bluuebunny/crossref_metadata_2025 huggingface dataset:
Guinness-Bound
Authors: Beckman Mary
Published: December 2003

Living a long time means nothing without good health, and until now, the nematode worms with the greatest life spans have been sickly. Now, researchers have created extremely long-lived worms that are spry to boot. Neutering worms after slashing the amount of a particular protein makes them last six times longer than normal. The work suggests that animals don't have to give up good health to achieve remarkably long life.
Detangling Alzheimer's Disease
Authors: Helmuth Laura
Published: December 2003

Alzheimer's disease afflicts 4.5 million people in the United States, and the number is expected to rise to 16 million by the year 2050, as the population ages. Researchers are scrambling to find genetic risk factors, decipher disease mechanisms, and develop reliable diagnostic tests that detect the illness at its earliest, potentially most treatable stage. Using these findings, they hope to devise new therapeutic approaches. Current clinical trials are assessing novel techniques that stall or reverse Alzheimer-like neuropathology in mice.
JNK-ing Cellular Poisons
Authors: Leslie Mitch
Published: December 2003

Adults who are under pressure at work or harried by bill collectors don't usually need their childhood pacifiers or stuffed animals to cope. But organisms do seem to revisit their youth when they need to battle stress caused by metabolic byproducts, according to a new study. The work suggests that fruit flies reuse a molecular pathway that's crucial during embryonic development to orchestrate the adult's defenses against cell-wounding reactive oxygen species.
Crypt-ic Message
Authors: Davenport R. John
Published: December 2003

Dark crypts swarming with cloned mutants: It's not the premise for a grade B horror film but the result of a new study. The work is the first to reveal that intestinal stem cells amass DNA mutations in their mitochondria, and it raises the possibility that the accumulation of such defects as we age could choke stem cells.
Strategies for Engineered Negligible Senescence
Authors: Gray Douglas A.
Published: December 2003

This Perspective summarizes presentations at the 10th Congress of the International Association of Biomedical Gerontology, which was held in Cambridge, England in September 2003. The seminar topics ranged from telomeres to ethics, stem cells to limb regeneration. The meeting organizer, Aubrey de Grey, is a leading proponent of life extension through biological engineering.
Brain Lubricant
Authors: Davenport R. John
Published: December 2003

When the body loses control of cholesterol, the heart isn't the only victim; the brain also suffers (see "
Greasing Aging's Downward Slide
"). In preliminary work presented here on 6 November at the Neurobiology of Aging meeting, researchers revealed new details of how a protein that fights fat buildup in blood vessels helps keep the brain healthy.
Just a Little Bit
Authors: Davenport R. John
Published: December 2003

Cholesterol fighters, heart medicine, arthritis pills--as people age, they need more and more medications to battle an escalating number of ailments. But in the elderly, drugs often lose effectiveness or gain toxicity. Changes in the production of drug-deactivating machinery might be partly to blame. A new study shows that nudging growth-hormone concentrations gives young rats an old complement of enzymes.
Dense Matter
Authors: Beckman Mary
Published: December 2003

SAN DIEGO, CALIFORNIA
--Irregularly placed studs can weaken a house's frame. A small change in a bone protein similarly disrupts the skeleton and dramatically reduces its strength, according to new research presented here 23 November 2003 at the Gerontological Society of America annual meeting. The results reveal that a characteristic other than density enfeebles bone.
Does the Road to Muscle Rejuvenation Go Through Notch?
Authors: Miller Jeffrey Boone , Emerson Jr. Charles P.
Published: December 2003

The capacity of skeletal muscles to repair and regenerate declines during aging in humans, and this decline may lead to muscle loss and frailty. Conboy
et al.
show that injured muscles of aging mice are defective in Notch signaling, because up-regulation of the Notch ligand, Delta-1, is impaired. Delta-1 promotes proliferation of the satellite cells that repair damaged muscles, and Conboy
et al.
show that experimental activation of Notch signaling is sufficient to reverse the age-related decline in muscle regenerative capacity. Extension of these important findings to humans could lead to the development of new therapeutic approaches to maintain muscle function during aging.
Mission (Not) Impossible
Authors: Chen Ingfei
Published: December 2003

Before he became an endocrinologist, Nir Barzilai served as a medic in the Israeli Defense Forces and spent time in Cambodia and South Africa treating victims of war, poverty, and disease. Today, 13 years after immigrating to the United States, he directs the Institute for Aging Research at Albert Einstein College of Medicine in New York City, where he explores the metabolic consequences of aging in rodents and the genetics of longevity in extremely old Ashkenazi Jews. Barzilai credits his military training with giving him the confidence and drive to pursue risky research proposals that are initially unlikely to win major funding. His top priority these days is mentoring young scientists in gerontology.
Can New Neurons Replace Memories Lost?
Authors: Shors Tracey J.
Published: December 2003

It now appears that new neurons are produced not only in the healthy adult brain but also in the brains of patients with Alzheimer's disease (AD). Although the function of new neurons in the healthy brain is unknown, there is evidence that they are involved in certain types of memory formation and that their survival is enhanced dramatically and persistently by learning experiences. The evidence that neurogenesis, or at least the expression of proteins unique to immature neurons, occurs during the late stages of AD raises the possibility that their production could be enhanced earlier in the disease process before so many neurons and memories are lost.
Out, Out, Damned Zinc
Authors: Leslie Mitch
Published: December 2003

Michael Jordan returned from retirement to lead the Chicago Bulls to three straight NBA championships, and researchers hope that a chemical can pull off a similarly splashy comeback and challenge Alzheimer's disease. The first clinical trials of the drug, an antibiotic that was retired more than 30 years ago, suggest that it slows progression of the illness in the most severely affected patients. The work also supports the hypothesis that jumbled metal metabolism in the older brain promotes deterioration.
Fear Factor
Authors: Davenport R. John
Published: December 2003

Shrinking violets, beware: An aversion to novelty presages an early death, according to new work. The research reveals that fearless rodents outlive their nervous brothers, perhaps because trepidation triggers the release of stress molecules.
Editing of CD1d-Bound Lipid Antigens by Endosomal Lipid Transfer Proteins
Authors: Zhou Dapeng , Cantu Carlos , Sagiv Yuval , Schrantz Nicolas , Kulkarni Ashok B. , Qi Xiaoyang , Mahuran Don J. , Morales Carlos R. , Grabowski Gregory A. , Benlagha Kamel , Savage Paul , Bendelac Albert , Teyton Luc
Published: December 2003

It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Vα14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a “tug-of-war” model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.
PDE4: Arrested at the Border
Authors: Brunton Laurence L.
Published: December 2003

Compartmentation plays a critical role in determining the specificity and efficacy of signaling by adenosine 3′,5′-monophosphate (cAMP) and other second messengers. It has become apparent that all of the protein components of the cAMP signaling pathway are subject to regulation and may be localized to particular subcellular compartments. This Perspective discusses recent research concerning the importance of cAMP phosphodiesterase localization in cAMP signaling, functional implications of the organization of this pathway's protein components into macromolecular complexes, and common themes in the compartmentation of cAMP- and Ca
2+
-mediated signaling pathways.
Coupling the Iron-Responsive Element to GFP--An Inducible System to Study Translation in a Single Living Cell
Authors: Goetze Bernhard , Grunewald Barbara , Kiebler Michael A. , Macchi Paolo
Published: December 2003

Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3′-untranslated region of calcium/calmodulin-dependent protein kinase IIα mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.
Signal Transduction and Genes-to-Behaviors Pathways in Psychiatric Diseases
Authors: Manji Husseini K. , Gottesman Irving I. , Gould Todd D.
Published: December 2003

Although psychiatric diseases are among the most common and destructive of all human illnesses, the molecular and cellular mechanisms underlying their complex origins remain to be elucidated. Dysfunction of critical intracellular signaling pathways is very likely to be involved. This conclusion is based on a number of observations, including the short- and long-term cellular effects of psychiatric drugs; the critical role signaling pathways play in neurotransmitter, neuropeptide, and neurohormone communication; and the fact that signaling pathways are principle regulators of the diverse array of behavioral symptoms experienced by patients. The genomics era has brought to psychiatry an abundance of genetic linkage and candidate gene findings. The difficult task--now under way--is to discern the functional relevance of these results. Recent evidence suggests the involvement of the ubiquitous protein phosphatase 2B (calcineurin), a critical regulator of many signal transduction pathways, as a schizophrenia susceptibility gene. It is likely that genetic findings in severe psychiatric disorders will continue to implicate direct and indirect modulation of critical intracellular signaling pathways.
Organization and Functions of Interacting Domains for Signaling by Protein-Protein Interactions
Authors: Buck Elizabeth , Iyengar Ravi
Published: December 2003

Information transfer from activated heterotrimeric guanine nucleotide-binding proteins (G proteins) to downstream effectors occurs through noncovalent protein-protein interactions. Such interactions involve multiple regions of contact between the G protein and the effector. Some of these regions mediate information transfer, as defined by their ability to change the activity of their downstream binding partners, whereas other interactions appear to contribute solely to binding affinity. Such modular configurations occur in functionally diverse proteins such as myosin and a regulator of the double-stranded DNA stimulated protein kinase (PKR) called PACT. In most cases, it appears that both charge complementarity and the architecture of the interacting surfaces provide the appropriate balance between specificity of interactions and their reversibility. Information transfer regions appear to display conformational flexibility in interactions. Such flexible interactions may be essential for the local conformational changes necessary to induce change in activity by an induced fit-type mechanism. Thus, a general mechanism for information transfer by protein-protein interactions could use specific regions that induce conformation changes in the downstream partner. Other binding regions may be arranged within the protein to impart specificity of recognition and thereby maintain overall contact between the partners during the conformational dynamics that occur in the signal-transfer process.
Spine-Tingling Excitement from Glutamate Receptors
Authors: Huettner James E.
Published: December 2003

Ionotropic glutamate receptors excite nerve cells by forming cation-selective pores in the membrane. Recent work, however, provides evidence that atypical signaling by glutamate receptors regulates the production and maintenance of dendritic spines, the short outgrowths that receive most central excitatory synapses. The control of spine formation involves the amino-terminal extracellular domain of the GluR2 subunit of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptors. How interactions with this domain elicit signals to downstream effectors remains to be elucidated, but ion flux through the channel may not be required. This Perspective discusses the possibility that regulation of spines by GluR2 may be one of a growing collection of cases in which ionotropic glutamate receptors can elicit biochemical changes that are conventionally attributed to metabotropic receptors. It is suggested that proteins in contact with specific glutamate receptor subunits may directly sense the conformational changes produced by agonist binding.
Miniature Transmitter Release: Accident of Nature or Careful Design?
Authors: Otsu Yo , Murphy Timothy H.
Published: December 2003

Miniature transmitter release results from the constitutive low-level release of individual vesicles of neurotransmitter. Since the 1950s, this form of synaptic transmission has largely been thought to reflect a leaky evoked-release mechanism, and it was not clear whether it had a function of its own. Recent data challenge this view and suggest that miniature release can affect both the local chemistry of synapses and the network properties of neurons.
Finding Partners: Emerging Protein Interaction Technologies Applied to Signaling Networks
Authors: Stagljar Igor
Published: December 2003

Signal transduction pathways play essential roles in cell differentiation, proliferation, and survival. Their precise regulation is achieved through specific protein-protein interactions that mediate the assembly of protein complexes in response to different signals. Disturbances of the normal protein-protein interactions within signaling pathways can lead to many diseases, including cancer. In this review, Stagljar aims to highlight the emerging protein interaction technologies that are advancing the field of signal transduction. These approaches include stable isotope labeling by amino acids in cell culture, SH2 profiling, target-assisted iterative screening, and the split-ubiquitin membrane yeast two-hybrid system. Although still at an early stage, these technologies show promise as useful methods for the characterization of novel components of various signal transduction pathways.
Isolation and Characterization ofBurkholderia cenocepaciaMutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability
Authors: Farmer Kate L. , Thomas Mark S.
Published: December 2003

The opportunistic pathogenBurkholderia cenocepaciaproduces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenizedB. cenocepaciawith the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. ThecysWgene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.
Osmoregulatory Systems ofEscherichia coli:Identification of Betaine-Carnitine-Choline Transporter Family MemberBetU and Distributions ofbetUandtrkGamongPathogenic and NonpathogenicIsolates
Authors: Ly Anh , Henderson James , Lu Annie , Culham Doreen E. , Wood Janet M.
Published: December 2003

Multiple transporters mediate osmoregulatory solute accumulation inEscherichia coliK-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K+(micromolar concentrations) and retained locustrkH, which encodes an osmoregulatory K+transporter. Both lacked thetrkHhomologuetrkG. Transporters ProP and ProU account for all glycine-betaine uptake activity inE. coliK-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. GenebetUwas isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence oftrkGandbetUwithin the ECOR collection, representatives of theE. colipathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. GenetrkGwas present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. GenebetUwas more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). GenestrkGandbetUmay have been acquired by lateral gene transfer, sincetrkGis part of theracprophage andbetUis flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the speciesE. coli, but they are not characteristic of a particular phylogenetic group or pathotype.
The BH1999 Protein of
Bacillus halodurans
C-125 Is Gentisyl-Coenzyme A Thioesterase
Authors: Zhuang Zhihao , Song Feng , Takami Hideto , Dunaway-Mariano Debra
Published: December 2003

In this study, we have shown that recombinant BH1999 from
Bacillus halodurans
catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a
k
cat
/

K
m

of 1.6 × 10
6
M
−1
s
−1
and the hydrolysis of 3-hydroxybenzoyl-CoA with a
k
cat
/

K
m

of 3.0 × 10
5
M
−1
s
−1
. All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.
The Genome-Sequenced Variant of
Campylobacter jejuni
NCTC 11168 and the Original Clonal Clinical Isolate Differ Markedly in Colonization, Gene Expression, and Virulence-Associated Phenotypes
Authors: Gaynor Erin C. , Cawthraw Shaun , Manning Georgina , MacKichan Joanna K. , Falkow Stanley , Newell Diane G.
Published: December 2003

The genome sequence of the enteric bacterial pathogen
Campylobacter jejuni
NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of
C. jejuni
-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods. These observations highlight the capacity of
C. jejuni
to adapt to multiple environmental niches, the likelihood that this adaptation involves genetic evolution, and provides the first whole-genome molecular exploration of the effect of laboratory culture and storage on colonization and virulence properties of this pathogen.
Sequence Analysis of the Mobile Genome Island pKLC102 of
Pseudomonas aeruginosa
C
Authors: Klockgether Jens , Reva Oleg , Larbig Karen , Tümmler Burkhard
Published: December 2003

The
Pseudomonas aeruginosa
plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with
P. aeruginosa
clone K chromosomes at one of the two tRNA
Lys
genes, pKLC102 is incorporated into the tRNA
Lys
gene only close to the
pilA
locus. Targeting of the other tRNA
Lys
copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred
oriV
and genes for replication, partitioning, and conjugation, including a
pil
cluster encoding type IV thin sex pili and an 8,524-bp
chvB
glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase,
att
, and a syntenic set of conserved hypothetical genes also observed in the tRNA
Gly
-associated genome islands of
P. aeruginosa
clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS
6100
elements. Intramolecular transposition of a copy of IS
6100
led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in
P. aeruginosa
clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.
Pseudomonas syringae
Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter
Authors: Schechter Lisa M. , Roberts Kathy A. , Jamir Yashitola , Alfano James R. , Collmer Alan
Published: December 2003

Pseudomonas syringae
pv. tomato strain DC3000 is a pathogen of tomato and
Arabidopsis
. The
hrp
-
hrc
-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most
avr
or
hop
genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of
P. syringae
TTSS effectors into plant cells. This system includes a cloned
P. syringae hrp
gene cluster and the model plant
Nicotiana benthamiana
. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the
P. syringae
Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes
hopPtoS4
::
tnpA
and
hopPtoAG
::
tnpA
. We also confirmed that HopPtoK, HopPtoC, and AvrPphE
Pto
are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in
P. syringae.
Surface Layer Protein EA1 Is Not a Component of
Bacillus anthracis
Spores but Is a Persistent Contaminant in Spore Preparations
Authors: Williams David D. , Turnbough Charles L.
Published: December 2003

EA1 is an abundant, highly antigenic, surface layer protein of
Bacillus anthracis
vegetative cells. Recent studies indicate that EA1 is also a component of
B. anthracis
spores and a potential marker for spore detection. We show here that EA1 is not a spore component but a persistent contaminant in spore preparations.
Expression and Regulation of a Silent Operon,
hyf
, Coding for Hydrogenase 4 Isoenzyme in
Escherichia coli
Authors: Self William T. , Hasona Adnan , Shanmugam K. T.
Published: December 2003

On the basis of
hyf-lacZ
fusion studies, the
hyf
operon of
Escherichia coli
, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the
hyc
-encoded hydrogenase 3 isoenzyme) activated
hyf-lacZ
. HyfR, an FhlA homolog encoded by the
hyfR
gene present at the end of the
hyf
operon, also activated transcription of
hyf-lacZ
but did so only when
hyfR
was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H
2
production. Optimum expression of
hyf-lacZ
required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.
Increased G→A Transition Frequencies Displayed by Primer Grip Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Cases-González Clara E. , Menéndez-Arias Luis
Published: December 2003

A genetic screen based on the blue-white β-galactosidase complementation assay designed to detect G→A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.
Impacts of Epitope Expression Kinetics and Class I Downregulation on the Antiviral Activity of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes
Authors: Ali Ayub , Lubong Rachel , Ng Hwee , Brooks David G. , Zack Jerome A. , Yang Otto O.
Published: December 2003

The determinants of CD8
+
cytotoxic T-lymphocyte (CTL) antiviral activity against human immunodeficiency virus type 1 (HIV-1) remain poorly defined. Although recent technological advances have markedly enhanced the ability to detect HIV-1-specific T cells, commonly used assays do not reveal their direct interaction with virus. We investigated two determinants of CTL antiviral efficiency by manipulating HIV-1 and measuring the effects on CTL suppression of viral replication in acutely infected cells. Translocation of a Gag epitope into the early protein Nef markedly increased the activity of CTL recognizing that epitope, in comparison to HIV-1 expressing the epitope normally in the late protein Gag. Because this epitope translocation resulted not only in earlier expression but also in loss of major histocompatibility complex class I downregulation by Nef, the activities of CTL against a panel of viral constructs differing in kinetics of epitope expression and class I downmodulation were compared. The results indicated that both the timing of epitope expression and the reduction of class I have profound effects on the ability of CTL to suppress HIV-1 replication in acutely infected cells. The epitope targeting of CTL and viral control of class I therefore likely play important roles in the ability of CTL to exert pressure on HIV-1.
Identification of an Extracellular Domain within the Human PiT2 Receptor That Is Required for Amphotropic Murine Leukemia Virus Binding
Authors: Feldman Steven A , Farrell Karen B. , Murthy Ravi K. , Russ Jill L. , Eiden Maribeth V.
Published: December 2003

Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.
Competitive Fitness of Nevirapine-Resistant Human Immunodeficiency Virus Type 1 Mutants
Authors: Collins Jennifer A. , Thompson M. Gregory , Paintsil Elijah , Ricketts Melisa , Gedzior Joanna , Alexander Louis
Published: December 2003

Determining the fitness of drug-resistant human immunodeficiency virus type 1 (HIV-1) strains is necessary for the development of population-based studies of resistance patterns. For this purpose, we have developed a reproducible, systematic assay to determine the competitive fitness of HIV-1 drug-resistant mutants. To demonstrate the applicability of this assay, we tested the fitness of the five most common nevirapine-resistant mutants (103N, 106A, 181C, 188C, and 190A), with mutations in HIV-1 reverse transcriptase (RT), singly and in combination (for a total of 31 variants) in a defined HIV-1 background. For these experiments, the 27 RT variants that produced viable virus were cocultured with wild-type virus without nevirapine. The ratios of the viral species were determined over time by utilization of a quantitative real-time RT-PCR-based assay. These experiments revealed that all of the viable variants were less fit than the wild type and demonstrated that the order of relative fitness of the single mutants tested was as follows: 103N > 181C > 190A > 188C > 106A. This order correlated with the commonality of these mutants as a result of nevirapine monotherapy. These investigations also revealed that, on average, the double mutants were less fit than the single mutants and the triple mutants were less fit than the double mutants. However, the fitness of the single and double mutants was often not predictive of the fitness of the derivative triple mutants, suggesting the presence of complex interactions between the closely aligned residues that confer nevirapine resistance. This complexity was also evident from the observation that all three of the replication-competent quadruple mutants were fitter than most of the triple mutants, and in some cases, even the double mutants. Our data suggest that, in many cases, viral fitness is the determining factor in the evolution of nevirapine-resistant mutants in vivo, that interactions between the residues that confer nevirapine resistance are complex, and that these interactions substantially affect reverse transcriptase structure and/or function.
Genetic and Biochemical Analysis of
cis
Regulatory Elements within the Keratinocyte Enhancer Region of the Human Papillomavirus Type 31 Upstream Regulatory Region during Different Stages of the Viral Life Cycle
Authors: Sen Ellora , Alam Samina , Meyers Craig
Published: December 2003

Using linker scanning mutational analysis, we recently identified potential
cis
regulatory elements contained within the 5′ upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the viral life cycle. For the present study, we extended the linker scanning mutational analysis to identify potential
cis
elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the viral life cycle. The linker scanning mutational analysis identified viral
cis
elements located in the KE region that regulate transcription in the presence and absence of any viral gene products or viral DNA replication and determine the role of host tissue differentiation on viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same
cis
regulatory elements at different stages of the HPV differentiation-dependent life cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify
cis
regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the viral life cycle. The variable recruitment of transcription factors to the same
cis
element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.
cis
-Acting Sequences That Contribute to the Synthesis of Relaxed-Circular DNA of Human Hepatitis B Virus
Authors: Liu Ning , Ji Lin , Maguire Megan L. , Loeb Daniel D.
Published: December 2003

Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of
cis
-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about
cis
-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for
cis
-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5′r, and 3′r were not analyzed in the study. Results from Southern blotting showed that multiple
cis
-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of
cis
-acting sequences that contribute to the synthesis of HBV RC DNA.
Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages
Authors: Lu Richard , Nakajima Noriko , Hofmann Wolfgang , Benkirane Monsef , Teh-Jeang Kuan , Sodroski Joseph , Engelman Alan
Published: December 2003

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into thenefposition of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.
Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition
Authors: Wang Wenyan , Lahser Frederick C. , Yi MinKyung , Wright-Minogue Jacquelyn , Xia Ellen , Weber Patricia C. , Lemon Stanley M. , Malcolm Bruce A.
Published: December 2003

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative
trans
cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.
Expansion of a Unique Region in the Marek's Disease Virus Genome Occurs Concomitantly with Attenuation but Is Not Sufficient To Cause Attenuation
Authors: Silva R. F. , Reddy S. M. , Lupiani B.
Published: December 2003

Pathogenic Marek's disease viruses (MDVs) have two head-to-tail copies of a 132-bp repeat. As MDV is serially passaged in cell culture, the virus becomes attenuated and the number of copies of the 132-bp repeat increases from 2 to often more than 20 copies. To determine the role of the repeats in attenuation, we used five overlapping cosmid clones that spanned the MDV genome to reconstitute infectious virus (rMd5). By mutating the appropriate cosmids, we generated clones of infectious MDVs that contained zero copies of the 132-bp repeats, rMd5(Δ132); nine copies of the 132-bp repeats, rMd5(9-132); and nine copies of the 132-bp repeats inserted in the reverse orientation, rMd5(rev9-132). After two passages in cell culture, wild-type Md5, rMd5, and rMd5(Δ132) were stable. However, rMd5(9-132) and rMd5(rev9-132) contained a population of viruses that contained from 3 to over 20 copies of the repeats. A major 1.8-kb mRNA, containing two copies of the 132-bp repeat, was present in wild-type Md5 and rMd5 but was not present in rMd5(Δ132), rMd5(9-132), rMd5(rev9-132), or an attenuated MDV. Instead, the RNAs transcribed from the 132-bp repeat region in rMd5(9-132) and rMd5(rev9-132) closely resembled the pattern of RNAs transcribed in attenuated MDVs. When inoculated into susceptible day-old chicks, all viruses produced various lesions. Thus, expansion of the number of copies of 132-bp repeats, which accompanies attenuation, is not sufficient in itself to attenuate pathogenic MDVs.
Neutrality of the Canonical NF-κB-Dependent Pathway for Human and Murine Cytomegalovirus Transcription and Replication In Vitro
Authors: Benedict Chris A. , Angulo Ana , Patterson Ginelle , Ha Sukwon , Huang Huang , Messerle Martin , Ware Carl F. , Ghazal Peter
Published: December 2003

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor κB (NF-κB) after infection of fibroblast and macrophage cells. NF-κB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-κB in transient-transfection assays. Here we investigate whether the NF-κB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-κB-alpha (IκBαM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-κB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-κB pathway and binding of NF-κB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.