet Hale-Bopp (C/1995 O1)
Authors: Weaver H. A. , Feldman P. D. , A'Hearn M. F. , Arpigny C. , Brandt J. C. , Festou M. C. , Haken M. , McPhate J. B. , Stern S. A. , Tozzi G. P.
Published: July 2002

Analysis of Hubble Space Telescope (HST) images of comet Hale-Bopp (C/1995 O1) suggests that the effective diameter of the nucleus is between 27 to 42 kilometers, which is at least three times larger than that of comet P/Halley. The International Ultraviolet Explorer and HST spectra showed emissions from OH (a tracer of H
2
O) and CS (a tracer of CS
2
) starting in April 1996, and from the CO Cameron system (which primarily traces CO
2
) starting in June 1996. The variation of the H
2
O production rate with heliocentric distance was consistent with sublimation of an icy body near its subsolar point. The heliocentric variation in the production rates of CS
2
and dust was different from that of H
2
O, which implies that H
2
O sublimation did not control the CS
2
or dust production during these observations.
Optical Observations of Comet Hale-Bopp (C/1995 O1) at Large Heliocentric Distances Before Perihelion
Authors: Rauer Heike , Arpigny Claude , Boehnhardt Hermann , Colas François , Crovisier Jacques , Jorda Laurent , Küppers Michael , Manfroid Jean , Rembor Kai , Thomas Nicolas
Published: July 2002

The activity of comet Hale-Bopp (C/1995 O1) was monitored monthly by optical imaging and long-slit spectroscopy of its dust and gas distribution over heliocentric distances of 4.6 to 2.9 astronomical units. The observed band intensities of the NH
2
radical and the H
2
O
+
ion cannot be explained by existing models of fluorescence excitation, warranting a reexamination of the corresponding production rates, at least at large heliocentric distances. Comparing the production rate of the CN radical to its proposed parent, HCN, shows no evidence for the need of a major additional source for CN in Hale-Bopp at large heliocentric distances. The dust and CN production rates are consistent with a significant amount of sublimation occurring from icy dust grains surrounding Hale-Bopp.
Type II Supernovae SN 1987A and SN 1993J
Authors: Chevalier Roger A.
Published: July 2002

The optical and ultraviolet emission from the type II supernova SN 1987A is currently powered by the radioactive decay of titanium-44. In contrast, the emission from SN 1993J is dominated by radiation from the interaction of the supernova shock wave with a dense, stellar wind released before the explosion. The dense wind around SN 1987A was displaced by the fast wind from the compact progenitor star, and the interaction of the supernova with dense gas is now being detected at x-ray and radio wavelengths. The expected neutron stars or black holes at the centers of these supernovae are not yet significant power sources.
Measurements of
12
C/
13
C,
14
N/
15
N, and
32
S/
34
S Ratios in Comet Hale-Bopp (C/1995 O1)
Authors: Jewitt David C. , Matthews Henry E. , Owen Tobias , Meier Roland
Published: July 2002

The
12
C/
13
C,
14
N/
15
N, and
32
S/
34
S isotope ratios in comet Hale-Bopp (C/1995 O1) were determined through observations taken with the James Clerk Maxwell Telescope. Measurements of rare isotopes in HCN and CS revealed isotope ratios of H
12
CN/H
13
CN = 111 ± 12, HC
14
N/HC
15
N = 323 ± 46, and C
32
S/C
34
S = 27 ± 3. Within the measurement uncertainties, the isotopic ratios are consistent with solar system values. The cometary volatiles thus have an origin in the solar system and show no evidence for an interstellar component.
Seismic Evidence for an Inner Core Transition Zone
Authors: Song Xiaodong , Helmberger Don V.
Published: July 2002

Seismic waves that traverse Earth's inner core along north-south paths produce unusually broad pulse shapes at long periods (compared with waves along east-west paths) and reflections from below the inner core boundary at short periods. The observations provide compelling evidence for a seismic velocity discontinuity along north-south paths about 200 kilometers below the inner core boundary separating an isotropic upper inner core from an anisotropic lower inner core. The triplication associated with such a structure might be responsible for reported waveform complexity of short-period inner core arrivals along north-south paths and, if the depth of the boundary is laterally variable, their large travel-time variation.
Spontaneous Bubble Domain Formation in a Layered Ferromagnetic Crystal
Authors: Fukumura T. , Sugawara H. , Hasegawa T. , Tanaka K. , Sakaki H. , Kimura T. , Tokura Y.
Published: July 2002

Magnetic domain structure on the surface of the layer-structured ferromagnet La
1.4
Sr
1.6
Mn
2
O
7
was observed in the temperature range from 37 to 97 kelvin with a scanning Hall probe microscope. The sensitivity to temperature of the domain structure changes was large relative to that in conventional ferromagnets. The stable and spontaneous appearance of magnetic bubble domains without an external magnetic field was observed in the neighborhood of 70 kelvin. The phenomenon observed could provide a potential route toward magnetic bubble memory.
Mechanisms of Artemisinin Resistance in the Rodent Malaria Pathogen
Plasmodium yoelii
Authors: Walker Daniel J. , Pitsch Jessica L. , Peng Michael M. , Robinson Brian L. , Peters Wallace , Bhisutthibhan Jamaree , Meshnick Steven R.
Published: July 2002

Artemisinin and its derivatives are important new antimalarials which are now used widely in Southeast Asia. Clinically relevant artemisinin resistance has not yet been reported but is likely to occur. In order to understand how the malaria parasite might become resistant to this drug, we studied artemisinin resistance in the murine malaria parasite
Plasmodium yoelii
. The artemisinin-resistant strain (ART), which is approximately fourfold less sensitive to artemisinin than the sensitive NS strain, accumulated 43% less radiolabeled drug in vitro (
P
< 0.01). Within the parasite, the drug appeared to react with the same parasite proteins in both strains. The translationally controlled tumor protein, one of the artemisinin target proteins, did not differ between the strains. No DNA sequence difference was found, but the resistant strain was found to express 2.5-fold-more protein than the sensitive strain (
P
< 0.01). Thus, the phenotype of artemisinin resistance in
P. yoelii
appears to be multifactorial.
Tolerance and Pharmacokinetic Interactions of Rifabutin and Azithromycin
Authors: Hafner Richard , Bethel James , Standiford Harold C. , Follansbee Stephen , Cohn David L. , Polk Ronald E. , Mole Larry , Raasch Ralph , Kumar Princy , Mushatt David , Drusano George
Published: July 2002

This multicenter study evaluated the tolerance and potential pharmacokinetic interactions between azithromycin and rifabutin in volunteers with or without human immunodeficiency virus infection. Daily dosing with the combination of azithromycin and rifabutin was poorly tolerated, primarily because of gastrointestinal symptoms and neutropenia. No significant pharmacokinetic interactions were found between these drugs.
Correlation between Antifungal Susceptibilities of
Coccidioides immitis
In Vitro and Antifungal Treatment with Caspofungin in a Mouse Model
Authors: González Gloria M. , Tijerina Rolando , Najvar Laura K. , Bocanegra Rosie , Luther Michael , Rinaldi Michael G. , Graybill John R.
Published: July 2002

Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of
Coccidoides immitis.
In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two
C. immitis
isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 μg/ml; 48-h MEC, 0.125 μg/ml) showed 100% survival to day 50 when treated with caspofungin at ≥1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 μg/ml; 48-h MEC, 0.125 μg/ml) displayed ≥80% survival when the treatment was caspofungin at ≥5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.
Isolation and Characterization of a New Denitrifying Spirillum Capable of Anaerobic Degradation of Phenol
Authors: Shinoda Yoshifumi , Sakai Yasuyoshi , Ué Makiko , Hiraishi Akira , Kato Nobuo
Published: July 2002

Two kinds of phenol-degrading denitrifying bacteria,
Azoarcus
sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a
Magnetospirillum
sp., a member of the α subclass of the class
Proteobacteria
, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described
Magnetospirillum
strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.
Secretion of Recombinant Proteins via the Chaperone/Usher Pathway in
Escherichia coli
Authors: Zavialov Anton V. , Batchikova Natalia V. , Korpela Timo , Petrovskaya Lada E. , Korobko Vyacheslav G. , Kersley Joanne , MacIntyre Sheila , Zav'yalov Vladimir P.
Published: July 2002

F1 antigen (Caf1) of
Yersinia pestis
is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in
Escherichia coli
. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1β (hIL-1β), and mature Caf1, the processed product (hIL-1β:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1β:Caf1 in the periplasm. Soluble hIL-1β:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1β. The results indicate that Caf1M-induced release of hIL-1β:Caf1 from the inner membrane promotes folding of the hIL-1β domain. Similar results were obtained with the fusion of Caf1 to hIL-1β receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1β:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1β:Caf1 could be detected on the cell surface of
E. coli
. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.
Cloning and Characterization of
linR
, Involved in Regulation of the Downstream Pathway for γ-Hexachlorocyclohexane Degradation in
Sphingomonas paucimobilis
UT26
Authors: Miyauchi Keisuke , Lee Haeng-Seog , Fukuda Masao , Takagi Masamichi , Nagata Yuji
Published: July 2002

In
Sphingomonas paucimobilis
UT26, LinD and LinE activities, which are responsible for the degradation of γ-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the
linE
gene was determined, and an open reading frame (ORF) was found in divergent orientation from
linE
. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it
linR
. The fragment containing the putative LTTR recognition sequence (a palindromic TN
11
A sequence), which exists immediately upstream of
linE
, was ligated with the reporter gene
lacZ
and was inserted into the plasmid expressing LinR under the control of the
lac
promoter. When the resultant plasmid was introduced into
Escherichia coli
, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in
linR
, revealed that the expression of the
linD
and
linE
genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the
linR
gene is directly involved in the inducible expression of the
linD
and
linE
genes.
Influence of Host Factors on Immunoglobulin G Concentration in Oral Fluid Specimens
Authors: Granade Timothy C. , Phillips Susan K. , Kitson-Piggott Wendy , Gomez Perry , Mahabir Bisram , Oleander Herbert , George J. Richard , Baggs James , Parekh Bharat
Published: July 2002

The influence of host factors (tobacco use, dentition, bleeding gums, oral rinsing, nasal medications, and time since the last meal) on immunoglobulin G (IgG) concentration in oral fluids (OF) was determined by univariate and multivariate analysis. Significant differences in IgG concentration were found to be associated with human immunodeficiency virus (HIV) status (HIV antibody positive, +16.60 μg/ml,
P
= 0.0001), sex (female, +1.23 μg/ml,
P
= 0.004), dentition (+2.83 μg/ml, edentulous versus dentulous,
P
= 0.0001), bleeding gums (+6.35 μg/ml,
P
= 0.0001), and time since the last meal (+3.55 μg/ml, >6 h,
P
= 0.0001). These factors could impact diagnostic methods that rely on the immunoglobulin concentration in OF specimens.
Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis
Authors: Berner Reinhard , Fürll Birgitt , Stelter Felix , Dröse Jana , Müller Hans-Peter , Schütt Christine
Published: July 2002

No data on lipopolysaccharide-binding protein (LBP) in newborns with sepsis have been available up to now. We therefore determined levels of LBP and soluble CD14 (sCD14) in plasma of healthy and septic neonates in order to evaluate their potential diagnostic role. The study included prospectively collected patient samples of two recently published studies on cytokine expression in neonatal sepsis. Twenty-nine septic patients were enrolled in the present analysis. Samples—either cord blood or peripheral blood—from patients admitted within the first 24 h of life for suspicion of sepsis and cord blood samples of a control group of 40 healthy mature infants delivered spontaneously were analyzed. For seven patients of the septic group, a second sample collected between 24 and 48 h of life was available. Levels of sCD14 and LBP in plasma were determined by an enzyme immunoassay using recombinant CD14 and LBP as standards. LBP and sCD14 were correlated to cytokine plasma levels. In septic neonates, LBP (median, 36.6 versus 7.8 μg/ml;P< 0.001) and sCD14 (median, 0.42 versus 0.28 μg/ml;P< 0.001) levels were highly elevated when compared to those of healthy neonates and strongly correlated to granulocyte colony-stimulating factor (G-CSF), interleukin-1β (IL-1β), IL-6, and IL-8 levels. LBP levels in septic neonates analyzed between 24 and 48 h of life even increased when compared to samples obtained at or shortly after delivery (median, 36.6 versus 60 μg/ml;P= 0.038). In summary, levels of LBP in plasma of neonates with early-onset sepsis are significantly elevated; the elevated plasma levels seem to persist for more than 24 h, which could provide the clinician with a prolonged time period to identify the newborn with bacterial sepsis.
Metalloprotease Is Not Essential for
Vibrio vulnificus
Virulence in Mice
Authors: Shao Chung-Ping , Hor Lien-I
Published: July 2002

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of
Vibrio vulnificus
, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of
Vibrio vulnificus
by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.
Temperature-Regulated Protein Synthesis by
Leptospira interrogans
Authors: Nally Jarlath E. , Timoney John F. , Stevenson Brian
Published: July 2002

Leptospira interrogans
is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in
L. interrogans
. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with
L. interrogans
serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.
Cellular and Humoral Immune Responses toBorrelia burgdorferiAntigens in Patients with Culture-Positive Early Lyme Disease
Authors: Vaz Austin , Glickstein Lisa , Field Jodie A. , McHugh Gail , Sikand Vijay K. , Damle Nitin , Steere Allen C.
Published: July 2002

We determined cellular and humoral immune responses toBorrelia burgdorferilysate and to recombinant flagellin (FlaB), OspC, and OspA in acute- and convalescent-phase samples from 39 culture-positive patients with erythema migrans and in 20 healthy control subjects. During the acute illness, a median of 4 days after the onset of erythema migrans, 51% of the patients had proliferative cellular responses and 72% had antibody responses to at least one of the borrelial antigens tested. During convalescence, at the conclusion of antibiotic therapy, 64% of the patients had proliferative cellular reactivity and 95% had antibody reactivity with at least one of the spirochetal antigens tested. In both acute- and convalescent-phase samples, cellular immune responses were found as frequently to OspA as to OspC and FlaB. Although antibody responses were also frequently seen to OspC and FlaB, only a few patients had marginal antibody reactivity with OspA. The percentage of patients with proliferative responses was similar in those with clinical evidence of localized or disseminated infection, whereas humoral reactivity was found more often in those with disseminated disease. We conclude that cellular and humoral responses toB. burgdorferiantigens are often found among patients with early Lyme disease. In contrast with the other antigens tested, cellular but not humoral reactivity was often found with OspA.
Counter-Protective Role for Interleukin-5 during AcuteToxoplasma gondiiInfection
Authors: Nickdel M. B. , Roberts F. , Brombacher F. , Alexander J. , Roberts C. W.
Published: July 2002

The role of interleukin-5 (IL-5) duringToxoplasma gondiiinfection was investigated by comparing disease progression in IL-5 gene deficient (IL-5−/−) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5−/− mice infected orally withT. gondiiwere less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5−/− mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5−/− mice produced levels of IL-12, interferon-γ (IFN-γ), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated withToxoplasmalysate antigen. However, peak serum IL-12 and IFN-γ levels (at days 6 and 8, respectively) were significantly higher in IL-5−/− mice than in WT mice. In addition, WT mice but not IL-5−/− mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration ofNω-nitro-l-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5−/− and WT mice, indicating a protective role for nitric oxide during the early stages of oralT. gondiiinfection. In comparison with oral infection, no difference in mortality was observed between IL-5−/− and WT mice following intraperitoneal infection withT. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection withT. gondiiwhich is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-γ levels.
Prolonged Survival and Cytoplasmic pH Homeostasis of
Helicobacter pylori
at pH 1
Authors: Stingl Kerstin , Uhlemann Eva-Maria , Deckers-Hebestreit Gabriele , Schmid Roland , Bakker Evert P. , Altendorf Karlheinz
Published: July 2002

In the presence of urea,
Helicobacter pylori
survived for at least 3 h at pH 1. Under these conditions, the cells maintained their cytoplasmic pH at 5.8. De novo protein synthesis during acid shock was not essential for survival of
H. pylori
at pH 1.
Complete Nucleotide Sequence and Analysis of the Locus of Enterocyte Effacement from Rabbit Diarrheagenic
Escherichia coli
RDEC-1
Authors: Zhu Chengru , Agin Tonia S. , Elliott Simon J. , Johnson Laura A. , Thate Timothy E. , Kaper James B. , Boedeker Edgar C.
Published: July 2002

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic
Escherichia coli
strain RDEC-1 (O15:H−) and compared it with those from human enteropathogenic
E. coli
(EPEC, O127:H6) and enterohemorrhagic
E. coli
(EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at
selC
and is flanked by an IS2 element and the
lifA
toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC.
orf3
and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of
LEE1, LEE2, LEE3, tir
, and
LEE4
operons are highly conserved among the LEEs, although the upstream regions varied considerably for
tir
and the crucial
LEE1
promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.
Identification of Continuous B-Cell Epitopes on the Protein Moiety of the 58-Kilodalton Cell Wall Mannoprotein ofCandida albicansBelonging to a Family of Immunodominant Fungal Antigens
Authors: Viudes Angel , Perea Sofia , Lopez-Ribot Jose L.
Published: July 2002

The 58-kiloDalton mannoprotein (mp58) on the surface ofCandida albicansis highly immunogenic, is expressed by allC. albicansisolates tested, and elicits strong antibody responses during candidiasis. It belongs to a family of immunodominant fungal antigens with representatives also in different species ofAspergillus. The amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) was used to synthesize a complete set of overlapping dodecapeptides (overlap, 7; offset, 5) covalently attached to the surface of derivatized polyethylene pins. The pin-coupled peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by a number of antiserum preparations containing anti-mp58 antibodies. This comprehensive epitope-scanning study revealed the presence of multiple immunoreactive continuous B-cell epitopes within the protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein (encompassing amino acid residues 16 to 50 and 286 to 299, respectively) and four internal regions spanning amino acids at positions 66 to 92, 121 to 142, 148 to 192, and 211 to 232. Further delineation of epitopic regions and identification of the boundaries of the antigenic sites was performed upon ELISA testing with a second Pepset consisting of completely overlapping 8-mer peptides spanning these reactive regions in the protein moiety of mp58. The highly reactive epitopic region at the C terminus of the protein was further evaluated using both window net and replacement net analyses. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein was immunogenic when injected into mice after being coupled to a carrier protein. Moreover, antibodies in the resulting sera specifically recognized the homologus mp58 in ELISAs and immunoblot assays. Delineation of the antibody responses to mp58 could provide the basis for the development of novel immunity-based prophylactic, therapeutic, and diagnostic techniques for the management of candidiasis.
Fas-Mediated Apoptosis of Neutrophils in Sera of Patients with Infection
Authors: Nwakoby Izuchukwu E. , Reddy Krishna , Patel Puja , Shah Neena , Sharma Saroj , Bhaskaran Madhu , Gibbons Nora , Kapasi Aditi A. , Singhal Pravin C.
Published: July 2002

In the presence of infection, neutropenia is considered to be a marker of poor prognosis; conversely, neutrophilia may not be a determinant of a better prognosis. Since apoptotic neutrophils are compromised functionally, we evaluated the effect of infection on neutrophil apoptosis. The rate of apoptosis was greater for neutrophils isolated from patients with infection than for healthy controls.Escherichia colidid not directly modulate the rate of neutrophil apoptosis. However, sera from infected patients promoted (P< 0.001) neutrophil apoptosis. Interestingly, the sera of patients with different types of infection (gram negative, gram positive, or culture negative) exerted a more or less identical response on neutrophil apoptosis. Sera of infected patients showed a fivefold greater content of FasL compared to controls. Moreover, anti-FasL antibody partly attenuated the infected-serum-induced neutrophil apoptosis. In in vitro studies,E. colienhanced monocyte FasL expression. Moreover, conditioned media prepared from activated macrophages from control mice showed enhanced apoptosis of human as well as mouse neutrophils. On the contrary, conditioned media prepared from activated macrophages isolated from FasL-deficient mice induced only a mild degree of neutrophil apoptosis. These results suggest that neutrophils in patients with infection undergo apoptosis at an accelerated rate. Infection not only promoted monocyte expression of FasL but also increased FasL content of the serum. Because the functional status of apoptotic cells is compromised, a significant number of neutrophils may not be participating in the body's defense. Since neutrophils play the most important role in innate immunity, their compromised status in the presence of infection may transfer the host defense burden from an innate response to acquired immunity. The present study provides some insight into the lack of correlation between neutrophilia and the outcome of infection.
Lysine 2,3-Aminomutase from
Clostridium subterminale
SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene
kamA
in
Escherichia coli
Authors: Ruzicka Frank J. , Lieder Kafryn W. , Frey Perry A.
Published: July 2002

Lysine 2,3-aminomutase (KAM, EC
5.4.3.2
.) catalyzes the interconversion of
l
-lysine and
l
-β-lysine, the first step in lysine degradation in
Clostridium subterminale
SB4. KAM requires
S
-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5′-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene
kamA
for
C. subterminale
SB4 KAM and conditions for its expression in
Escherichia coli
. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and
C. subterminale
SB4 chromosomal DNA to produce a portion of
kamA
containing 1,029 base pairs of the gene. The complete gene was obtained from a genomic library of
C. subterminale
SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47,173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of
kamA
. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An
E. coli
expression system consisting of pET 23a(+) plus
kamA
yielded unsatisfactory expression and bacterial growth. Codon usage in
kamA
includes the use of AGA for all 29 arginine residues. AGA is rarely used in
E. coli
, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of
E. coli argU
dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from
C. subterminale
SB4.
Regulation of the
rulAB
Mutagenic DNA Repair Operon of
Pseudomonas syringae
by UV-B (290 to 320 Nanometers) Radiation and Analysis of
rulAB
-Mediated Mutability In Vitro and In Planta
Authors: Kim Jae J. , Sundin George W.
Published: July 2002

The effects of the
rulAB
operon of
Pseudomonas syringae
on mutagenic DNA repair and the transcriptional regulation of
rulAB
following irradiation with UV-B wavelengths were determined. For a
rulB
::Km insertional mutant constructed in
P. syringae
pv. syringae B86-17, sensitivity to UV-B irradiation increased and UV mutability decreased by 12- to 14-fold.
rulAB
-induced UV mutability was also tracked in phyllosphere populations of B86-17 for up to 5 days following plant inoculation. UV mutability to rifampin resistance (Rif
r
) was detected at all sampling points at levels which were significantly greater than in nonirradiated controls. In
P. aeruginosa
PAO1, the cloned
rulAB
determinant on pJJK17 conferred a 30-fold increase in survival and a 200-fold increase in mutability following a UV-B dose of 1,900 J m
−2
. In comparative studies using defined genetic constructs, we determined that
rulAB
restored mutability to the
Escherichia coli umuDC
deletion mutant RW120 at a level between those of its homologs
mucAB
and
umuDC
. Analyses using a
rulAB
::
inaZ
transcriptional fusion in
Pseudomonas fluorescens
Pf5 showed that
rulAB
was rapidly induced after UV-B irradiation, with expression levels peaking at 4 h. At the highest UV-B dose administered, transcriptional activity of the
rulAB
promoter was elevated as much as 261-fold compared to that of a nonirradiated control. The importance of
rulAB
for survival of
P. syringae
in its phyllosphere habitat, coupled with its wide distribution among a broad range of
P. syringae
genotypes, suggests that this determinant would be appropriate for continued investigations into the ecological ramifications of mutagenic DNA repair.
Bacillus subtilis
SMC Is Required for Proper Arrangement of the Chromosome and for Efficient Segregation of Replication Termini but Not for Bipolar Movement of Newly Duplicated Origin Regions
Authors: Graumann Peter L.
Published: July 2002

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in
Bacillus subtilis
. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an
smc
mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in
smc
mutant cells. This analysis was extended to germinating spores of an
smc
mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4′,6′-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.