bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant
Authors: Revello Maria Grazia , Gerna Giuseppe
Published: October 2002

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy.
Clinical and Taxonomic Status of Pathogenic Nonpigmented or Late-Pigmenting Rapidly Growing Mycobacteria
Authors: Brown-Elliott Barbara A. , Wallace Richard J.
Published: October 2002

The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing.
Current Consensus Guidelines for Treatment of Neurocysticercosis
Authors: García Hector H. , Evans Carlton A. W. , Nash Theodore E. , Takayanagui Osvaldo M. , White A. Clinton , Botero David , Rajshekhar Vedantam , Tsang Victor C. W. , Schantz Peter M. , Allan James C. , Flisser Ana , Correa Dolores , Sarti Elsa , Friedland Jon S. , Martinez S. Manuel , Gonzalez Armando E. , Gilman Robert H. , Del Brutto Oscar H.
Published: October 2002

Taenia solium neurocysticercosis is a common cause of epileptic seizures and other neurological morbidity in most developing countries. It is also an increasingly common diagnosis in industrialized countries because of immigration from areas where it is endemic. Its clinical manifestations are highly variable and depend on the number, stage, and size of the lesions and the host's immune response. In part due to this variability, major discrepancies exist in the treatment of neurocysticercosis. A panel of experts in taeniasis/cysticercosis discussed the evidence on treatment of neurocysticercosis for each clinical presentation, and we present the panel's consensus and areas of disagreement. Overall, four general recommendations were made: (i) individualize therapeutic decisions, including whether to use antiparasitic drugs, based on the number, location, and viability of the parasites within the nervous system; (ii) actively manage growing cysticerci either with antiparasitic drugs or surgical excision; (iii) prioritize the management of intracranial hypertension secondary to neurocysticercosis before considering any other form of therapy; and (iv) manage seizures as done for seizures due to other causes of secondary seizures (remote symptomatic seizures) because they are due to an organic focus that has been present for a long time.
Effects of Global Climate on Infectious Disease: the Cholera Model
Authors: Lipp Erin K. , Huq Anwar , Colwell Rita R.
Published: October 2002

Recently, the role of the environment and climate in disease dynamics has become a subject of increasing interest to microbiologists, clinicians, epidemiologists, and ecologists. Much of the interest has been stimulated by the growing problems of antibiotic resistance among pathogens, emergence and/or reemergence of infectious diseases worldwide, the potential of bioterrorism, and the debate concerning climate change. Cholera, caused by
Vibrio cholerae
, lends itself to analyses of the role of climate in infectious disease, coupled to population dynamics of pathogenic microorganisms, for several reasons. First, the disease has a historical context linking it to specific seasons and biogeographical zones. In addition, the population dynamics of
V. cholerae
in the environment are strongly controlled by environmental factors, such as water temperature, salinity, and the presence of copepods, which are, in turn, controlled by larger-scale climate variability. In this review, the association between plankton and
V. cholerae
that has been documented over the last 20 years is discussed in support of the hypothesis that cholera shares properties of a vector-borne disease. In addition, a model for environmental transmission of cholera to humans in the context of climate variability is presented. The cholera model provides a template for future research on climate-sensitive diseases, allowing definition of critical parameters and offering a means of developing more sophisticated methods for prediction of disease outbreaks.
Toll-Like Receptor 4 (TLR4)-Deficient Murine Macrophage Cell Line as an In Vitro Assay System To Show TLR4-Independent Signaling ofBacteroides fragilisLipopolysaccharide
Authors: Lorenz Eva , Patel Dhavalkumar D. , Hartung Thomas , Schwartz David A.
Published: October 2002

Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS fromEscherichia coliwhile responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) fromStaphylococcus aureus, which signal TLR4 independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine were found when cells were stimulated withE. coliLPS.Bacteroides fragilis-derived LPS, however, can effectively stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate immune response.
Control of Mycobacterial Replication in Human Macrophages: Roles of Extracellular Signal-Regulated Kinases 1 and 2 and p38 Mitogen-Activated Protein Kinase Pathways
Authors: Blumenthal Antje , Ehlers Stefan , Ernst Martin , Flad Hans-Dieter , Reiling Norbert
Published: October 2002

Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control ofMycobacterium aviumisolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype ofM. aviumstrain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of theM. aviumstrain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicativeM. aviumstrains, although it did not increase survival of the poorly replicatingM. aviumisolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-α) secretion irrespective of the virulence of theM. aviumisolate used for infection, revealing that TNF-α could have been only partially responsible for the control of intracellularM. aviumgrowth. In conclusion, ERK1/2- and TNF-α-independent pathways are sufficient to limit intramacrophage growth of less-virulentM. aviumstrains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicativeM. aviumstrains.
Tolerance to Self Gangliosides Is the Major Factor Restricting the Antibody Response to Lipopolysaccharide Core Oligosaccharides inCampylobacter jejuniStrains Associated with Guillain-Barré Syndrome
Authors: Bowes Tyrone , Wagner Eric R. , Boffey Judith , Nicholl Dawn , Cochrane Lynne , Benboubetra Mustapha , Conner Joe , Furukawa Keiko , Furukawa Koichi , Willison Hugh J.
Published: October 2002

Guillain-Barré syndrome followingCampylobacter jejuniinfection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority ofCampylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.
Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197
Authors: Mawas Fatme , Niggemann Jutta , Jones Christopher , Corbel Michael J. , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: October 2002

Oligosaccharides (OSs) related to the pneumococcal type 14 capsular polysaccharide (Pn14PS) were studied for their ability to inhibit the binding between anti-PS14 antisera and native PS14. A synthetic tetrasaccharide corresponding to the repeating unit of the Pn14PS, a hexasaccharide mimic, and an octasaccharide fragment obtained by Pn14PS depolymerization were good inhibitors. CRM197 conjugates of the tetrasaccharide and an octasaccharide mimic were prepared by using either adipic acid diester or diethyl squarate linkers. The conjugate with the tetrasaccharide chains induced anti-Pn14PS antibodies when injected subcutaneously into mice, as determined by an enzyme-linked immunosorbent assay, and antibody titers increased with oligosaccharide loading. The adipic acid-linked tetrasaccharide conjugates elicited higher antibody titers than those prepared with a squarate spacer. The lower anti-Pn14PS antibody response of the octasaccharide mimic conjugate indicates the importance of the backbone galactose residue for an appropriate antibody response. The OS-CRM197 conjugate prepared from a single repeat unit of the Pn14PS is a potential vaccine candidate.
Effects of Ageing on the Immunoregulation of Parasitic Infection
Authors: Humphreys Neil E. , Grencis Richard K.
Published: October 2002

The plethora of changes associated with immunosenescence radically alters virtually all aspects of immune responsiveness. How this transformation effects resolution of an infectious challenge is addressed in this study. A well-established infection model was used;Trichuris muris, a cecum-dwelling helminth, is natural to mice, and infection in different strains results in clearly polarized responses. A dominating T helper 2 (Th2) response orchestrates immunity, whereas a Th1 response will result in susceptibility. Mice between 19 and 28 months old were more susceptible to infection, whereas 3-month-old mice of the same strain demonstrated the resistant phenotype. The cytokine response made by these aged mice was clearly altered at the site of infection, and within the local draining lymph nodes higher Th1 and lower Th2 cytokine levels were found, both at the protein and RNA level. Confirming these changes, aged mice also showed a delayed parasite-specific immunoglobulin G1 response and intestinal mastocytosis, both of which are driven by Th2 cytokines. To address possible causes of the observed immune deviation, purified CD4 cells from both young and aged mice were stimulated in vitro. Cells from aged mice did not respond to stimulation via CD28 and in vitro were less able to proliferate and polarize into Th2 cells; Th1 polarization was found to be normal. Together these data suggest that changes in cytokine phenotype, particularly CD4 cells, contribute to the observed age-associated switch fromT. murisresistance to susceptibility.
Susceptibility of Nontypeable
Haemophilus influenzae
to Human β-Defensins Is Influenced by Lipooligosaccharide Acylation
Authors: Starner Timothy D. , Swords W. Edward , Apicella Michael A. , McCray Paul B.
Published: October 2002

Nontypeable
Haemophilus influenzae
(NTHI) lipooligosaccharide
htrB
mutants exhibited greater than 45-fold-increased sensitivity to human β-defensin 2 (HBD-2) compared to the wild type. Complementation by
htrB in trans
to acylation competence reversed this increased sensitivity. In contrast, NTHI was more susceptible to HBD-3 and showed no changes in sensitivity as a result of lipooligosaccharide mutations in oligosaccharide and lipid A biosynthesis genes.
Cluster IIcheGenes fromPseudomonas aeruginosaAre Required for an Optimal Chemotactic Response
Authors: Ferrández Abel , Hawkins Andrew C. , Summerfield Douglas T. , Harwood Caroline S.
Published: October 2002

Pseudomonas aeruginosa, a γ-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate.P. aeruginosahas multiple homologues ofEscherichia colichemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set ofchegenes, as well as two genes,mcpAandmcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster IIchegenes and in themcpgenes to examine their possible contributions toP. aeruginosachemotaxis. AcheB2mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. ProvidingcheB2intranscomplemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I,cheB-deficient strain to near wild-type levels. AnmcpAmutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. AnmcpBmutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition ofmcpBintrans. Overexpression of either McpA or McpB inP. aeruginosaorEscherichia coliresulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression ofP. aeruginosa cheA2,cheB2, orcheW2inE. coliK-12 completely disrupted wild-type chemotaxis, while expression ofcheY2had no effect. These results indicate thatchecluster II genes are expressed inP. aeruginosaand are required for an optimal chemotactic response.
N
-Acyl-Homoserine Lactone Inhibition of Rhizobial Growth Is Mediated by Two Quorum-Sensing Genes That Regulate Plasmid Transfer
Authors: Wilkinson A. , Danino V. , Wisniewski-Dyé F. , Lithgow J. K. , Downie J. A.
Published: October 2002

The growth of some strains of
Rhizobium leguminosarum
bv. viciae is inhibited by
N
-(3-hydroxy-7-
cis
tetradecenoyl)-
l
-homoserine lactone (3OH-C
14:1
-HSL), which was previously known as the
small
bacteriocin before its characterization as an
N
-acyl homoserine lactone (AHL). Tn
5
-induced mutants of
R. leguminosarum
bv. viciae resistant to 3OH-C
14:1
-HSL were isolated, and mutations in two genes were identified. These genes,
bisR
and
triR
, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing
trb
genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is
traI
, which encodes an AHL synthase, and the
trbBCDEJKLFGHI
genes were found between
traI
and
bisR
. Mutations in
bisR
,
triR
,
traI
, or
trbL
blocked plasmid transfer. Using gene fusions, it was demonstrated that
bisR
regulates
triR
in response to the presence of 3OH-C
14:1
-HSL. In turn,
triR
is then required for the induction of the
traI-trb
operon required for plasmid transfer.
bisR
also represses expression of
cinI
, which is chromosomally located and determines the level of production of 3OH-C
14:1
-HSL. The cloned
bisR
and
triR
genes conferred 3OH-C
14:1
-HSL sensitivity to strains of
R. leguminosarum
bv. viciae normally resistant to this AHL. Furthermore,
bisR
and
triR
made
Agrobacterium tumefaciens
sensitive to
R. leguminosarum
bv. viciae strains producing 3OH-C
14:1
-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C
14:1
-HSL, the presence of other AHLs such as
N
-octanoyl
-l-
homoserine lactone and/or
N
-(3-oxo-octanoyl)
-l-
homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of
R. leguminosarum
bv. viciae carrying cloned
bisR
and
triR
was treated with an AHL extract containing 3OH-C
14:1
-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.
Genome-Scale Metabolic Model of
Helicobacter pylori
26695
Authors: Schilling Christophe H. , Covert Markus W. , Famili Iman , Church George M. , Edwards Jeremy S. , Palsson Bernhard O.
Published: October 2002

A genome-scale metabolic model of
Helicobacter pylori
26695 was constructed from genome sequence annotation, biochemical, and physiological data. This represents an in silico model largely derived from genomic information for an organism for which there is substantially less biochemical information available relative to previously modeled organisms such as
Escherichia coli
. The reconstructed metabolic network contains 388 enzymatic and transport reactions and accounts for 291 open reading frames. Within the paradigm of constraint-based modeling, extreme-pathway analysis and flux balance analysis were used to explore the metabolic capabilities of the in silico model. General network properties were analyzed and compared to similar results previously generated for
Haemophilus influenzae
. A minimal medium required by the model to generate required biomass constituents was calculated, indicating the requirement of eight amino acids, six of which correspond to essential human amino acids. In addition a list of potential substrates capable of fulfilling the bulk carbon requirements of
H. pylori
were identified. A deletion study was performed wherein reactions and associated genes in central metabolism were deleted and their effects were simulated under a variety of substrate availability conditions, yielding a number of reactions that are deemed essential. Deletion results were compared to recently published in vitro essentiality determinations for 17 genes. The in silico model accurately predicted 10 of 17 deletion cases, with partial support for additional cases. Collectively, the results presented herein suggest an effective strategy of combining in silico modeling with experimental technologies to enhance biological discovery for less characterized organisms and their genomes.
The NorR Protein of
Escherichia coli
Activates Expression of the Flavorubredoxin Gene
norV
in Response to Reactive Nitrogen Species
Authors: Hutchings Matthew I. , Mandhana Neeraj , Spiro Stephen
Published: October 2002

The
Escherichia coli norVW
genes encode a flavorubredoxin and NADH:(flavo)rubredoxin reductase, respectively, which are involved in nitric oxide detoxification under anaerobic growth conditions. Here it is shown that the
norVW
genes also have a role in protection against reactive nitrogen intermediates generated from nitroprusside. Transcription from the
norV
promoter is activated by the presence of nitroprusside in the growth medium; activation requires the product of a divergently transcribed regulatory gene,
norR
.
Organization and Regulation of Pentachlorophenol-Degrading Genes in
Sphingobium chlorophenolicum
ATCC 39723
Authors: Cai Mian , Xun Luying
Published: October 2002

The first three enzymes of the pentachlorophenol (PCP) degradation pathway in
Sphingobium chlorophenolicum
(formerly
Sphingomonas chlorophenolica
) ATCC 39723 have been characterized, and the corresponding genes,
pcpA
,
pcpB
, and
pcpC
, have been individually cloned and sequenced. To search for new genes involved in PCP degradation and map the physical locations of the
pcp
genes, a 24-kb fragment containing
pcpA
and
pcpC
was completely sequenced. A putative LysR-type transcriptional regulator gene,
pcpM
, and a maleylacetate reductase gene,
pcpE
, were identified upstream of
pcpA. pcpE
was found to play a role in PCP degradation.
pcpB
was not found on the 24-kb fragment. The four gene products PcpB, PcpC, PcpA, and PcpE were responsible for the metabolism of PCP to 3-oxoadipate in ATCC 39723, and inactivational mutation of each gene disrupted the degradation pathway. The organization of the
pcp
genes is unusual because the four PCP-degrading genes,
pcpA
,
pcpB
,
pcpC
, and
pcpE
, were found to be located at four discrete locations. Two hypothetical LysR-type regulator genes,
pcpM
and
pcpR,
have been identified;
pcpM
was not required, but
pcpR
was essential for the induction of
pcpB
,
pcpA
, and
pcpE
. The coinducers of PcpR were PCP and other polychlorinated phenols. The expression of
pcpC
was constitutive. Thus, the organization and regulation of the genes involved in PCP degradation to 3-oxoadipate were documented.
Pseudomonas aeruginosa
Synthesizes Phosphatidylcholine by Use of the Phosphatidylcholine Synthase Pathway
Authors: Wilderman Paula J. , Vasil Adriana I. , Martin Wesley E. , Murphy Robert C. , Vasil Michael L.
Published: October 2002

Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes. Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt). Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline. The CDP-choline then condenses with diacylglycerol (DAG) to form PC. In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs). PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in
Sinorhizobium meliloti
was characterized. The completed release of the
Pseudomonas aeruginosa
PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from
Rhodobacter sphaeroides
. Moreover, the
P. aeruginosa
PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of
S. meliloti
. Both the
pcs
and
pmtA
homologues were cloned from PAO1, and homologous sequences were found in almost all of the
P. aeruginosa
strains examined. Although the pathway for synthesizing PC by use of Pcs is functional in
P. aeruginosa
, it does not appear that this organism uses the PmtA pathway for PC synthesis. We demonstrate that the PC synthesized by
P. aeruginosa
PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.
LasR, a Transcriptional Activator of
Pseudomonas aeruginosa
Virulence Genes, Functions as a Multimer
Authors: Kiratisin Pattarachai , Tucker Kenneth D. , Passador Luciano
Published: October 2002

The
Pseudomonas aeruginosa
LasR protein functions in concert with
N
-3-oxo-dodecanoyl-
l
-homoserine lactone (3O-C
12
-HSL) to coordinate the expression of target genes, including many genes that encode virulence factors, with cell density. We used a LexA-based protein interaction assay to demonstrate that LasR forms multimers only when 3O-C
12
-HSL is present. A series of LasR molecules containing internal deletions or substitutions in single, conserved amino acid residues indicated that the N-terminal portion of LasR is required for multimerization. Studies performed with these mutant versions of LasR demonstrated that the ability of LasR to multimerize correlates with its ability to function as a transcriptional activator of
lasI
, a gene known to be tightly regulated by the LasR-3O-C
12
-HSL regulatory system. A LasR molecule that carries a C-terminal deletion can function as a dominant-negative mutant in
P. aeruginosa
, as shown by its ability to decrease expression of
lasB,
another LasR-3O-C
12
-HSL target gene. Taken together, our data strongly support the hypothesis that LasR functions as a multimer in vivo.
The
metD
d
-Methionine Transporter Locus of
Escherichia coli
Is an ABC Transporter Gene Cluster
Authors: Gál József , Szvetnik Attila , Schnell Róbert , Kálmán Miklós
Published: October 2002

The
metD
d
-methionine transporter locus of
Escherichia coli
was identified as the
abc
-
yaeE-yaeC
cluster (now renamed
metNIQ
genes). The
abc
open reading frame is preceded by tandem MET boxes bracketed by the −10 and −35 boxes of a promoter. The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.
The Spectrum of Spontaneous Rifampin Resistance Mutations in the
rpoB
Gene of
Bacillus
subtilis
168 Spores Differs from That of Vegetative Cells and Resembles That of
Mycobacterium tuberculosis
Authors: Nicholson Wayne L. , Maughan Heather
Published: October 2002

Mutations causing rifampin resistance in vegetative cells of
Bacillus subtilis
168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the
rpoB
gene encoding the β subunit of RNA polymerase. In this study, we demonstrated that spores of
B. subtilis
168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the
rpoB
mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of
rpoB
, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in
rpoB
mutations sequenced from rifampin-resistant clinical isolates of
Mycobacterium tuberculosis.
The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.
Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases fromClostridium cellulovorans
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H.
Published: October 2002

Clostridium cellulovoransproduces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.
ExbB and ExbD Do Not Function Independently in TonB-Dependent Energy Transduction
Authors: Held Kiara G. , Postle Kathleen
Published: October 2002

ExbB and ExbD proteins are part of the TonB-dependent energy transduction system and are encoded by theexboperon inEscherichia coli. TonB, the energy transducer, appears to go through a cycle during energy transduction, with the absence of both ExbB and ExbD creating blocks at two points: (i) in the inability of TonB to respond to the cytoplasmic membrane proton motive force and (ii) in the conversion of TonB from a high-affinity outer membrane association to a high-affinity cytoplasmic membrane association. The recent observation that ExbB exists in 3.5-fold molar excess relative to the molarity of ExbD inE. colisuggests the possibility of two types of complexes, those containing both ExbB and ExbD and those containing only ExbB. Such distinct complexes might individually manifest one of the two activities described above. In the present study this hypothesis was tested and rejected. Specifically, both ExbB and ExbD were found to be required for TonB to conformationally respond to proton motive force. Both ExbB and ExbD were also required for association of TonB with the cytoplasmic membrane. Together, these results support an alternative model where all of the ExbB in the cell occurs in complex with all of the ExbD in the cell. Based on recently determined cellular ratios of TonB system proteins, these results suggest the existence of a cytoplasmic membrane complex that may be as large as 520 kDa.
Global Expression Profile of
Bacillus subtilis
Grown in the Presence of Sulfate or Methionine
Authors: Auger Sandrine , Danchin Antoine , Martin-Verstraete Isabelle
Published: October 2002

DNA arrays were used to investigate the global transcriptional profile of
Bacillus subtilis
grown in the presence of sulfate or methionine as the sole sulfur source. The expression of at least 56 genes differed significantly under the two growth conditions. The expression of several genes belonging to the S-box regulon was repressed in the presence of methionine probably in response to
S
-adenosylmethionine availability. The expression of genes encoding transporters (
yhcL
,
ytmJKLMN
, and
yxeMO
) was high when the sulfur source was methionine or taurine and reduced when it was sulfate.
Amplification of the Tetracycline Resistance Determinant of pAMα1 in
Enterococcus faecalis
Requires a Site-Specific Recombination Event Involving Relaxase
Authors: Francia M. Victoria , Clewell Don B.
Published: October 2002

The small multicopy plasmid pAMα1 (9.75 kb) encoding tetracycline resistance in
Enterococcus faecalis
is known to generate tandem repeats of a 4.1-kb segment carrying
tet
(L) when cells are grown extensively in the presence of tetracycline. Here we show that the initial (rate-limiting) step involves a site-specific recombination event involving plasmid-encoded relaxase activity acting at two recombination sequences (RS1 and RS2) that flank the
tet
determinant. We also present the complete nucleotide sequence of pAMα1.
Rapid Detection of Methicillin-Resistant Staphylococci from Blood Culture Bottles by Using a Multiplex PCR Assay
Authors: Louie L. , Goodfellow J. , Mathieu P. , Glatt A. , Louie M. , Simor A. E.
Published: October 2002

Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the
nuc
,
mecA
, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant
Staphylococcus
spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.
Diagnosis of Community-Acquired Pertussis Infection: Comparison of Both Culture and Fluorescent-Antibody Assays with PCR Detection Using Electrophoresis or Dot Blot Hybridization
Authors: Lingappa Jairam R. , Lawrence William , West-Keefe Sheyla , Gautom Romesh , Cookson Brad T.
Published: October 2002

Diagnosis ofBordetella pertussisinfection has been difficult due to the low sensitivity of culture. PCR tests have been shown to be more sensitive than culture, but the reported sensitivity of PCR is variable. We evaluated PCR product detection by using either agarose gel electrophoresis (PCR-gel) or dot blot hybridization with32P-labeled oligonucleotide probes, and we compared these methods to both culture and direct fluorescent-antibody (DFA) assays with microscopy for the detection of pertussis. This was done with 225 nasopharyngeal swab specimens collected in community clinic settings. The multiplexed PCR amplified the multiply repeated IS481 B. pertussissequence and a sequence from the human globin gene as a positive control for specimen adequacy. Of 225 specimens, 179 were judged to be adequate for PCR analysis. Among the adequate specimens, 9, 4, and 10 were culture, DFA, and PCR-gel positive, respectively. The sensitivity of PCR-gel versus culture was 89% while the sensitivity of culture versus PCR-gel was 80%. DFA had the lowest sensitivity. Thirty specimens were positive by PCR with dot blot hybridization; no negative control specimens showed a signal above the background. Among the 79 (44%) adequate specimens with clinical data available, the rates of reported cough or persistent cough were similar for persons who were pertussis positive by each assay. The IS481PCR, with either electrophoresis or dot blot hybridization, is a sensitive assay; however, at this time it cannot completely replace culture without an overall loss in sensitivity for the detection of pertussis. Further study is required to understand the clinical significance ofB. pertussisPCR products detected by dot blot hybridization alone.
Cytomegalovirus Assemblin (pUL80a): Cleavage at Internal Site Not Essential for Virus Growth; Proteinase Absent from Virions
Authors: Chan Chee-Kai , Brignole Edward J. , Gibson Wade
Published: October 2002

The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.
Hemagglutinin Protein of Wild-Type Measles Virus Activates Toll-Like Receptor 2 Signaling
Authors: Bieback Karen , Lien Egil , Klagge Ingo M. , Avota Elita , Schneider-Schaulies Jürgen , Duprex W. Paul , Wagner Herrmann , Kirschning Carsten J. , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: October 2002

Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2−/−mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.
Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor on Chang C Cells
Authors: Arnberg Niklas , Pring-Åkerblom Patricia , Wadell Göran
Published: October 2002

Epidemic keratoconjunctivitis (EKC) is a severe eye infection caused mainly by adenovirus type 8 (Ad8), Ad19, and Ad37. We have shown that the EKC-causing adenoviruses use sialic acid as a cellular receptor on A549 cells instead of the coxsackie-adenovirus receptor, which is used by most adenoviruses. Recently, Wu et al. (Virology 279:78-89, 2001) proposed that Ad37 uses a 50-kDa protein as a receptor on Chang C conjunctival cells and that this interaction is independent of sialic acid. According to the American Type Culture Collection, this cell line carries HeLa cell markers and should be considered to be a genital cell line. This prompted us to investigate the function of sialic acid as a cellular receptor for Ad37 in Chang C cells. In this study, we demonstrate that enzymatic removal or lectin-mediated blocking of cell surface sialic acid inhibits the binding of Ad37 virions to Chang C cells, as does soluble, virion-interacting sialic acid-containing substances. The binding was Ca
2+
or Mg
2+
ion independent and mediated by the knob domain of the trimeric viral fiber polypeptide. Moreover, Ad37 virions infected Chang C cells and two other genital cell lines (HeLa and SiHa) as well as a corneal cell line in a strictly sialic acid-dependent manner. From these results, we conclude that Ad37 uses sialic acid as a major receptor in cell lines derived from both genital and corneal tissues.
Transgenic Expression in Mouse Lung Reveals Distinct Biological Roles for the Adenovirus Type 5 E1A 243- and 289-Amino-Acid Proteins
Authors: Yang Yongping , McKerlie Colin , Borenstein Steven H. , Lu Zhan , Schito Marco , Chamberlain John W. , Buchwald Manuel
Published: October 2002

Little is known about the biological significance of human adenovirus type 5 (Ad5) E1A in vivo. However, Ad5 E1A is well defined in vitro and can be detected frequently in the lungs of patients with pulmonary disease. Transgenic expression of the Ad5E1Agene targeted to the mouse lung reveals distinct biological effects caused by two Ad5 E1A products. Either of two Ad5 E1A proteins was preferentially expressed in vivo in the transgenic lungs. The preferential expression of the Ad5 E1A 243-amino-acid (aa) protein at a moderate level was associated with cellular hyperplasia, nodular lesions of proliferating lymphocyte-like cells, and a low level of p53-dependent apoptosis in the lungs of transgenic mice. In contrast, the preferential expression of the Ad5 E1A 289-aa protein at a moderate level resulted in a proapoptotic injury and an acute pulmonary proinflammation in the lungs of transgenic mice, mediated by multiple apoptotic pathways, as well as an enhancement of the host immune cell response. Expression of the Ad5 E1A 243-aa protein resulted in proliferation-stimulated p53 upregulation, while expression of the Ad5 E1A 289-aa protein led to DNA damage-induced p53 activation. These data suggest that the Ad5 E1A 243- and 289-aa proteins lead to distinct biological roles in vivo.
Decelerating Decay of Latently Infected Cells during Prolonged Therapy for Human Immunodeficiency Virus Type 1 Infection
Authors: Müller Viktor , Vigueras-Gómez Javier Flavio , Bonhoeffer Sebastian
Published: October 2002

Antiviral therapy induces a rapid drop in human immunodeficiency virus type 1 viremia, but the decline of virus levels decelerates over time. Mathematical modeling demonstrates that the source of residual virus production might be a single compartment of latently infected cells with an extended distribution of activation rates.
Ahi-1
, a Novel Gene Encoding a Modular Protein with WD40-Repeat and SH3 Domains, Is Targeted by the
Ahi-1
and
Mis-2
Provirus Integrations
Authors: Jiang Xiaoyan , Hanna Zaher , Kaouass Mohammadi , Girard Luc , Jolicoeur Paul
Published: October 2002

The
Ahi-1
locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-
myb
proto-oncogene. Since no significant alteration of c-
myb
expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the
Ahi-1
locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (
Ahi-1
) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3′ end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the
Ahi-1
gene. The
Ahi-1
cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The
Ahi-1
gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The
Ahi-1
gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in
Ahi-1
, truncated
Ahi-1
/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore,
Ahi-1
is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus,
Ahi-1
may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-
abl
and c-
myc
) or with a tumor suppressor gene (
Nf1
), since Ahi-1 insertion sites were identified in tumors harboring v-
abl
defective retroviruses or a c-
myc
transgene or in tumors exhibiting deletion of
Nf1
.