bluuebunny/crossref_metadata_2025 huggingface dataset:
Reference Command Shaping Using Specified-Negative-Amplitude Input Shapers for Vibration Reduction
Authors: Singhose William , Ooten Biediger Erika , Chen Ye-Hwa , Mills Bart
Published: April 2004

Residual vibrations can be greatly reduced by using specially-shaped reference command signals. Input shaping is one such technique that reduces vibration by convolving a sequence of impulses with any desired reference command. Several types of useful impulse sequences have been developed. Most of these have contained only positively valued impulses. However, rise time can be improved by using some negative impulses in the sequence. Unfortunately, the use of negative impulses can excite unmodeled high modes. A new type of impulse sequence containing negative impulses is proposed. These sequences are designed to fill the performance gap between all-positive impulse sequences and the negative sequences previously developed. A proof governing the worst case scenario provides an upper bound on high-mode excitation. The resulting class of impulse sequences allows the designer to make a precise trade off between rise time and vibration reduction.
A Performance Enhancement Scheme for Robust Repetitive Control System
Authors: Kim Byung-Sub , Tsao Tsu-Chin
Published: April 2004

This paper presents a method for enhancing the performance of a digital repetitive control system. The performance at the fundamental frequency and its harmonics of repetitive exogenous signals is improved by applying a modified low-pass qz,z−1 filter structure in the repetitive signal generator of the internal model. Stability and robust performance is achieved through μ-synthesis. The modified low-pass filter is motivated by the attempt to reduce the sensitivity function by squaring it. The magnitude of the sensitivity function is substantially reduced at the periodic reference frequencies since the value is already much less than unity. The proposed approach is demonstrated by its implementation on an electrohydraulic actuator for tracking periodic cam-like profiles.
Extended Influence Coefficient Method for Rotor Active Balancing During Acceleration
Authors: Zhou Shiyu , Dyer Stephen W. , Shin Kwang-keun , Shi Jianjun , Ni Jun
Published: April 2004

Imbalance-induced vibration of rotating machineries is an important factor limiting the performance and fatigue life of a rotor system. Particularly, the severe resonant vibration of a rotor when it passes through its critical speeds could damage the rotor system. To avoid this peak vibration, this paper presents an active balancing method to offset the imbalance of the rotor system during acceleration by using an electromagnetic balancer. In this method, “instantaneous” influence coefficients at different speeds are obtained and stored in a look-up table. Then, a gain scheduling strategy is adopted to suppress the imbalance-induced vibration during acceleration based on the “instantaneous” influence coefficient table. A comprehensive testbed is built to validate this scheme, and the validation results are presented.
Robust Adaptive Control of Nonlinear Output Feedback Systems Under Disturbances With Unknown Bounds
Authors: Kim Dong H. , Wang Hua O. , Yang Hai-Won
Published: April 2004

This paper describes a systematic procedure to design robust adaptive controllers for a class of nonlinear systems with unknown functions of unknown bounds based on backstepping and sliding mode techniques. These unknown functions can be unmodeled system nonlinearities, uncertainties and disturbances with unknown bounds. Both state feedback and output feedback designs are addressed. In the design procedure, the upper bounds of the unknown functions are estimated using an adaptation strategy, and the estimates are used to design stabilizing functions and control inputs based on the backstepping design methodology. The proposed controllers guarantee that the tracking errors converge to a residual set close to zero exponentially for both state feedback and output feedback designs, while maintaining the boundedness of all other variables.
Precision Control of a Piezoceramic Actuator Using Neural Networks
Authors: Kung Ying-Shieh , Fung Rong-Fong
Published: April 2004

In this paper, a control method combining the feedforward and feedback controllers is proposed to precisely control the dynamic performance of the piezoceramic actuator (PA). In the feedforward controller design, the hysteresis nonlinearity of the PA is modeled by using Preisach model first. Then a database of switching input/output values and a neural networks architecture treated as the inverse function of Preisach model are utilized in the feedforward controller. In the feedback controller design, a PI controller is used to regulate the output error. Finally, some experimental results are validated the excellent tracking performances of the proposed controller.
Jerk Limited Input Shapers
Authors: Singh Tarunraj
Published: April 2004

The focus of this paper is on the design of jerk limited input shapers (time-delay filters). Closed form solutions for the jerk limited time-delay filter for undamped systems is derived followed by the formulation of the problem for damped systems. Since the jerk limited filter involves concatenating an integrator to a time-delay filter, a general filter design technique is proposed where smoothing of the shaped input can be achieved by concatenating transfer functions of first order, harmonic systems, etc.
Modeling the Pneumatic Subsystem of an S-cam Air Brake System
Authors: Subramanian Shankar C. , Darbha Swaroop , Rajagopal K. R.
Published: April 2004

This paper deals with the development of a fault-free model of the pneumatic subsystem of an air brake system that is used in commercial vehicles. Our objective is to use this model in brake control and diagnostic applications. The development of a diagnostic system would be useful in automating enforcement inspections and also in monitoring the condition of the brake system in real-time. This paper presents a detailed description of the development of this model and of the experimental setup used to corroborate this model for various realistic test runs.
Robotic Observations of Enhanced Carbon Biomass and Export at 55°S During SOFeX
Authors: Bishop James K. B. , Wood Todd J. , Davis Russ E. , Sherman Jeffrey T.
Published: April 2004

Autonomous floats profiling in high-nitrate low-silicate waters of the Southern Ocean observed carbon biomass variability and carbon exported to depths of 100 m during the 2002 Southern Ocean Iron Experiment (SOFeX) to detect the effects of iron fertilization of surface water there. Control and “in-patch” measurements documented a greater than fourfold enhancement of carbon biomass in the iron-amended waters. Carbon export through 100 m increased two- to sixfold as the patch subducted below a front. The molar ratio of iron added to carbon exported ranged between 10
4
and 10
5
. The biomass buildup and export were much higher than expected for iron-amended low-silicate waters.
Removal of Meteoric Iron on Polar Mesospheric Clouds
Authors: Plane John M. C. , Murray Benjamin J. , Chu Xinzhao , Gardner Chester S.
Published: April 2004

Polar mesospheric clouds are thin layers of nanometer-sized ice particles that occur at altitudes between 82 and 87 kilometers in the high-latitude summer mesosphere. These clouds overlap in altitude with the layer of iron (Fe) atoms that is produced by the ablation of meteoroids entering the atmosphere. Simultaneous observations of the Fe layer and the clouds, made by lidar during midsummer at the South Pole, demonstrate that essentially complete removal of Fe atoms can occur inside the clouds. Laboratory experiments and atmospheric modeling show that this phenomenon is explained by the efficient uptake of Fe on the ice particle surface.
Smoke No Longer Gets in Your Eyes
Authors: Henderson A. Scott
Published: April 2004

Devastation and Renewal
An Environmental History of Pittsburgh and Its Region. by Joel A. Tarr, Ed. University of Pittsburgh Press, Pittsburgh, 2003. 291 pp. $32. ISBN 0-8229-4156-2.

The contributors consider the effects of industrialization on Pittsburgh's air, water, and landscape along with the actions that have been taken to recover from that environmental degradation.
Human Impact on the Chinese Landscape
Authors: McNeill J. R.
Published: April 2004

The Retreat of the Elephants
An Environmental History of China. by Mark Elvin. Yale University Press, New Haven, 2004. 592 pp. $39.95, £25. ISBN 0-300-10111-2.

Drawing on some 3000 years of written records, Elvin chronicles the impact of humans on the environment of China, paying particular attention to views and feelings of the Chinese toward their landscape.
Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin
Authors: Onn Itay , Milman-Shtepel Neta , Shlomai Joseph
Published: April 2004

Kinetoplast DNA, the mitochondrial DNA of the trypanosomatidCrithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region.C. fasciculatatrypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.
Role of Micronucleus-Limited DNA in Programmed Deletion of mse2.9 during Macronuclear Development of
Tetrahymena thermophila
Authors: Fillingham Jeffrey S. , Pearlman Ronald E.
Published: April 2004

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan
Tetrahymena thermophila
. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important
cis
-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the
cis
-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the
ARP1
gene. We show that the expression of
ARP1
is not essential for the growth of
Tetrahymena
. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.
pdf1
, a Palmitoyl Protein Thioesterase 1 Ortholog in
Schizosaccharomyces pombe
: a Yeast Model of Infantile Batten Disease
Authors: Cho Steve K. , Hofmann Sandra L.
Published: April 2004

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human
PPT1
(palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins.
PPT1
has orthologs in many species, including lower organisms and plants, but not in
Saccharomyces cerevisiae.
The fission yeast
Schizosaccharomyces pombe
contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the
S. pombe
Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as
pdf1
, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the
pdf1
sequence. Strains bearing a deletion of the entire
pdf1
open reading frame are nonviable and are rescued by a
pdf1
expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (
vps
) mutants and vacuolar membrane H
+
-ATPase (
vma
) mutants in
S. cerevisiae
. Importantly, the Ppt1p-deficient phenotype is complemented by the human
PPT1
gene. These results indicate that the function of
PPT1
has been widely conserved throughout evolution and that
S. pombe
may serve as a genetically tractable model for the study of human infantile Batten disease.
Nrg1 and Nrg2 Transcriptional Repressors Are Differently Regulated in Response to Carbon Source
Authors: Berkey Cristin D. , Vyas Valmik K. , Carlson Marian
Published: April 2004

The Nrg1 and Nrg2 repressors of
Saccharomyces cerevisiae
have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that
NRG1
and
NRG2
are differently regulated in response to carbon source at both the RNA and protein levels. Expression of
NRG1
RNA is glucose repressed, whereas
NRG2
RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.
Translational Accuracy during Exponential, Postdiauxic, and Stationary Growth Phases in
Saccharomyces cerevisiae
Authors: Stahl Guillaume , Salem Samia N. Ben , Chen Lifeng , Zhao Bing , Farabaugh Philip J.
Published: April 2004

When the yeast
Saccharomyces cerevisiae
shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.
Role of a Mitogen-Activated Protein Kinase Pathway during Conidial Germination and Hyphal Fusion in
Neurospora crassa
Authors: Pandey Amita , Roca M. Gabriela , Read Nick D. , Glass N. Louise
Published: April 2004

Mitogen-activated protein (MAP) kinase signaling pathways are ubiquitous and evolutionarily conserved in eukaryotic organisms. MAP kinase pathways are composed of a MAP kinase, a MAP kinase kinase, and a MAP kinase kinase kinase; activation is regulated by sequential phosphorylation. Components of three MAP kinase pathways have been identified by genome sequence analysis in the filamentous fungus
Neurospora crassa
. One of the predicted MAP kinases in
N. crassa
, MAK-2, shows similarity to Fus3p and Kss1p of
Saccharomyces cerevisiae
, which are involved in sexual reproduction and filamentation, respectively. In this study, we show that an
N. crassa
mutant disrupted in
mak
-
2
exhibits a pleiotropic phenotype: derepressed conidiation, shortened aerial hyphae, lack of vegetative hyphal fusion, female sterility, and autonomous ascospore lethality. We assessed the phosphorylation of MAK-2 during conidial germination and early colony development. Peak levels of MAK-2 phosphorylation were most closely associated with germ tube elongation, branching, and hyphal fusion events between conidial germlings. A MAP kinase kinase kinase (NRC-1) is the predicted product of
N. crassa nrc
-
1
locus and is a homologue of
STE11
in
S. cerevisiae.
An
nrc
-
1
mutant shares many of the same phenotypic traits as the
mak
-
2
mutant and, in particular, is a hyphal fusion mutant. We show that MAK-2 phosphorylation during early colony development is dependent upon the presence of NRC-1 and postulate that phosphorylation of MAK-2 is required for hyphal fusion events that occur during conidial germination.
Genetic Dissection of the
Kluyveromyces lactis
Telomere and Evidence for Telomere Capping Defects in
TER1
Mutants with Long Telomeres
Authors: Underwood Dana H. , Carroll Coleen , McEachern Michael J.
Published: April 2004

In the yeast
Kluyveromyces lactis
, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the
K. lactis
telomere was performed by using mutant telomerase RNA (
TER1
) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3′ of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3′ terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number
RAP1
. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.
Polarized Hyphal Growth in
Candida albicans
Requires the Wiskott-Aldrich Syndrome Protein Homolog Wal1p
Authors: Walther A. , Wendland J.
Published: April 2004

The yeast-to-hypha transition is a key feature in the cell biology of the dimorphic human fungal pathogen
Candida albicans
. Reorganization of the actin cytoskeleton is required for this dimorphic switch in
Candida
. We show that
C. albicans WAL1
mutants with both copies of the Wiskott-Aldrich syndrome protein (WASP) homolog deleted do not form hyphae under all inducing conditions tested. Growth of the wild-type and
wal1
mutant strains was monitored by in vivo time-lapse microscopy both during yeast-like growth and under hypha-inducing conditions. Isotropic bud growth produced round
wal1
cells and unusual mother cell growth. Defects in the organization of the actin cytoskeleton resulted in the random localization of actin patches. Furthermore,
wal1
cells exhibited defects in the endocytosis of the lipophilic dye FM4-64, contained increased numbers of vacuoles compared to the wild type, and showed defects in bud site selection. Under hypha-inducing conditions
wal1
cells were able to initiate polarized morphogenesis, which, however, resulted in the formation of pseudohyphal cells. Green fluorescent protein (GFP)-tagged Wal1p showed patch-like localization in emerging daughter cells during the yeast growth phase and at the hyphal tips under hypha-inducing conditions. Wal1p-GFP localization largely overlapped with that of actin. Our results demonstrate that Wal1p is required for the organization of the actin cytoskeleton and hyphal morphogenesis in
C. albicans
as well as for endocytosis and vacuole morphology.
Cryptosporidium parvum
Mitochondrial-Type HSP70 Targets Homologous and Heterologous Mitochondria
Authors: Šlapeta Jan , Keithly Janet S.
Published: April 2004

A mitochondrial HSP70 gene (
Cp-mtHSP70
) is described for the apicomplexan
Cryptosporidium parvum
, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH
2
-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR,
Cp-mtHSP70
mRNA was observed in
C. parvum
intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a ∼70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH
2
-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan
Toxoplasma gondii
and the mitochondrial network of the yeast
Saccharomyces cerevisiae
. When this presequence was truncated, the predicted amphiphilic α-helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in
C. parvum
.
A
Theileria annulata
DNA Binding Protein Localized to the Host Cell Nucleus Alters the Phenotype of a Bovine Macrophage Cell Line
Authors: Shiels Brian R. , McKellar Sue , Katzer Frank , Lyons Kim , Kinnaird Jane , Ward Chris , Wastling Jonathan M. , Swan David
Published: April 2004

The apicomplexan parasite
Theileria annulata
is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (
SuAT1
) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that
Theileria
parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.
LaeA, a Regulator of Secondary Metabolism inAspergillusspp
Authors: Bok Jin Woo , Keller Nancy P.
Published: April 2004

Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We present here a novelAspergillusnuclear protein, LaeA, as a global regulator of secondary metabolism in this genus. Deletion oflaeA (ΔlaeA) blocks the expression of metabolic gene clusters, including the sterigmatocystin (carcinogen), penicillin (antibiotic), and lovastatin (antihypercholesterolemic agent) gene clusters. Conversely, overexpression oflaeAtriggers increased penicillin and lovastatin gene transcription and subsequent product formation.laeAexpression is negatively regulated by AflR, a sterigmatocystin Zn2Cys6transcription factor, in a unique feedback loop, as well as by two signal transduction elements, protein kinase A and RasA. Although these last two proteins also negatively regulate sporulation, ΔlaeAstrains show little difference in spore production compared to the wild type, indicating that the primary role of LaeA is to regulate metabolic gene clusters.
Inactivation of Transcription Factor Gene
ACE2
in the Fungal Pathogen
Candida glabrata
Results in Hypervirulence
Authors: Kamran Mohammed , Calcagno Ana-Maria , Findon Helen , Bignell Elaine , Jones Michael D. , Warn Peter , Hopkins Philip , Denning David W. , Butler Geraldine , Rogers Thomas , Mühlschlegel Fritz A. , Haynes Ken
Published: April 2004

During an infection, the coordinated orchestration of many factors by the invading organism is required for disease to be initiated and to progress. The elucidation of the processes involved is critical to the development of a clear understanding of host-pathogen interactions. For
Candida
species, the inactivation of many fungal attributes has been shown to result in attenuation. Here we demonstrate that the
Candida glabrata
homolog of the
Saccharomyces cerevisiae
transcription factor gene
ACE2
encodes a function that mediates virulence in a novel way. Inactivation of
C. glabrata ACE2
does not result in attenuation but, conversely, in a strain that is hypervirulent in a murine model of invasive candidiasis.
C. glabrata ace2
null mutants cause systemic infections characterized by fungal escape from the vasculature, tissue penetration, proliferation in vivo, and considerable overstimulation of the proinflammatory arm of the innate immune response. Compared to the case with wild-type fungi, mortality occurs much earlier in mice infected with
C. glabrata ace2
cells, and furthermore, 200-fold lower doses are required to induce uniformly fatal infections. These data demonstrate that
C. glabrata ACE2
encodes a function that plays a critical role in mediating the host-
Candida
interaction. It is the first virulence-moderating gene to be described for a
Candida
species.
Strand-Specific RNA Synthesis Determinants in the RNA-Dependent RNA Polymerase of Poliovirus
Authors: Cornell Christopher T. , Perera Rushika , Brunner Jo Ellen , Semler Bert L.
Published: April 2004

The viral RNA-dependent RNA polymerase (3D
pol
) is highly conserved between the closely related enteroviruses poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3). In this study, we generated PV1/CVB3 chimeric polymerase sequences in the context of full-length poliovirus transcripts to determine the role of different subdomains within the RNA-dependent RNA polymerase of PV1 that are required for functions critical for RNA replication in vitro and in cell culture. The substitution of CVB3 sequences in the carboxy-terminal portion (thumb subdomain) of the polymerase resulted in transcripts incapable of RNA replication. In contrast, three of the seven chimeras were capable of synthesizing RNA, albeit to reduced levels compared to that of wild-type PV1 RNA. Interestingly, one of the replication-competent chimeras (CPP) displayed an inability to generate positive strands, indicating the presence of amino-terminal sequences within the 3D polymerase and/or the 3D domain of the 3CD precursor polypeptide that are necessary for the assembly of strand-specific RNA synthesis complexes. In some constructs, the partial reestablishment of PV1 amino acid sequences in this region was capable of rescuing RNA replication in vitro and in cell culture.
Human Immunodeficiency Virus (HIV) Type 1 Vpu Induces the Expression of CD40 in Endothelial Cells and Regulates HIV-Induced Adhesion of B-Lymphoma Cells
Authors: Henderson Winnie W. , Ruhl Rebecca , Lewis Paul , Bentley Matthew , Nelson Jay A. , Moses Ashlee V.
Published: April 2004

AIDS-related B-cell non-Hodgkin's lymphoma (AIDS-NHL) is a significant cause of morbidity and mortality among individuals infected with human immunodeficiency virus type 1 (HIV-1). AIDS-NHL is clinically and histologically heterogeneous, but common features include an aggressive clinical course and frequent extranodal presentation. HIV-1 infection of nonimmune cells that interact with malignant B cells at extranodal sites may influence both the development and the clinical presentation of disease. Our previous studies have shown that coculture of B-lymphoma (BL) cells with HIV-1-infected endothelial cells (EC) leads to contact activation of EC and firm BL-cell adhesion. The key event promoting EC-BL-cell adhesion was HIV-1 upregulation of endothelial CD40, which allowed induction of vascular cell adhesion molecule 1 (VCAM-1) in a CD40-dependent manner. The present study was designed to identify the HIV-1 protein(s) that influence EC-BL-cell adhesion. When HIV-1 proteins were individually expressed in EC by using recombinant adenoviruses, cultured BL cells adhered exclusively to Vpu-transduced EC. As with HIV-infected EC, adhesive properties were linked to the capacity of Vpu to upregulate CD40, which in turn allowed efficient expression of VCAM-1. When EC were infected with an HIV-1 pseudotype lacking the Vpu gene, CD40 upregulation and BL-cell adhesive properties were lost, indicating an essential role for Vpu in EC-BL-cell interactions. Thus, these data reveal a novel function for HIV-1 Vpu and further suggest a role for Vpu in the development of AIDS-NHL at EC-rich extranodal sites.
Cross-Dressing the Virion: the Transcapsidation of Adeno-Associated Virus Serotypes Functionally Defines Subgroups
Authors: Rabinowitz Joseph E. , Bowles Dawn E. , Faust Susan M. , Ledford Julie G. , Cunningham Scott E. , Samulski R. Jude
Published: April 2004

For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this observed synergy: (i) heparin-mediated binding from AAV2 and (ii) an unidentified enhancement activity from AAV1 structural proteins. Using this procedure of mixing different AAV helper plasmids to generate “cross-dressed” AAV virions, we propose an additional means of classifying new AAV serotypes into subgroups based on functional approaches to analyze AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom-designed AAV vectors should be of significant value to the field of gene therapy.
Smallpox DNA Vaccine Protects Nonhuman Primates against Lethal Monkeypox
Authors: Hooper J. W. , Thompson E. , Wilhelmsen C. , Zimmerman M. , Ichou M. Ait , Steffen S. E. , Schmaljohn C. S. , Schmaljohn A. L. , Jahrling P. B.
Published: April 2004

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.
Human Cytomegalovirus Induces Monocyte Differentiation and Migration as a Strategy for Dissemination and Persistence
Authors: Smith M. Shane , Bentz Gretchen L. , Alexander J. Steven , Yurochko Andrew D.
Published: April 2004

Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.
The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3
Authors: Sirena Dominique , Lilienfeld Benjamin , Eisenhut Markus , Kälin Stefan , Boucke Karin , Beerli Roger R. , Vogt Lorenz , Ruedl Christiane , Bachmann Martin F. , Greber Urs F. , Hemmi Silvio
Published: April 2004

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.
Comprehensive Analysis of Human Immunodeficiency Virus Type 1-Specific CD4 Responses Reveals Marked Immunodominance ofgagandnefand the Presence of Broadly Recognized Peptides
Authors: Kaufmann Daniel E. , Bailey Paul M. , Sidney John , Wagner Bradford , Norris Philip J. , Johnston Mary N. , Cosimi Lisa A. , Addo Marylyn M. , Lichterfeld Mathias , Altfeld Marcus , Frahm Nicole , Brander Christian , Sette Alessandro , Walker Bruce D. , Rosenberg Eric S.
Published: April 2004

Increasing evidence suggests that human immunodeficiency virus type 1 (HIV-1)-specific CD4 T-cell responses contribute to effective immune control of HIV-1 infection. However, the breadths and specificities of these responses have not been defined. We screened fresh CD8-depleted peripheral blood mononuclear cells (PBMC) from 36 subjects at different stages of HIV-1 infection for virus-specific CD4 responses by gamma interferon enzyme-linked immunospot assay, using 410 overlapping peptides spanning all HIV-1 proteins (based on the clade B consensus sequence). HIV-1-specific CD4 responses were identified in 30 of the 36 individuals studied, with the strongest and broadest responses detected in persons treated in acute infection who underwent treatment interruption. In individuals with identified responses, the total number of recognized HIV-1 peptides ranged from 1 to 36 (median, 7) and the total magnitude of responses ranged from 80 to >14,600 (median, 990) spot-forming cells/106CD8-depleted PBMC. Neither the total magnitude nor the number of responses correlated with viremia. The most frequent and robust responses were directed against epitopes within the Gag and Nef proteins. Peptides targeted by ≥25% of individuals were then tested for binding to a panel of common HLA-DR molecules. All bound broadly to at least four of the eight alleles tested, and two bound to all of the HLA-DR molecules studied. Fine mapping and HLA restriction of the responses against four of these peptides showed a combination of clustering of epitopes and promiscuous presentation of the same epitopes by different HLA class II alleles. These findings have implications for the design of immunotherapeutic strategies and for testing candidate HIV vaccines.
The Hypervariable Region 1 of the E2 Glycoprotein of Hepatitis C Virus Binds to Glycosaminoglycans, but This Binding Does Not Lead to Infection in a Pseudotype System
Authors: Basu Arnab , Beyene Aster , Meyer Keith , Ray Ranjit
Published: April 2004

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein is a 27-amino-acid sequence located at its N terminus. In this study, we investigated the functional role of HVR1 for interaction with the mammalian cell surface. The C-terminal truncated E2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein for generation of the chimeric E2-G gene construct. A deletion of the HVR1 sequence from E2 was created for the construction of E2ΔHVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2ΔHVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2ΔHVR1-G had a reduced plaquing efficiency (∼50%) in HepG2 cells compared to that for the E2-G virus. Cells prior treated with pronase (0.5 U/ml) displayed a complete inhibition of infectivity of the E2ΔHVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2ΔHVR1-G pseudotypes were not sensitive to heparin (6 to 50 μg/ml) as an inhibitor of plaque formation compared to the E2-G pseudotype virus. Although the HVR1 sequence itself does not match with the known heparin-binding domain, a synthetic peptide representing 27 amino acids of the E2 HVR1 displayed a strong affinity for heparin in an enzyme-linked immunosorbent assay. This binding was competitively inhibited by a peptide from the V3 loop of a human immunodeficiency virus glycoprotein subunit (gp120) known to bind with cell surface heparin. Taken together, our results suggest that the HVR1 of E2 glycoprotein binds to the cell surface proteoglycans and may facilitate virus-host interaction for replication cycle of HCV.
Functional and Structural Similarities between the Internal Ribosome Entry Sites of Hepatitis C Virus and Porcine Teschovirus, a Picornavirus
Authors: Pisarev Andrey V. , Chard Louisa S. , Kaku Yoshihiro , Johns Helen L. , Shatsky Ivan N. , Belsham Graham J.
Published: April 2004

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5′ untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA
i
Met
. Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.
Activation of the NF-κB Pathway in Human Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major Immediate-Early Promoter
Authors: DeMeritt Ian B. , Milford Liesl E. , Yurochko Andrew D.
Published: April 2004

We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-κB. Here, we investigate the mechanism for the HCMV-induced NF-κB activation and the role that the induced NF-κB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-κB, the IκB-superrepressor, we demonstrated that active NF-κB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-κB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-κB, IκBα and IκBβ. Because the IκB kinases (IKKs) regulate the degradation of the IκBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-κB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-κB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.
A Chimeric Protein of Simian Immunodeficiency Virus Envelope Glycoprotein gp140 andEscherichia coliAspartate Transcarbamoylase
Authors: Chen Bing , Cheng Yifan , Calder Lesley , Harrison Stephen C. , Reinherz Ellis L. , Skehel John J. , Wiley Don C.
Published: April 2004

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) ofEscherichia coliaspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R2) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C6R6ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.