bluuebunny/crossref_metadata_2025 huggingface dataset:
A Methodology for Validating Numerical Ground Water Models
Authors: Hassan Ahmed E.
Published: December 2005

Ground water validation is one of the most challenging issues facing modelers and hydrogeologists. Increased complexity in ground water models has created a gap between model predictions and the ability to validate or build confidence in predictions. Specific procedures and tests that can be easily adapted and applied to determine the validity of site‐specific ground water models do not exist. This is true for both deterministic and stochastic models, with stochastic models posing the more difficult validation problem. The objective of this paper is to propose a general validation approach that addresses important issues recognized in previous validation studies, conferences, and symposia. The proposed method links the processes for building, calibrating, evaluating, and validating models in an iterative loop.

The approach focuses on using collected validation data to reduce uncertainty in the model and narrow the range of possible outcomes. This method is designed for stochastic numerical models utilizing Monte Carlo simulation approaches, but it can be easily adapted for deterministic models. The proposed methodology relies on the premise that absolute validity is not theoretically possible, nor is it a regulatory requirement. Rather, the proposed methodology highlights the importance of testing various aspects of the model and using diverse statistical tools for rigorous checking and confidence building in the model and its predictions. It is this confidence that will encourage regulators and the public to accept decisions based on the model predictions. This validation approach will be applied to a model, described in this paper, dealing with an underground nuclear test site in rural Nevada.
Magnetic Resonance Sounding Applied to Aquifer Characterization
Authors: Legchenko Anatoly , Baltassat Jean‐Michel , Bobachev Alexey , Martin Charlotte , Robain Henri , Vouillamoz Jean‐Michel
Published: December 2005

Magnetic resonance sounding (MRS) is distinguished from other geophysical tools used for ground water investigation by the fact that it measures a magnetic resonance signal generated directly from subsurface water molecules. An alternating current pulse energizes a wire loop on the ground surface and the MRS signal is generated; subsurface water is indicated, with a high degree of reliability, by nonzero amplitude readings. Measurements with varied pulse magnitudes then reveal the depth and thickness of water saturated layers. The hydraulic conductivity of aquifers can also be estimated using boreholes for calibration. MRS can be used for both predicting the yield of water supply wells and for interpolation between boreholes, thereby reducing the number of holes required for hydrogeological modeling. An example of the practical application of MRS combined with two‐dimensional electrical imaging, in the Ker‐bernez and Kerien catchments area of France, demonstrates the efficiency of the technique.
Effects of Model Sensitivity and Nonlinearity on Nonlinear Regression of Ground Water Flow
Authors: Yager Richard M.
Published: December 2005

Nonlinear regression is increasingly applied to the calibration of hydrologic models through the use of perturbation methods to compute the Jacobian or sensitivity matrix required by the Gauss‐Newton optimization method. Sensitivities obtained by perturbation methods can be less accurate than those obtained by direct differentiation, however, and concern has arisen that the optimal parameter values and the associated parameter covariance matrix computed by perturbation could also be less accurate.

Sensitivities computed by both perturbation and direct differentiation were applied in nonlinear regression calibration of seven ground water flow models. The two methods gave virtually identical optimum parameter values and covariances for the three models that were relatively linear and two of the models that were relatively nonlinear, but gave widely differing results for two other nonlinear models. The perturbation method performed better than direct differentiation in some regressions with the nonlinear models, apparently because approximate sensitivities computed for an interval yielded better search directions than did more accurately computed sensitivities for a point. The method selected to avoid overshooting minima on the error surface when updating parameter values with the Gauss‐Newton procedure appears for nonlinear models to be more important than the method of sensitivity calculation in controlling regression convergence.
Temporal Changes in Water Quality at a Childhood Leukemia Cluster
Authors: Seller Ralph L.
Published: December 2005

Since 1997, 15 cases of acute lymphocytic leukemia and one case of acute myelocytic leukemia have been diagnosed in children and teenagers who live, or have lived, in an area centered on the town of Fallon, Nevada. The expected rate for the population is about one case every five years. In 2001, 99 domestic and municipal wells and one industrial well were sampled in the Fallon area. Twenty‐nine of these wells had been sampled previously in 1989. Statistical comparison of concentrations of major ions and trace elements in those 29 wells between 1989 and 2001 using the nonparametric Wilcoxon signed‐rank test indicate water quality did not substantially change over that period; however, short‐term changes may have occurred that were not detected. Volatile organic compounds were seldom detected in ground water samples and those that are regulated were consistently found at concentrations less than the maximum contaminant level (MCL). The MCL for gross‐alpha radioactivity and arsenic, radon, and uranium concentrations were commonly exceeded, and sometimes were greatly exceeded. Statistical comparisons using the nonparametric Wilcoxon rank‐sum test indicate gross‐alpha and ‐beta radioactivity, arsenic, uranium, and radon concentrations in wells used by families having a child with leukemia did not statistically differ from the remainder of the domestic wells sampled during this investigation. Isotopic measurements indicate the uranium was natural and not the result of a 1963 underground nuclear bomb test near Fallon. In arid and semiarid areas where trace‐element concentrations can greatly exceed the MCL, household reverse‐osmosis units may not reduce their concentrations to safe levels. In parts of the world where radon concentrations are high, water consumed first thing in the morning may be appreciably more radioactive than water consumed a few minutes later after the pressure tank has been emptied because secular equilibrium between radon and its immediate daughter progeny is attained in pressure tanks overnight.
Genetic Transplantation:
Salmonella enterica
Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems
Authors: Karlinsey Joyce E. , Hughes Kelly T.
Published: December 2005

In
Salmonella enterica
serovar Typhimurium,σ
28
and anti-sigma factor FlgM are regulatory
proteins crucial for flagellar biogenesis and motility. In this study,
we used
S. enterica
serovar Typhimurium as an in vivo
heterologous system to study σ
28
and
anti-σ
28
interactions in organisms where genetic
manipulation poses a significant challenge due to special growth
requirements. The chromosomal copy of the
S. enterica
serovar
Typhimurium σ
28
structural gene
fliA
was
exchanged with homologs of
Aquifex aeolicus
(an extreme
thermophile) and
Chlamydia trachomatis
(an obligate
intracellular pathogen) by targeted replacement of a
tetRA
element in the
fliA
gene location using λ-Red-mediated
recombination. The
S. enterica
serovar Typhimurium hybrid
strains showed σ
28
-dependent gene expression,
suggesting that σ
28
activities from diverse species
are preserved in the heterologous host system.
A. aeolicus
mutants defective for σ
28
/FlgM interactions were
also isolated in
S. enterica
serovar Typhimurium. These
studies highlight a general strategy for analysis of protein function
in species that are otherwise genetically intractable and a
straightforward method of chromosome restructuring usingλ
-Red-mediated
recombination.
CcpC-Dependent Regulation ofcitBand lmo0847 inListeria monocytogenes
Authors: Kim Hyun-Jin , Mittal Meghna , Sonenshein Abraham L.
Published: December 2005

InBacillus subtilis, the catabolite control protein C (CcpC) plays a critical role in regulating the genes encoding the enzymes of the tricarboxylic acid branch of the Krebs citric acid cycle. A gene encoding a potential CcpC homolog and two potential target genes were identified in theListeria monocytogenesgenome. In vitro gel mobility shift assays and DNase I footprinting experiments showed thatL. monocytogenesCcpC (CcpCLm) interacts with the promoter regions ofcitBLm(the gene that is likely to encode aconitase) and lmo0847 (encoding a possible glutamine transporter) and that citrate is a specific inhibitor of this interaction. To study in vivo promoter activity, a newlacZreporter system was developed. This system allows stable integration into the chromosome of a promoter region transcriptionally fused to a promoterlesslacZgene at a nonessential, ectopic locus. Analysis of strains carrying acitBLm-lacZor lmo0847-lacZfusion revealed that CcpCLmrepressescitBLmand lmo0847 in media containing an excess of glucose and glutamine. In addition, regulation ofcitBLmexpression in rich medium was growth phase dependent; during exponential growth phase, expression was very low even in the absence of CcpCLm, but a higher level ofcitBLmexpression was induced in stationary phase, suggesting the involvement of another, as yet unidentified regulatory factor.
Transposon Disruption of the Complex I NADH Oxidoreductase Gene (
snoD
) in
Staphylococcus aureus
Is Associated with Reduced Susceptibility to the Microbicidal Activity of Thrombin-Induced Platelet Microbicidal Protein 1
Authors: Bayer Arnold S. , McNamara Peter , Yeaman Michael R. , Lucindo Natalie , Jones Tiffanny , Cheung Ambrose L. , Sahl Hans-Georg , Proctor Richard A.
Published: December 2005

The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against
Staphylococcus aureus
. We previously reported that a Tn
551 S. aureus
transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1
r
) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun.
65:
3293-3299, 1997). In the current study, the genetic basis for tPMP-1
r
in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn
551
mutant strains showed that the fourth gene in the staphylococcal
mnh
operon (
mnhABCDEFG
) was insertionally inactivated. This operon was previously reported to encode a Na
+
/H
+
antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (ΔΨ) during postexponential growth, suggested that the
mnh
gene products are not functioning as a secondary (i.e., passive) Na
+
/H
+
antiporter. Moreover, we identified protein homologies between
mnhD
and the
nuo
genes of
Escherichia coli
that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1
s
) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1
r
mutants. To reflect the energization of the gene products encoded by the
mnh
operon, we have renamed the locus
sno
(
S. aureus nuo
orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in
S. aureus
.
The AidB Component of the
Escherichia coli
Adaptive Response to Alkylating Agents Is a Flavin-Containing, DNA-Binding Protein
Authors: Rohankhedkar Mukta S. , Mulrooney Scott B. , Wedemeyer William J. , Hausinger Robert P.
Published: December 2005

Upon exposure to alkylating agents,
Escherichia coli
increases expression of
aidB
along with three genes (
ada
,
alkA
, and
alkB
) that encode DNA repair proteins. In order to begin to identify the role of AidB in the cell, the protein was purified to homogeneity, shown to possess stoichiometric amounts of flavin adenine dinucleotide (FAD), and confirmed to have low levels of isovaleryl-coenzyme A (CoA) dehydrogenase activity. A homology model of an AidB homodimer was constructed based on the structure of a four-domain acyl-CoA oxidase. The predicted structure revealed a positively charged groove connecting the two active sites and a second canyon of positive charges in the C-terminal domain, both of which could potentially bind DNA. Three approaches were used to confirm that AidB binds to double-stranded DNA. On the basis of its ability to bind DNA and its possession of a redox-active flavin, AidB is predicted to catalyze the direct repair of alkylated DNA.
Cold-Induced Putative DEAD Box RNA Helicases CshA and CshB Are Essential for Cold Adaptation and Interact with Cold Shock Protein B in
Bacillus subtilis
Authors: Hunger Karen , Beckering Carsten L. , Wiegeshoff Frank , Graumann Peter L. , Marahiel Mohamed A.
Published: December 2005

The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from
Bacillus subtilis
during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both
cshA
and
cshB
genes could be deleted only in the presence of a
cshB
copy in
trans
, showing that the presence of one
csh
gene is essential for viability. The combined gene deletion of
cshB
and
cspD
resulted in a cold-sensitive phenotype that was not observed for either helicase or
csp
single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in
B. subtilis
.
Genomic Changes during Chronic
Helicobacter pylori
Infection
Authors: Kraft Christian , Stack Allison , Josenhans Christine , Niehus Eike , Dietrich Guido , Correa Pelayo , Fox James G. , Falush Daniel , Suerbaum Sebastian
Published: December 2005

The gastric pathogen
Helicobacter pylori
shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of
H. pylori
to the host individual may principally occur through sequence changes rather than loss or gain of genes.
Divergence and Mosaicism among Virulent Soil Phages of the
Burkholderia cepacia
Complex
Authors: Summer Elizabeth J. , Gonzalez Carlos F. , Bomer Morgan , Carlile Thomas , Embry Addie , Kucherka Amalie M. , Lee Jonte , Mebane Leslie , Morrison William C. , Mark Louise , King Maria D. , LiPuma John J. , Vidaver Anne K. , Young Ry
Published: December 2005

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the
Burkholderia cepacia
complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in
Photorhabdus luminescens
. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.
A Little Gene with Big Effects: a
serT
Mutant Is Defective in
flgM
Gene Translation
Authors: Chevance Fabienne F. V. , Karlinsey Joyce E. , Wozniak Christopher E. , Hughes Kelly T.
Published: December 2005

A conditional-lethal mutant was isolated as having a flagellar regulatory phenotype at 30°C and being unable to grow at 42°C. Chromosomal mapping localized the mutation to the
serT
gene, which encodes an essential serine tRNA species (

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{_{cmo}5\mathbf{UGA}}^{Ser}\) \end{document}

). DNA sequence analysis revealed the mutation to be a single base change in G:A at position 10 of the
serT
gene that lies within the D-stem of the essential

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{_{cmo}5}\mathbf{UGA}^{Ser}\) \end{document}

species.

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{_{cmo}5}\mathbf{UGA}^{Ser}\) \end{document}

recognizes UCA, UCG, and UCU codons, but UCU is also recognized by

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA_{GGA}^{Ser}}\) \end{document}

and UCG by

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA_{CGA}^{Ser}}\) \end{document}

. No other tRNAs are known to read the UCA codon. Thus, the UCA codon is specifically recognized by

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{_{cmo}5}\mathbf{UGA}^{Ser}\) \end{document}

. We show that the anti-σ
28
activity of FlgM is defective in the
serT
mutant strain. The
serT
allele causes a 10-fold increase in σ
28
-dependent
fliC
promoter transcription, indicating a defect in FlgM anti-σ
28
activity in the presence of the
serT
mutation. The
flgM
gene contains only one UCA codon. Changing the UCA of
flgM
to ACG reversed the effect of the
serT
allele. Implications for context effects in regulation of gene expression are discussed.
Autoinducer 2 Controls Biofilm Formation in
Escherichia coli
through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)
Authors: González Barrios Andrés F. , Zuo Rongjun , Hashimoto Yoshifumi , Yang Li , Bentley William E. , Wood Thomas K.
Published: December 2005

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from
S-
adenosylhomocysteine, directly increased
Escherichia coli
biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (
lsr
null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in
trans
. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in
trans
). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of
qseBC
,
flhD
,
fliA
, and
motA
, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces
crl
(26-fold) and
flhCD
(8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022,
qseBC
,
fliA
, and
motA
mutants. Adding AI-2 also increased
motA
transcription for the wild-type strain but did not stimulate
motA
transcription for the b3022 and
qseB
mutants. Together, these results indicate AI-2 induces biofilm formation in
E. coli
through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the
mqsR
gene).
Comparative and Functional Genomic Analysis of Prokaryotic Nickel and Cobalt Uptake Transporters: Evidence for a Novel Group of ATP-Binding Cassette Transporters
Authors: Rodionov Dmitry A. , Hebbeln Peter , Gelfand Mikhail S. , Eitinger Thomas
Published: December 2005

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B
12
riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B
12
biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the
Salmonella enterica
serovar Typhimurium transporter.
Mutations in
Flavobacterium johnsoniae secDF
Result in Defects in Gliding Motility and Chitin Utilization
Authors: Nelson Shawn S. , McBride Mark J.
Published: December 2005

secDF
mutants of
Flavobacterium johnsoniae
were deficient in gliding motility and chitin utilization. Cells of the mutants had reduced levels of GldJ protein, which is required for both processes. SecDF is similar to
Escherichia coli
SecD and SecF, which are involved in protein secretion.
Characterization of thePseudomonas putidaMobile Genetic Element ISPpu10: an Occupant of Repetitive Extragenic Palindromic Sequences
Authors: Ramos-González María Isabel , Campos María Jesús , Ramos Juan Luis , Espinosa-Urgel Manuel
Published: December 2005

We have characterized thePseudomonas putidaKT2440 insertion element ISPpu10. This insertion sequence encodes a transposase which exhibits homology to the transposases and specific recombinases of the Piv/Moov family, and no inverted repeats are present at the borders of its left and right ends, thus constituting a new member of the atypical IS110/IS492family. ISPpu10was found in at least seven identical loci in the KT2440 genome, and variants were identified having an extra insertion at distinct loci. ISPpu10always appeared within the core of specific repetitive extragenic palindromic (REP) sequences TCGCGGGTAAACCCGCTCCTAC, exhibiting high target stringency. One intragenic target was found associated with the truncation of a GGDEF/EAL domain protein. After active in vitro transposition to a plasmid-borne target, a duplication of the CT (underlined above) at the junction as a consequence of the ISPpu10insertion was experimentally demonstrated for the first time in the IS110/IS492family. The same duplication was observed after transposition of ISPpu10from a plasmid to the chromosome ofP. putidaDOT-T1E, an ISPpu10-free strain with REPs similar to those of strain KT2440. Plasmid ISPpu10-mediated rearrangements were observed in vivo under laboratory conditions and in the plant rhizosphere.
Identification of the Lower Baseplate Protein as the Antireceptor of the Temperate Lactococcal Bacteriophages TP901-1 and Tuc2009
Authors: Vegge Christina S. , Vogensen Finn K. , Mc Grath Stephen , Neve Horst , van Sinderen Douwe , Brøndsted Lone
Published: December 2005

The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate
Lactococcus lactis
phage TP901-1 belongs to the P335 species of the
Siphoviridae
family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (

bppL
TP901-1

) was exchanged with the analogous gene (

orf53
2009

) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the

orf53
2009

gene in exchange for

bppL
TP901-1

; however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF53
2009
and not the lower baseplate protein BppL
TP901-1
, and it was concluded that BppL
TP901-1
and ORF53
2009
constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.
Identification and Characterization of the Genes Involved in Glycosylation Pathways of Mycobacterial Glycopeptidolipid Biosynthesis
Authors: Miyamoto Yuji , Mukai Tetsu , Nakata Noboru , Maeda Yumi , Kai Masanori , Naka Takashi , Yano Ikuya , Makino Masahiko
Published: December 2005

Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such asMycobacterium avium. In this study, we identified four genes (gtf1,gtf2,gtf3, andgtf4) in the genome ofMycobacterium smegmatis. These genes were independently inactivated by homologous recombination inM. smegmatis, and the structures of GPLs from each gene disruptant were analyzed. Thin-layer chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses revealed that the mutants Δgtf1 and Δgtf2 accumulated the fatty acyl-tetrapeptide core havingO-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant Δgtf4 possessed the same GPLs as the wild type, whereas the mutant Δgtf3 lacked two minor GPLs, consisting of 3-O-methyl-rhamnose attached toO-methyl-rhamnose of the fatty acyl-tetrapeptide core. These results indicate that thegtf1andgtf2genes are responsible for the early glycosylation steps of GPL biosynthesis and thegtf3gene is involved in transferring a rhamnose residue not to 6-deoxy-talose but to anO-methyl-rhamnose residue. Moreover, a complementation experiment showed thatM. avium gtfAandgtfB, which are deduced glycosyltransferase genes of GPL biosynthesis, restore complete GPL production in the mutants Δgtf1 and Δgtf2, respectively. Our findings propose that bothM. smegmatisandM. aviumhave the common glycosylation pathway in the early steps of GPL biosynthesis but differ at the later stages.
Association of Herpesvirus Saimiri Tip with Lipid Raft Is Essential for Downregulation of T-Cell Receptor and CD4 Coreceptor
Authors: Cho Nam-Hyuk , Kingston Dior , Chang Heesoon , Kwon Eun-Kyung , Kim Jo-Min , Lee Jung Hee , Chu Hyuk , Choi Myung-Sik , Kim Ik-Sang , Jung Jae Ung
Published: December 2005

Lipid rafts are membrane microdomains that are proposed to function as platforms for both receptor signaling and trafficking. Our previous studies have demonstrated that Tip of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, is constitutively targeted to lipid rafts and interacts with cellular Lck tyrosine kinase and p80 WD repeat-containing endosomal protein. Through the interactions with Lck and p80, HVS Tip modulates diverse T-cell functions, which leads to the downregulation of T-cell receptor (TCR) and CD4 coreceptor surface expression, the inhibition of TCR signal transduction, and the activation of STAT3 transcription factor. In this study, we investigated the functional significance of Tip association with lipid rafts. We found that Tip expression remarkably increased lipid raft fractions in human T cells by enhancing the recruitment of lipid raft-resident proteins. Genetic analysis showed that the carboxyl-terminal transmembrane, but not p80 and Lck interaction, of Tip was required for the lipid raft localization and that lipid raft localization of Tip was necessary for the efficient downregulation of TCR and CD4 surface expression. Correlated with this, treatment with Filipin III, a lipid raft-disrupting agent, effectively reversed the downregulation of CD3 and CD4 surface expression induced by Tip. On the other hand, Tip mutants that were no longer present in lipid rafts were still capable of inhibiting TCR signaling and activating STAT3 transcription factor activity as efficiently as wild-type (wt) Tip. These results indicate that the association of Tip with lipid rafts is essential for the downregulation of TCR and CD4 surface expression but not for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor. These results also suggest that the signaling and targeting activities of HVS Tip rely on functionally and genetically separable mechanisms, which may independently modulate T-cell function for viral persistence or pathogenesis.
CD8+T Cells Require Perforin To Clear West Nile Virus from Infected Neurons
Authors: Shrestha Bimmi , Samuel Melanie A. , Diamond Michael S.
Published: December 2005

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+T cells use a perforin-dependent mechanism to clear WNV from infected neurons.
Gamma Interferon Blocks Gammaherpesvirus Reactivation from Latency
Authors: Steed Ashley L. , Barton Erik S. , Tibbetts Scott A. , Popkin Daniel L. , Lutzke Mary L. , Rochford Rosemary , Virgin Herbert W.
Published: December 2005

Establishment of latent infection and reactivation from latency are critical aspects of herpesvirus infection and pathogenesis. Interfering with either of these steps in the herpesvirus life cycle may offer a novel strategy for controlling herpesvirus infection and associated disease pathogenesis. Prior studies show that mice deficient in gamma interferon (IFN-γ) or the IFN-γ receptor have elevated numbers of cells reactivating from murine gammaherpesvirus 68 (γHV68) latency, produce infectious virus after the establishment of latency, and develop large-vessel vasculitis. Here, we demonstrate that IFN-γ is a powerful inhibitor of reactivation of γHV68 from latency in tissue culture. In vivo, IFN-γ controls viral gene expression during latency. Importantly, depletion of IFN-γ in latently infected mice results in an increased frequency of cells reactivating virus. This demonstrates that IFN-γ is important for immune surveillance that limits reactivation of γHV68 from latency.
The Pseudorabies Virus VP1/2 Tegument Protein Is Required for Intracellular Capsid Transport
Authors: Luxton G. W. Gant , Lee Joy I-Hsuan , Haverlock-Moyns Sarah , Schober Joseph Martin , Smith Gregory Allan
Published: December 2005

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.
Changes in Soluble Factor-Mediated CD8+Cell-Derived Antiviral Activity in Cynomolgus Macaques Infected with Simian Immunodeficiency Virus SIVmac251: Relationship to Biological Markers of Progression
Authors: Dioszeghy Vincent , Benlhassan-Chahour Kadija , Delache Benoit , Dereuddre-Bosquet Nathalie , Aubenque Celine , Gras Gabriel , Le Grand Roger , Vaslin Bruno
Published: December 2005

Cross-sectional studies have shown that the capacity of CD8+cells from human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV) SIVmac-infected macaques to suppress the replication of human and simian immunodeficiency viruses in vitro depends on the clinical stage of disease, but little is known about changes in this antiviral activity over time in individual HIV-infected patients or SIV-infected macaques. We assessed changes in the soluble factor-mediated noncytolytic antiviral activity of CD8+cells over time in eight cynomolgus macaques infected with SIVmac251 to determine the pathophysiological role of this activity. CD8+cell-associated antiviral activity increased rapidly in the first week after viral inoculation and remained detectable during the early phase of infection. The net increase in antiviral activity of CD8+cells was correlated with plasma viral load throughout the 15 months of follow-up. CD8+cells gradually lost their antiviral activity over time and acquired virus replication-enhancing capacity. Levels of antiviral activity correlated with CD4+T-cell counts after viral set point. Concentrations of β-chemokines and interleukin-16 in CD8+cell supernatants were not correlated with this antiviral activity, and α-defensins were not detected. The soluble factor-mediated antiviral activity of CD8+cells was neither cytolytic nor restricted to major histocompatibility complex. This longitudinal study strongly suggests that the increase in noncytolytic antiviral activity from baseline and the maintenance of this increase over time in cynomolgus macaques depend on both viral replication and CD4+T cells.
Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation
Authors: Hrdličková Radmila , Nehyba Jiří , Liss Andrew S. , Bose Henry R.
Published: December 2005

Telomerase
is activated during the transformation of lymphoid cells and
fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB
family of transcription factors. v-Rel-transformed cell lines have
longer telomeres than untransformed chicken lymphoid cells and have
high levels of telomerase activity. v-Rel-mediated activation of
telomerase is achieved by multiple mechanisms. The expression of the
gene encoding the catalytic subunit of telomerase (TERT) was directly
upregulated by v-Rel. Moreover, the expression of v-Rel altered the
ratio of alternatively spliced and full-length TERT transcripts in
favor of the full-length forms. The activation of telomerase by v-Rel
in lymphocytes was also accompanied by inactivation of nuclear
inhibitors. The inhibition of telomerase activity in v-Rel-transformed
cell lines led to apoptosis within 24 h. The expression of
v-Rel in a macrophage cell line resulted in elevated levels of reactive
oxygen species (ROS), increased telomerase activity, and increased
sensitivity to telomerase inhibitors. In contrast, the ectopic
expression of TERT decreased the extent of apoptosis induced by ROS.
The activation of telomerase by v-Rel may, therefore, partially protect
the transformed cells from apoptosis induced by
ROS.
Expanded Tropism and Altered Activation of a Retroviral Glycoprotein Resistant to an Entry Inhibitor Peptide
Authors: Amberg Sean M. , Netter Robert C. , Simmons Graham , Bates Paul
Published: December 2005

The envelope of class I viruses can be a target for potent viral inhibitors, such as the human immunodeficiency virus type 1 (HIV-1) inhibitor enfuvirtide, which are derived from the C-terminal heptad repeat (HR2) of the transmembrane (TM) subunit. Resistance to an HR2-based peptide inhibitor of a model retrovirus, subgroup A of the
Avian Sarcoma and Leukosis Virus
genus (ASLV-A), was studied by examining mutants derived by viral passage in the presence of inhibitor. Variants with reduced sensitivity to inhibitor were readily selected in vitro. Sensitivity determinants were identified for 13 different isolates, all of which mapped to the TM subunit. These determinants were identified in two regions: (i) the N-terminal heptad repeat (HR1) and (ii) the N-terminal segment of TM, between the subunit cleavage site and the fusion peptide. The latter class of mutants identified a region outside of the predicted HR2-binding site that can significantly alter sensitivity to inhibitor. A subset of the HR1 mutants displayed the unanticipated ability to infect nonavian cells. This expanded tropism was associated with increased efficiency of envelope triggering by soluble receptor at low temperatures, as measured by protease sensitivity of the surface subunit (SU) of envelope. In addition, expanded tropism was linked for the most readily triggered mutants with increased sensitivity to neutralization by SU-specific antiserum. These observations depict a class of HR2 peptide-selected mutations with a reduced activation threshold, thereby allowing the utilization of alternative receptors for viral entry.
Cytokine Responses in Gnotobiotic Pigs after Infection with Virulent or Attenuated Human Rotavirus
Authors: Azevedo M. S. P. , Yuan L. , Pouly S. , Gonzales A. M. , Jeong K. I. , Nguyen T. V. , Saif L. J.
Published: December 2005

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-γ) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor β (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-γ, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-α levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-γ responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-γ responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-γ-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-γ and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-γ (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.
Role of the Mitochondrial Signaling Pathway in Murine Coronavirus-Induced Oligodendrocyte Apoptosis
Authors: Liu Yin , Pu Yinghui , Zhang Xuming
Published: December 2005

A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol.
77:
11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome
c
release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome
c
release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.
Hepatitis C Virus Subgenomic Replicon Requires an Active NS3 RNA Helicase
Authors: Lam Angela M. I. , Frick David N.
Published: December 2005

Mutations were introduced into the NS3 helicase region of a hepatitis C virus (HCV) Con1 subgenomic replicon to ascertain the role of the helicase in viral replication. One new replicon lacked two-thirds of the NS3 helicase (Δhel), and six others contained one of the following six amino acid substitutions in NS3: R393A, F438A, T450I, E493K, W501A, and W501F. It has been previously reported that purified R393A, F438A, and W501A HCV helicase proteins do not unwind RNA but unwind DNA, bind RNA, and hydrolyze ATP. On the other hand, previous data suggest that a W501F protein retains most of its unwinding abilities and that purified T450I and E493K HCV helicase proteins have enhanced unwinding abilities. In a hepatoma cell line that has been cured of HCV replicons using interferon, the T450I and W501F replicons synthesized both negative-sense and positive-sense viral RNA and formed colonies after selection with similar efficiencies as the parent replicon. However, the Δhel, R393A, F438A, and W501A replicons encoded and processed an HCV polyprotein but did not synthesize additional viral RNA or form colonies. Surprisingly the same phenotype was seen for the E493K replicon. The inability of the E493K replicon to replicate might point to a role of pH in viral replication because a previous analysis has shown that, unlike the wild-type NS3 protein, the helicase activity of an E493K protein is not sensitive to pH changes. These results demonstrate that the RNA-unwinding activity of the HCV NS3 helicase is needed for RNA replication.