bluuebunny/crossref_metadata_2025 huggingface dataset:
Digital micromirror device and its application to projection displays
Authors: Sampsell Jeffrey B.
Published: July 2002

The digital micromirror device (DMD) is a monolithic, micromechanical spatial light modulator. The DMD has been used to implement the first truly digital projection display systems. In these systems, discrete, tilting mirror elements are fabricated from sputter deposited aluminum directly on top of arrays of complementary metal–oxide semiconductor memory cells. The mirrors are switched between two stable tilted states according to whether a ‘‘1’’ or a ‘‘0’’ is stored in the underlying memory location. An optical system illuminates the DMD and projects its image in such a way that the image of each mirror, which represents a single pixel in the projected image, is at full brightness when the mirror is tilted in the ‘‘1’’ state and full darkness when the mirror is tilted in the ‘‘0’’ state. The refresh rate of the memory and the response rate of the mirrors are high enough so that hundreds of memory frames can be displayed during one video frame, and so that each pixel can be on or off in a binary fashion for a portion of the video frame proportional to that pixel’s individual intensity. The digital-to-analog conversion of this intensity occurs in the eye/brain of the viewer. The mirrors are typically square, 16 μm on a side, and placed on 17 μm centers. Each mirror tilts 10° from horizontal in each of its two addressed states, so that the ‘‘1’’ state and ‘‘0’’ state are 20° apart. Arrays of mirrors ranging from resolutions of 768×576 mirrors up to 2048×1152 mirrors have been fabricated.The article will describe the fabrication process for the DMD, the optical system used to project the DMD image, and the electronic method of addressing the device. Prototype projection systems will be described and preliminary performance measurements will be presented.
Growth mode and surface structures of the Pb/Si(001) system observed by scanning tunneling microscopy
Authors: Itoh H. , Tanabe H. , Winau D. , Schmid A. K. , Ichinokawa T.
Published: July 2002

Growth mode and surface structures of the Pb/Si(001) system have been investigated by scanning tunneling microscopy. The growth mode is the Stranski–Krastanov type and Pb grows layer by layer up to three layers before three dimensional (3D) nucleation of Pb islands occurs. The structure of the first layer changes from 2×2 and 2×1, passing through intermediate phases of 2×4 and c(8×4) with mixing by increasing Pb coverage. The second layer is a 2×1 structure, and the third layer is a c(4×4) structure. 3D islands of triangular shapes grow on the third layer of c(4×4) in an orientation of Pb(111)∥Si(001) at coverages higher than 2 monolayers.
Solvophobically Driven Folding of Nonbiological Oligomers
Authors: Nelson James C. , Saven Jeffery G. , Moore Jeffrey S. , Wolynes Peter G.
Published: July 2002

In solution, biopolymers commonly fold into well-defined three-dimensional structures, but only recently has analogous behavior been explored in synthetic chain molecules. An aromatic hydrocarbon backbone is described that spontaneously acquires a stable helical conformation having a large cavity. The chain does not form intramolecular hydrogen bonds, and solvophobic interactions drive the folding transition, which is sensitive to chain length, solvent quality, and temperature.
Role of Sensory-Evoked NMDA Plateau Potentials in the Initiation of Locomotion
Authors: Di Prisco Gonzalo Viana , Pearlstein Edouard , Robitaille Richard , Dubuc Réjean
Published: July 2002

Reticulospinal (RS) neurons constitute the main descending motor system of lampreys. This study reports on natural conditions whereby
N
-methyl-
d
-aspartate (NMDA)–mediated plateau potentials were elicited and associated with the onset of locomotion. Reticulospinal neurons responded in a linear fashion to mild skin stimulation. With stronger stimuli, large depolarizing plateaus with spiking activity were elicited and were accompanied by swimming movements. Calcium imaging revealed sustained intracellular calcium rise upon sensory stimulation. Blocking NMDA receptors on RS neurons prevented the plateau potentials as well as the associated rise in intracellular calcium. Thus, the activation of NMDA receptors mediates a switch from sensory-reception mode to a motor command mode in RS neurons.
Oxygen Isotopic Abundances in Calcium- Aluminum-Rich Inclusions from Ordinary Chondrites: Implications for Nebular Heterogeneity
Authors: McKeegan Kevin D. , Leshin Laurie A. , Russell Sara S. , MacPherson Glenn J.
Published: July 2002

The oxygen isotopic compositions of two calcium-aluminum–rich inclusions (CAIs) from the unequilibrated ordinary chondrite meteorites Quinyambie and Semarkona are enriched in
16
O by an amount similar to that in CAIs from carbonaceous chondrites. This may indicate that most CAIs formed in a restricted region of the solar nebula and were then unevenly distributed throughout the various chondrite accretion regions. The Semarkona CAI is isotopically homogeneous and contains highly
16
O-enriched melilite, supporting the hypothesis that all CAI minerals were originally
16
O-rich, but that in most carbonaceous chondrite inclusions some minerals exchanged oxygen isotopes with an external reservoir following crystallization.
Gating of BDNF-Induced Synaptic Potentiation by cAMP
Authors: Boulanger Lisa , Poo Mu-ming
Published: July 2002

Neurotrophins have been implicated in activity-dependent synaptic plasticity, but the underlying intracellular mechanisms remain largely unknown. Synaptic potentiation induced by brain-derived neurotrophic factor (BDNF), but not neurotrophin 3, was prevented by blockers of adenosine 3',5'-monophosphate (cAMP) signaling. Activators of cAMP signaling alone were ineffective in modifying synaptic efficacy but greatly enhanced the potentiation effect of BDNF. Blocking cAMP signaling abolished the facilitation of BDNF-induced potentiation by presynaptic activity. Thus synaptic actions of BDNF are gated by cAMP. Activity and other coincident signals that modulate cAMP concentrations may specify the action of secreted neurotrophins on developing nerve terminals.
Activity-Induced Potentiation of Developing Neuromuscular Synapses
Authors: Wan Ji-jun , Poo Mu-ming
Published: July 2002

Electrical activity plays a critical role in shaping the structure and function of synaptic connections in the nervous system. In
Xenopus
nerve-muscle cultures, a brief burst of action potentials in the presynaptic neuron induced a persistent potentiation of neuromuscular synapses that exhibit immature synaptic functions. Induction of potentiation required an elevation of postsynaptic Ca
2+
and expression of potentiation appeared to involve an increased probability of transmitter secretion from the presynaptic nerve terminal. Thus, activity-dependent persistent synaptic enhancement may reflect properties characteristic of immature synaptic connections, and bursting activity in developing spinal neurons may promote functional maturation of the neuromuscular synapse.
A Cyclic Carbanionic Valence Isomer of a Carbocation: Diphosphino Analogs of Diaminocarbocations
Authors: Kato Tsuyoshi , Gornitzka Heinz , Baceiredo Antoine , Schoeller Wolfgang W. , Bertrand Guy
Published: July 2002

Diaminocarbocations (or amidinium salts) feature a three-center 4π electron system with an open planar structure. Their 2π electron three-membered cyclic valence isomers, in which the carbon atom bears a negative charge, are predicted to be about 541 kilojoules per mole higher in energy than the open form. This isomer has not been identified yet. In contrast, the attempted synthesis of a diphosphorus analog of amidinium salts leads to the cyclic carbanionic form. There is no precedent for such a transformation of a carbocationic center into a carbanionic center, but with the help of heavier main-group elements, numerous examples can be imagined. This approach will enable the preparation of many unknown structural moieties that are difficult or even impossible to access in the corresponding carbon and nitrogen series.
Ultrastructure of Achilles Tendons of Rats Treated with Ofloxacin and Fed a Normal or Magnesium-Deficient Diet
Authors: Shakibaei Mehdi , Pfister Kerstin , Schwabe Rudolf , Vormann Jürgen , Stahlmann Ralf
Published: July 2002

Fluoroquinolones can cause tendinitis and tendon rupture. However, toxicological as well as clinical information on quinolone-induced tendopathy is scarce. We performed extensive electron microscopic studies with Achilles tendon specimens from ofloxacin-treated rats. The drug was given at a dose of 1,200 mg/kg (body weight) orally. Juvenile Wistar rats received one or three oral doses each of 1,200 mg of ofloxacin/kg (body weight)/day. Three days after treatment, the tenocytes of their Achilles tendons showed degenerative alterations, such as multiple vacuoles and vesicles in the cytoplasm that had developed due to swellings and dilatations of cell organelles. Other indications of cell degradation were the occurrence of cell debris and cell detachment from the extracellular matrix accompanied by a loss of cell-matrix interaction. The tenocytes of juvenile Wistar rats that had been treated at day 36 with a single oral dose of 1,200 mg of ofloxacin/kg (body weight) and sacrificed either 3 or 6 months later exhibited similar degenerative alterations. The number of degenerative alterations of tenocytes after ofloxacin treatment was considerably higher in rats that had received a magnesium-deficient diet than in rats with normal magnesium status. Of the adult rats that had been treated once, 5 times, and 10 times with ofloxacin and killed 1 day later, only those with the 10-times treatment showed a significantly increased number of degeneratively altered tenocytes. In summary, effects observed in tendons show similar pathological features as described earlier in cartilage, indicating that quinolone-induced arthropathy and quinolone-induced tendopathy probably are different clinical manifestations of the same toxic effect on cellular components of connective tissue structures.
Potent Synergism of the Combination of Fluconazole and Cyclosporine in
Candida albicans
Authors: Marchetti Oscar , Moreillon Philippe , Glauser Michel P. , Bille Jacques , Sanglard Dominique
Published: July 2002

Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on
Candida albicans
. These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors. Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC. In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined. Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of <1). This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC. The MIC-0 values of FLC and Cy tested alone in
C. albicans
were >32 and >10 μg/ml, respectively, and decreased to 0.5 and 0.625 μg/ml when the two drugs were combined. The combination of 0.625 μg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments. This effect was fungicidal or fungistatic, depending on the
C. albicans
strain used. Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.
Nasopharyngeal Carriage of Antimicrobial-Resistant
Streptococcus pneumoniae
among Young Children Attending 79 Kindergartens and Day Care Centers in Hong Kong
Authors: Chiu Susan S. , Ho Pak Leung , Chow Frankie K. H. , Yuen Kwok Yung , Lau Yu Lung
Published: July 2002

Resistance to penicillin and multiple antimicrobial agents among
Streptococcus pneumoniae
strains is becoming an increasing problem worldwide and in Asia. To determine the prevalence of carriage of
S. pneumoniae
isolates not susceptible to penicillin in young children, we obtained nasopharyngeal swab specimens from 1,978 children (ages, 2 to 6 years) attending 79 day care centers or kindergartens. Three hundred eighty-three strains of
S. pneumoniae
were isolated from these children. Fifty-eight percent of these isolates had reduced susceptibility to penicillin, 123 (32.1%) were intermediate, and 100 (26.1%) were resistant. A very high penicillin MIC (4 μg/ml) was found in 3.3% of the isolates. The isolates also demonstrated high rates of resistance to other antimicrobial agents (51.2% to cefaclor, 50.2% to cefuroxime, 42.8% to cefotaxime, 80.7% to trimethoprim-sulfamethoxazole, 77% to erythromycin, 60% to clindamycin, and 33.7% to chloramphenicol). No isolate was resistant to fluoroquinolone. Multidrug resistance (not susceptible to the β-lactams and three or more other classes) was found in 39.4% of the isolates. Risk factors for the carriage of
S. pneumoniae
not susceptible to penicillin were multiple physician visits in the preceding 3 months and use of antimicrobial agents by the individual or by household members in the preceding 3 months. In the logistic regression analysis, only the use of antimicrobial agents in the preceding 3 months was an independent risk factor (
P
= 0.004; odds ratio, 2; 95% confidence interval, 1.2 to 3.2). This study demonstrated the high prevalence of antibiotic-resistant
S. pneumoniae
in healthy young children in the community in Hong Kong.
Effect of Potassium on
Saccharomyces cerevisiae
Resistance to Fluconazole
Authors: Stella Carlos A. , Burgos Hilda I.
Published: July 2002

Susceptibility of strain S288c of
Saccharomyces cerevisiae
to fluconazole was assayed in the presence and absence of KCl. Addition of 150 mM KCl renders the strain more sensitive to the antifungal agent. The effect is caused by the K
+
ion rather than the anion or the osmolarity of the medium. The increase in sensitivity does not modify the values of intracellular and extracellular pH established in the presence of KCl.
Inducible, but Not Constitutive, Resistance of Gonococci to Hydrophobic Agents Due to the MtrC-MtrD-MtrE Efflux Pump Requires TonB-ExbB-ExbD Proteins
Authors: Rouquette-Loughlin Corinne , Stojiljkovic Igor , Hrobowski Tara , Balthazar Jacqueline T. , Shafer William M.
Published: July 2002

The MtrC-MtrD-MtrE efflux pump possessed byNeisseria gonorrhoeaeis very similar to the MexA-MexB-OprM efflux pump ofPseudomonas aeruginosa. Because the antimicrobial resistance property afforded by the MexA-MexB-OprM efflux pump also requires the TonB protein, we asked whether a similar requirement exists for the gonococcal efflux pump. Unlike earlier studies withP. aeruginosa, we found that constitutive levels of gonococcal resistance to hydrophobic antimicrobial agents (i.e., Triton X-100 [TX-100]) did not require the TonB, ExbB, or ExbD protein. However, inducible levels of TX-100 resistance in gonococci had an absolute requirement for the TonB-ExbB-ExbD system, suggesting that such resistance in gonococci has an energy requirement above and beyond that required for constitutive pump activity.
Behavioral Responses of
Rhodobacter sphaeroides
to Linear Gradients of the Nutrients Succinate and Acetate
Authors: Packer Helen L. , Armitage Judith P.
Published: July 2002

Rhodobacter sphaeroides
cells were tethered by their flagella and subjected to increasing and decreasing nutrient gradients. Using motion analysis, changes in flagellar motor rotation were measured and the responses of the cells to the chemotactic gradients were determined. The steepness and concentration ranges of increasing and decreasing gradients were varied, and the bacterial responses were measured. This allowed the limits of gradients that would invoke changes in flagellar behavior to be determined and thus predicts the nature of gradients that would evoke chemotaxis in the environment. The sensory threshold was measured at 30 nM, and the response showed saturation at 150 μM. The study determined that cells detected and responded to changing concentration rates as low as 1 nM/s for acetate and 5 nM/s for succinate. The complex sensory system of
R. sphaeroides
responded to both increasing and decreasing concentration gradients of attractant with different sensitivities. In addition, transition phases involving changes in the motor speed and the smoothness of motor rotation were found.
Genetically Controlled Self-Aggregation of Cell-Surface-Engineered Yeast Responding to Glucose Concentration
Authors: Zou Wen , Ueda Mitsuyoshi , Tanaka Atsuo
Published: July 2002

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and α-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agα1p (α-agglutinin). The upstream region of the isocitrate lyase gene of
Candida tropicalis
(
UPR-ICL
), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and α-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.
Neurovirulence of Type 1 Polioviruses Isolated from Sewage in Japan
Authors: Horie Hitoshi , Yoshida Hiromu , Matsuura Kumiko , Miyazawa Miwako , Ota Yoshihiro , Nakayama Takashi , Doi Yutaka , Hashizume So
Published: July 2002

Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.
Differentiation of
Phytophthora infestans
Sporangia from Other Airborne Biological Particles by Flow Cytometry
Authors: Day Jennifer P. , Kell Douglas B. , Griffith Gareth W.
Published: July 2002

The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen
Phytophthora infestans
from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of
Alternaria
or
Botrytis
spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between
P. infestans
sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed.
Genomic Sequence and Evolution of Marine Cyanophage P60: a New Insight on Lytic and Lysogenic Phages
Authors: Chen Feng , Lu Jingrang
Published: July 2002

The genome of cyanophage P60, a lytic virus which infects marine
Synechococcus
WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, phi-YeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria.
Comparative Analysis of Human Cytomegalovirus-Specific CD4
+
T-Cell Frequency and Lymphoproliferative Response in Human Immunodeficiency Virus-Positive Patients
Authors: Piccinini Giampiero , Comolli Giuditta , Genini Emilia , Lilleri Daniele , Gulminetti Roberto , Maccario Rita , Revello Maria Grazia , Gerna Giuseppe
Published: July 2002

Evaluation of human cytomegalovirus (HCMV)-specific T-helper immunity could contribute in optimizing anti-HCMV therapy in human immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific CD4
+
T-cell frequency by flow cytometry detection of intracellular cytokine production after short-term antigen-specific activation of peripheral blood mononuclear cells. HCMV-specific LPR and CD4
+
T-cell frequency were compared in a group of 78 blood samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about half of patients the T-helper HCMV-specific immune response remained stable during highly active antiretroviral therapy (HAART), in the other half declining levels of the HCMV-specific CD4
+
-mediated immune response were determined by either one or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4
+
T-cell frequency can be considered a valuable alternative to the LPR test for the detection of HCMV-specific T-helper response in HIV-infected patients. It could facilitate wider screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of discontinuation or maintenance of anti-HCMV therapy.
Insecticide-Treated Bed Nets Reduce Plasma Antibody Levels and Limit the Repertoire of Antibodies to
Plasmodium falciparum
Variant Surface Antigens
Authors: Askjaer Nikolaj , Maxwell Caroline , Chambo William , Staalsoe Trine , Nielsen Morten , Hviid Lars , Curtis Chris , Theander Thor G.
Published: July 2002

The use of insecticide-treated bed nets (ITN) has been documented to reduce malaria morbidity and mortality in areas with endemic malaria, but concerns have been raised that ITN usage could affect the acquisition of malaria immunity. Several lines of evidence have indicated that antibodies against variant surface antigens (VSA) are important in the development of naturally acquired immunity to
Plasmodium falciparum
malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from children from a nearby village not using ITN. A total of 97 plasma samples were analyzed using 13 different
P. falciparum
isolates. We found that the children using ITN had significantly lower VSA antibody levels and recognized a smaller proportion of the VSA expressed by the tested parasite isolates than children not using ITN.
fbpABC
Gene Cluster in
Neisseria meningitidis
Is Transcribed as an Operon
Authors: Khun Heng H. , Deved Vinay , Wong Howard , Lee B. Craig
Published: July 2002

The neisserial
fbpABC
locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with
Neisseria meningitidis
. The presence of
fbpAB
and
fbpBC
transcripts obtained by priming cDNA synthesis with an
fbpC
-sequence-specific oligonucleotide indicates that
fbpABC
is organized as a single expression unit. The ratio of
fbpA
to
fbpABC
mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.
Macropinocytosis as a Mechanism of Entry into Primary Human Urethral Epithelial Cells by
Neisseria gonorrhoeae
Authors: Zenni Michael K. , Giardina Peter C. , Harvey Hillery A. , Shao Jianqiang , Ketterer Margaret R. , Lubaroff David M. , Williams Richard D. , Apicella Michael A.
Published: July 2002

Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with
M
r
70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.
Are B Lymphocytes of Importance in SevereStaphylococcus aureusInfections?
Authors: Gjertsson Inger , Hultgren Olof Hörnquist , Stenson Martin , Holmdahl Rikard , Tarkowski Andrzej
Published: July 2002

To investigate the role of B cells in experimental, superantigen-mediatedStaphylococcus aureusarthritis and sepsis, we used gene-targeted B-cell-deficient mice. The mice were inoculated intravenously with a toxic shock syndrome toxin 1 (TSST-1)-producingS. aureusstrain. The B-cell-deficient and thus agamma-globulinemic mice showed striking similarities to the wild-type control animals with respect to the development of arthritis, the mortality rate, and the rate of bacterial clearance. Surprisingly, we found that the levels of gamma interferon in serum were significantly lower (P< 0.0001) in B-cell-deficient mice than in the controls, possibly due to impaired superantigen presentation and a diminished expression of costimulatory molecules. In contrast, the levels of interleukin-4 (IL-4), IL-6, and IL-10 in serum were equal in both groups. Our findings demonstrate that neither mature B cells nor their products significantly contribute to the course ofS. aureus-induced septic arthritis.
Host and Tissue Specificity of
Trichomonas vaginalis
Is Not Mediated by Its Known Adhesion Proteins
Authors: Addis Maria Filippa , Rappelli Paola , Fiori Pier Luigi
Published: July 2002

Adhesion of
Trichomonas vaginalis
is believed to be dependent on four adhesion proteins, which are thought to bind to vaginal epithelial cells in a specific manner with a ligand-receptor type of interaction. However, the specific receptors on the host cell have not yet been identified. In this work, the ability of the
T. vaginalis
adhesins to bind to cells of different histologic derivations and from different species has been studied. HeLa, CHO, and Vero cell lines; erythrocytes from different species; and a prokaryote without a cell wall,
Mycoplasma hominis
, were employed in order to investigate the cell specificity of the
T. vaginalis
adhesins. We observed that the
T. vaginalis
adhesins are able to bind to the different cell types to the same extent, suggesting that the host and tissue specificity of
T. vaginalis
adhesion should not be due to specificity of the parasite adhesins. Our results suggest that the data published to date on the subject are probably artifactual and that the experiments reported in the literature are not appropriate for identification of protozoan adhesins.
Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic
Escherichia coli
Authors: Meysick K. C. , Mills M. , O'Brien A. D.
Published: July 2002

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic
Escherichia coli
belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.
Activation of Extracellular Signal-Related Protein Kinases 1 and 2 of the Mitogen-Activated Protein Kinase Family by Lipopolysaccharide Requires Plasma in Neutrophils from Adults and Newborns
Authors: Bonner S. , Yan S. R. , Byers D. M. , Bortolussi R.
Published: July 2002

Neutrophils exposed to low concentrations of gram-negative lipopolysaccharide (LPS) become primed and have an increased oxidative response to a second stimulus (e.g., formyl-methionyl-leucyl-phenylalanine [fMLP]). In studies aimed at understanding newborn sepsis, we have shown that neutrophils of newborns are not primed in response to LPS. To further understand the processes involved in LPS-mediated priming of neutrophils, we explored the role of extracellular signal-related protein kinases (ERK 1 and 2) of the mitogen-activated protein kinase family. We found that LPS activated ERK 1 and 2 in cells of both adults and newborns and that activation was plasma dependent (maximal at ≥5%) through LPS-binding protein. Although fibronectin in plasma is required for LPS-mediated priming of neutrophils of adults assessed by fMLP-triggered oxidative burst, it was not required for LPS-mediated activation of ERK 1 and 2. LPS-mediated activation was dose and time dependent; maximal activation occurred with approximately 5 ng of LPS per ml and at 10 to 40 min. We used the inhibitor PD 98059 to study the role of ERK 1 and 2 in the LPS-primed fMLP-triggered oxidative burst. While Western blotting showed that 100 μM PD 98059 completely inhibited LPS-mediated ERK activation, oxidative response to fMLP by a chemiluminescence assay revealed that the same concentration inhibited the LPS-primed oxidative burst by only 40%. We conclude that in neutrophils, LPS-mediated activation of ERK 1 and 2 requires plasma and that this activation is not dependent on fibronectin. In addition, we found that the ERK pathway is not responsible for the lack of LPS priming in neutrophils of newborns but may be required for 40% of the LPS-primed fMLP-triggered oxidative burst in cells of adults.
Induction of Protective Immunity toListeria monocytogenesby Immunization with Plasmid DNA Expressing a Helper T-Cell Epitope That Replaces the Class II-Associated Invariant Chain Peptide of the Invariant Chain
Authors: Nagata Toshi , Aoshi Taiki , Suzuki Mina , Uchijima Masato , Kim Yeung-Hyen , Yang Zhibo , Koide Yukio
Published: July 2002

Listeriaepitope-specific helper T (Th) cells were able to be primed and induced in vivo by immunization with a plasmid carrying an invariant chain (Ii) gene whose class II-associated invariant chain peptide (CLIP) region was replaced by aListeriaTh epitope. Immunization of C3H/He mice with an Ii-LLO 215-226 plasmid induced specific interferon-γ- and interleukin 2-producing Th cells and conferred significant protective immunity against listerial infection.
Transcriptional and Mutational Analyses of theStreptomyces lividans recXGene and Its Interference with RecA Activity
Authors: Vierling Silke , Weber Tilmann , Wohlleben Wolfgang , Muth Günther
Published: July 2002

The role of the 20,922-Da RecX protein and its interference with RecA activity were analyzed inStreptomyces lividans. TherecXgene is located 220 bp downstream ofrecA. Transcriptional analysis by reverse transcriptase PCR demonstrated thatrecXandrecAconstitute an operon. WhilerecAwas transcribed at a basal level even under noninducing conditions, arecA-recXcotranscript was only detectable after induction ofrecAfollowing DNA damage. TherecA-recXcotranscript was less abundant than therecAtranscript alone. TherecXgene was inactivated by gene replacement. The resulting mutant had a clearly diminished colony size, but was not impaired in recombination activity, genetic instability, and resistance against UV irradiation. Expression of an extra copy of theS. lividans recAgene under control of the thiostrepton-inducibletipApromoter was lethal to therecXmutant, demonstrating that RecX is required to overcome the toxic effects ofrecAoverexpression. Since inactivation of therecXgene did not influence transcription ofrecA, the putative function of the RecX protein might be the downregulation of RecA activity by interaction with the RecA protein or filament.
A Gene Cluster for the Mevalonate Pathway from
Streptomyces
sp. Strain CL190
Authors: Takagi Motoki , Kuzuyama Tomohisa , Takahashi Shunji , Seto Haruo
Published: July 2002

A biosynthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC
1.1.1.34
), the rate-limiting enzyme of the mevalonate pathway for isopentenyl diphosphate biosynthesis, had previously been purified from
Streptomyces
sp. strain CL190 and its corresponding gene (
hmgr
) had been cloned (S. Takahashi, T. Kuzuyama, and H. Seto, J. Bacteriol. 181:1256–1263, 1999). Sequence analysis of the flanking regions of the
hmgr
gene revealed five new open reading frames,
orfA
to -
E
, which showed similarity to those encoding eucaryotic and archaebacterial enzymes for the mevalonate pathway. Feeding experiments with [1-
13
C]acetate demonstrated that
Escherichia coli
JM109 harboring the
hmgr
gene and these open reading frames used the mevalonate pathway under induction with isopropyl β-
d
-thiogalactopyranoside. This transformant could grow in the presence of fosmidomycin, a potent and specific inhibitor of the nonmevalonate pathway, indicating that the mevalonate pathway, intrinsically absent in
E. coli
, is operating in the
E. coli
transformant. The
hmgr
gene and
orfABCDE
are thus unambiguously shown to be responsible for the mevalonate pathway and to form a gene cluster in the genome of
Streptomyces
sp. strain CL190.
Identification of an Ancillary Protein, YabF, Required for Activity of the KefC Glutathione-Gated Potassium Efflux System in
Escherichia coli
Authors: Miller S. , Ness L. S. , Wood C. M. , Fox B. C. , Booth I. R.
Published: July 2002

A new subunit, YabF, for the KefC K
+
efflux system in
Escherichia coli
has been identified. The subunit is required for maximum activity of KefC. Deletion of
yabF
reduces KefC activity 10-fold, and supply of YabF in
trans
restores activity. IS
2
and IS
10
R insertions in
yabF
can be isolated as suppressors of KefC activity consequent upon the V427A and D264A KefC mutations.
Expression of Different-Size Transcripts from the
clpP-clpX
Operon of
Escherichia coli
during Carbon Deprivation
Authors: Li Chin , Tao Yi Ping , Simon Lee D.
Published: July 2002

Transcription of the
clpP-clpX
operon of
Escherichia coli
leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entire
clpP-clpX
operon, whereas the shorter transcript contains the entire
clpP
gene but none of the
clpX
coding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς
32
-dependent promoter of the
clpP-clpX
operon within 15 min after the start of carbon starvation. Expression of the
clpP-clpX
operon from the promoters upstream of the
clpP
gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation.
E. coli rpoB
mutations that affect transcription termination efficiency alter the ratio of the shorter
clpP-clpX
transcript to the longer transcript. The
E. coli rpoB3595
mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the
rpoB8
mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter
clpP-clpX
transcript when
E. coli
is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.
Enzymatic Removal of Nitric Oxide Catalyzed by Cytochrome
c
′ in
Rhodobacter capsulatus
Authors: Cross Richard , Lloyd David , Poole Robert K. , Moir James W. B.
Published: July 2002

Cytochrome
c
′ from
Rhodobacter capsulatus
has been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome
c
′ synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome
c
′ is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N
2
O), thus showing that cytochrome
c
′ is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome
c
′ is distinct from the NO reductase pathway that is involved in denitrification. Cytochrome
c
′ is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome
c
′ synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of
R. capsulatus
by NO suggests that one role for cytochrome
c
′ is to maintain oxidase activity when both NO and O
2
are present.
SdeK, a Histidine Kinase Required for
Myxococcus xanthus
Development
Authors: Pollack Jeffrey S. , Singer Mitchell
Published: July 2002

The
sdeK
gene is essential to the
Myxococcus xanthus
developmental process. We reported previously, based on sequence analysis (A. G. Garza, J. S. Pollack, B. Z. Harris, A. Lee, I. M. Keseler, E. F. Licking, and M. Singer, J. Bacteriol. 180:4628–4637, 1998), that SdeK appears to be a histidine kinase. In the present study, we have conducted both biochemical and genetic analyses to test the hypothesis that SdeK is a histidine kinase. An SdeK fusion protein containing an N-terminal polyhistidine tag (His-SdeK) displays the biochemical characteristics of a histidine kinase. Furthermore, histidine 286 of SdeK, the putative site of phosphorylation, is required for both in vitro and in vivo protein activity. The results of these assays have led us to conclude that SdeK is indeed a histidine kinase. The developmental phenotype of a Δ
sdeK1
strain could not be rescued by codevelopment with wild-type cells, indicating that the defect is not due to the mutant's inability to produce an extracellular signal. Furthermore, the Δ
sdeK1
mutant was found to produce both A- and C-signal, based on A-factor and codevelopment assays with a
csgA
mutant, respectively. The expression patterns of several Tn
5lacZ
transcriptional fusions were examined in the Δ
sdeK1
-null background, and we found that all C-signal-dependent fusions assayed also required SdeK for full expression. Our results indicate that SdeK is a histidine kinase that is part of a signal transduction pathway which, in concert with the C-signal transduction pathway, controls the activation of developmental-gene expression required to progress past the aggregation stage.
Involvement of the TonB System in Tolerance to Solvents and Drugs inPseudomonas putidaDOT-T1E
Authors: Godoy Patricia , Ramos-González Marı́a Isabel , Ramos Juan L.
Published: July 2002

Pseudomonas putidaDOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM)p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5′phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with14C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted theexbDgene, which forms part of theexbBD tonBoperon. Complementation by theexbBDandtonBgenes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.
Negative Control of
rpoS
Expression by Phosphoenolpyruvate:Carbohydrate Phosphotransferase System in
Escherichia coli
Authors: Ueguchi Chiharu , Misonou Naoko , Mizuno Takeshi
Published: July 2002

The ς
S
(or ς
38
) subunit of RNA polymerase, encoded by the
rpoS
gene, is a crucial regulator in the transcriptional control of a set of genes under stressful conditions, such as nutrient starvation. The expression of
rpoS
is regulated in a complex manner at the levels of transcription, translation, and stability of the product. Although a number of factors involved in the regulation of
rpoS
expression have been identified, the underlying molecular mechanisms are not fully understood. In this study, we identified the Crr (or EIIA
Glc
) protein as a novel factor that plays an important role not only in the transcriptional control but also in the translational control of
rpoS
expression. Crr is an important component in glucose uptake through the well-characterized phosphoenolpyruvate:carbohydrate phosphotransferase system. The results of a series of genetic analyses revealed that Crr negatively controls
rpoS
translation and transcription. The observed transcriptional control by Crr appears to be mediated by cyclic AMP. However, it was found that Crr negatively controls
rpoS
translation rather directly. These results suggest a possible linkage between the control of
rpoS
expression and carbon metabolism.
Benzylsuccinate Synthase of
Azoarcus
sp. Strain T: Cloning, Sequencing, Transcriptional Organization, and Its Role in Anaerobic Toluene and
m
-Xylene Mineralization
Authors: Achong Gypsy R. , Rodriguez Ana M. , Spormann Alfred M.
Published: July 2002

Biochemical studies in
Azoarcus
sp. strain T have demonstrated that anaerobic oxidation of both toluene and
m
-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from
Azoarcus
sp. strain T and examined the role of this enzyme in both anaerobic toluene and
m
-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase,
bssCAB
, together with two additional genes,
bssD
and
bssE
, were organized in an operon in the order
bssDCABE. bssD
is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase.
bssE
shows homology to
tutH
from
Thauera aromatica
strain T1, whose function is currently unknown. A second operon that is upstream of
bssDCABE
and divergently transcribed contains two genes,
tdiS
and
tdiR
. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null
bssA
mutant was constructed (the
bssA
gene encodes the α-subunit of benzylsuccinate synthase). This
bssA
null mutant strain was unable to grow under denitrifying conditions on either toluene or
m
-xylene, while growth on benzoate was unaffected. The growth phenotype of the Δ
bssA
mutant could be rescued by reintroducing
bssA
in
trans
. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and
m
-xylene.