bluuebunny/crossref_metadata_2025 huggingface dataset:
Plant Hair Signals
Authors: Gough Nancy R.
Published: September 2008

Trichomes are epidermal structures that often resemble hairs that grow in regular patterns on various parts of plants. Although most models of trichome patterning invoke an activation-inhibition mechanism, Bouyer
et al
. suggest that a trapping-depletion mechanism may also be important. The activation-inhibition mechanisms involve the formation of an active trimeric complex of a single R2R3 MYB transcription factor (GL1 or MYB23), the WD40 repeat protein TTG1, and a basic helix-loop-helix (bHLH) transcription factor (GL3 or EGL3). The inhibitor is an R3 MYB-like transcription factor (TRY, CPC, ETC1, ETC2, TRICHOMELESS1, or CPL3) that competes with the R2R3 MYB-TTG1 complex for binding to the bHLH transcription factor. Bouyer
et al
. expressed a yellow fluorescent protein (YFP)-tagged TTG1 (TTG1-YFP) under control of the
TTG1
promoter and found that the protein was ubiquitously distributed in young leaves before trichome formation, but as trichome formation began, TTG1-YFP accumulated in the incipient trichome cells and was less abundant in the neighboring cells, with those closest to the trichome having the least and abundance increasing with distance from the trichome. Inhibition of the proteasome by exposure of the plants to epoxomicin did not prevent this pattern of high and low TTG1-YFP abundance in the leaf cells, but did allow TTG1 to accumulate in the cotyledons, in which TTG1-YFP was undetectable in the absence of proteasomal inhibition. TTG1 appeared to move between cells, likely through plasmodesmata (selective pores between plant cells). When TTG1-YFP was expressed by a promoter that did not drive expression in trichome cells, TTG1-YFP appeared in the initiating trichomes. In mosaic leaves in which the
TGG1
gene was selectively knocked out in some regions, trichomes formed even in the null areas. After microinjection of tagged TTG1 into a tobacco mesophyll cell, TTG1 appeared in neighboring cells, and TTG1-YFP appeared in epidermal cells when expression was forced in subepidermal cells, which are connected to the epidermal cells by plasmodesmata. In plants deficient for GL3, TTG1-YFP was slightly higher in the incipient trichome cell, but was not substantially depleted from the neighboring cells. The authors propose that GL3, which is more abundant in trichome cells, presumably through a transcriptional positive feedback mechanism, binds TTG1 in the initiating trichome; thus, as TTG1 moves out of the adjacent cells, it is trapped in the GL3-positive cells that form the trichome. Mathematical modeling of trapping-depletion-mediated trichome patterning provided a mechanism by which null alleles result in a glabrous (trichomeless) phenotype, but weak alleles lead to clustering of trichomes.

D. Bouyer, F. Geier, F. Kragler, A. Schnittger, M. Pesch, K. Wester, R. Balkunde, J. Timmer, C. Fleck, M. Hülskamp, Two-dimensional patterning by a trapping/depletion mechanism: The role of TTG1 and GL3 in
Arabidopsis
trichome formation.
PLoS Biol.
6
, e141 (2008).
[PubMed]
Expanding β-Arrestin’s Remit
Authors: Ray L. Bryan
Published: September 2008

The β-arrestin proteins help restore G protein-coupled receptors to an inactive state after stimulation. Recently, positive signaling roles for the arrestins have been revealed, as well as roles in signaling by other types of receptors, including the receptor Smoothened (Smo), which has structural similarity to G protein-coupled receptors but signals in a distinct manner. Kovacs
et al
. (see the Perspective by Rohatgi and Scott) report that β-arrestins 1 and 2 in cultured cells were detected in a complex with a tagged version of the Smo protein and with Kif 3A, a kinesin motor protein. Depletion of β-arrestin 1 or β-arrestin 2 inhibited localization of Smo to primary cilia, which function as "antenna"-like structures rich in receptor proteins. Smo-dependent transcriptional activation was also inhibited if the arrestins were depleted. This Smo-dependent interaction of β-arrestins with Kif 3A may link Smo to the transport apparatus that brings it to the primary cilium.

J. J. Kovacs, E. J. Whalen, R. Liu, K. Xiao, J. Kim, M. Chen, J. Wang, W. Chen, R. J. Lefkowitz, β-arrestin–mediated localization of Smoothened to the primary cilium.
Science
320
, 1777-1781 (2008).
[Abstract]
[Full Text]

R. Rohatgi, M. P. Scott, Arrestin' movement in cilia.
Science
320
, 1726-1727 (2008).
[Summary]
[Full Text]
Nuclear GAPDH Triggers Apoptosis
Authors: Gough Nancy R.
Published: September 2008

In addition to its functions in cellular metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is nitrosylated and translocated to the nucleus in response to stimuli that activate inducible nitric oxide synthase (iNOS). Sen
et al
. show that when nitrosylated GAPDH is translocated to the nucleus, it interacts with and is acetylated by the nuclear protein acetyltransferase p300 or its related homolog CBP (collectively p300/CBP). Lys
160
was the acetylated residue on GAPDH, and a K160R mutant was not acetylated and acted as a dominant-negative protein preventing acetylation of wild-type protein, but not inhibiting
S
-nitrosylation or nuclear translocation. When a nuclear localized version of GAPDH (NLS-GAPDH) was expressed in wild-type or
iNOS
-null macrophages, NLS-GAPDH bound to p300/CBP and was acetylated in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treatment, which stimulates iNOS. A nuclear-localized GAPDH that had a mutation at the nitrosylation site also interacted with p300/CBP and was acetylated. Thus, nitrosylation does not appear to be required for the interaction with p300/CBP; instead, it seems to promote the nuclear localization of GAPDH. When p300 and CBP are autoacetylated, their acetylating activities are increased. LPS/IFN-γ stimulated p300 acetylation, and this was blocked by inhibition of iNOS or if GAPDH was depleted from the cells by RNA interference (RNAi). In vitro GAPDH also increased the acetylation of p300, and this did not require nitrosylation of GAPDH and was not enhanced by nitrosylation of GAPDH. To gain insight into how the interaction between GAPDH and p300/CBP may contribute to apoptosis, Sen
et al
. showed that NO stimulated the acetylation of p53 in human embryonic kidney (HEK) 293 cells, and this was prevented if the cells were expressing the K160R mutant, iNOS was inhibited, or GAPDH was depleted by RNAi. Chromatin immunoprecipitation experiments revealed that p53, GAPDH, and p300 were all present at the promoter of
PUMA
, which encodes a p53-regulated protein implicated in apoptosis. When peritoneal macrophages or the neuroblastoma cell line SH-SY5Y were transfected with GAPDH, cell death induced by LPS/IFN-γ or an NO donor, respectively, was increased, and when the cells were transfected with the K160R, mutant cell death was prevented. Thus, GAPDH appears to contribute to cell death triggered by an NO cascade by functioning in the nucleus to stimulate the acetyltransferase activity of p300/CBP, leading to the activation of p53 and proapoptotic gene expression.

N. Sen, M. R. Hara, M. D. Kornberg, M. B. Cascio, B.-I. Bae, N. Shahani, B. Thomas, T. M. Dawson, V. L. Dawson, S. H. Snyder, A. Sawa, Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis.
Nat. Cell Biol.
10
, 866-873 (2008).
[PubMed]
Chromosomes Get Together
Authors: Foley John F.
Published: September 2008

The transcriptional activation of the genes encoding type I interferons (IFNs) in infected cells is critical to the antiviral response. Like some other cytokine genes,
IFN-
β expression is stochastic (not all infected cells express the gene) and mostly monoallelic. Secretion of type I IFNs and activation of their receptors on infected and uninfected cells triggers the activation of other antiviral genes. Transcriptional activation of
IFN-
β requires the temporally coordinated assembly of transcription factors to an enhancer element upstream of its promoter. Nuclear factor κB (NF-κB) binds first, followed by interferon regulatory factor 1 (IRF-1), activating transcription factor 2 (ATF-2), and c-Jun. Last to join this complex (the enhanceosome) is IRF-7, which binds just before transcription begins. Apostolou and Thanos speculated that the inherent complexity of enhanceosome formation might itself be responsible for the stochastic pattern of
IFN-
β expression. By comparing viral-induced
IFN-
β expression in control HeLa cells with that in cells overexpressing the individual transcription factors, the authors found that cellular concentrations of all components of the enhanceosome were limiting. Circular chromosome conformation capture (4C) assays, chromatin immunoprecipitations, and DNA fluorescence in situ hybridization (FISH) experiments demonstrated the viral-induced interaction of the
IFN-
β enhancer with three gene loci (only one of which was on the same chromosome as
IFN-
β), which was dependent on NF-κB binding to these loci. This led to the transfer of NF-κB to the
IFN-
β enhancer, enhanceosome formation, and monoallelic
IFN-
β expression. In a second phase, IFN-β signaling induced
IRF-7
expression in infected and uninfected cells, which promoted enhanceosome formation and
IFN-
β expression independently of interchromosomal interactions. As Schoenfelder and Fraser discuss, this study underlines the need to think of gene regulation in three-dimensional space.

E. Apostolou, D. Thanos, Virus infection induces NF-κB-dependent interchromosomal associations mediating monoallelic
IFN-β
gene expression.
Cell
134
, 85-96 (2008).
[PubMed]

S. Schoenfelder, P. Fraser, Interchromosomal huddle kickstarts antiviral defense.
Cell
134
, 14-16 (2008).
[PubMed]
Resist or Persist
Authors: Simpson Stephen J.
Published: September 2008

Many of the key immune pathways initiated in response to infection begin with the activation of the transcription factor NF-κB. Thus, it would not be unreasonable to expect that at least some pathogens would have evolved the means by which to impede this pathway. Kravchenko
et al
. demonstrate that the opportunistic bacteria
Pseudomonas aeruginosa
produces a small signaling protein, C12, which impairs the turnover of proteins critical to the regulation of NF-κB activity. The resulting reduction in transcription of key immune response genes could have significant influence on persistent infection by this and other bacteria.

V. V. Kravchenko, G. F. Kaufmann, J. C. Mathison, D. A. Scott, A. Z. Katz, D. C. Grauer, M. Lehmann, M. M. Meijler, K. D. Janda, R. J. Ulevitch, Modulation of gene expression via disruption of NF-κB signaling by a bacterial small molecule.
Science
321
, 259-263 (2008).
[Abstract]
[Full Text]
Facilitating Multicellularity
Authors: VanHook Annalisa M.
Published: September 2008

Tyrosine kinase (TK) signaling plays an integral role in intercellular communication in multicellular animals (metazoans). There are three molecular components essential for TK signaling: protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and SH2 domains (which mediate tyrosine phosphorylation-dependent protein-protein interactions). Once thought to be limited to metazoans, TK signaling components have also been identified in choanoflagellates, their closest unicellular relatives.
Monosiga brevicollis
, a colony-forming choanoflagellate, has emerged as a model in which to study the cell biological processes that correlate with the emergence of multicellularity. Manning
et al
. and Pincus
et al
. address the evolution of TK signaling. Both groups surveyed the
Monosiga
genome for genes that encode proteins containing PTK, PTP, and SH2 domains and report a large number, most of which have no metazoan orthologs, of each class. For example, only eight of the
Monosiga
TKs identified by Manning
et al
. have metazoan orthologs, and all of these are Src-related, which indicates that the majority of expansion and diversification of the PTP-, PTK-, and SH2-containing proteins occurred after the two lineages diverged. Both lineages have independently evolved similar repertoires of functionality through domain shuffling and convergent evolution. For example, receptor tyrosine kinases with cysteine-rich motifs have evolved in both lineages. Pincus
et al.
compared tyrosine kinase signaling components across 41 eukaryotes and proposed a model for the emergence of TK signaling systems. Fungi and slime molds, eukaryotes outside the metazoan-choanoflagellate lineage, have PTPs and SH2-containing proteins but not PTKs. Pincus
et al
. point out that even having only these two components could be advantageous because some serine-threonine kinases, which fungi and slime molds have in abundance, do perform tyrosine phosphorylation, albeit inefficiently. The authors propose that the first PTK arose from a spontaneous mutation that rendered one of the less selective serine-threonine kinases capable of phosphorylating only tyrosine residues. The number of types of PTPs, PTKs, and SH2 domains expanded in the metazoan-choanoflagellate lineage as cells exploited this new pathway to communicate with one another. Mayer provides a clear synthesis of the two studies’ combined findings.

G. Manning, S. L. Young, W. T. Miller, Y. Zhai, The protist,
Monosiga brevicollis
, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan.
Proc. Natl. Acad. Sci.
U.S.A.
105
, 9674-9679 (2008).
[Abstract]
[Full Text]

D. Pincus, I. Letunic, P. Bork, W. A. Lim, Evolution of the phospho-tyrosine signaling machinery in premetazoan lineages.
Proc. Natl. Acad. Sci.
U.S.A.
105
, 9680-9684 (2008).
[Abstract]
[Full Text]

B. J. Mayer, Clues to the evolution of complex signaling machinery.
Proc. Natl. Acad. Sci. U.S.A.
105
, 9453-9454 (2008).
[Full Text]
Doubled-Up Decision
Authors: Simpson Stephen J.
Published: September 2008

CD8
+
T cells are major contributors to cell-mediated immunity to virally infected cells and tumors. Like their helper CD4
+
counterparts, these cells rely on the transcriptional regulator T-bet for their correct development. Recently, a second factor called eomesodermin has also been found to control CD8 functions. Intlekofer
et al.
now find that without both factors, CD8
+
T cells fail to develop their normal cell-mediated functions and instead secrete the inflammatory cytokine IL-17, which has been characterized recently in helper T cells. This secretion of IL-17 caused significant pathology in a mouse model of viral infection, which suggests that both transcription factors play a crucial role in maintaining appropriate cell-mediated responses to infection.

A. M. Intlekofer, A. Banerjee, N. Takemoto, S. M. Gordon, C. S. DeJong, H. Shin, C. A. Hunter, E. J. Wherry, T. Lindsten, S. L. Reiner, Anomalous Type 17 response to viral infection by CD8
+
T cells lacking T-bet and eomesodermin.
Science
321
, 408-411 (2008).
[Abstract]
[Full Text]
iPS Cell Origins
Authors: VanHook Annalisa M.
Published: September 2008

Retroviral-driven overexpression of genes encoding four transcription factors (
Oct 3/4
,
Sox2
,
Klf4
, and
c-Myc
) is sufficient to generate pluripotent cells from adult fibroblasts. Whether these induced pluripotent stem (iPS) cells arise from preexisting stem cells or from reprogramming of differentiated cells has not been clear. The group that first developed the iPS cell method reports that iPS cells made from adult mouse epithelial cells from the stomach (gastric epithelia) and liver (primary hepatocytes) were very similar to embryonic stem (ES) cells in several respects, including morphology, proliferation rate, pluripotency, and gene expression profile. Lineage tracing experiments revealed that iPS cells generated from primary hepatocytes were derived from cells that expressed the liver marker
albumin
, illustrating that differentiated cells can indeed be reprogrammed to give rise to iPS cells. Liver-derived iPS cells expressed ES cell markers more robustly than did those derived from adult fibroblasts, and they exhibited more extensive, although not complete, promoter demethylation, suggesting that the epithelial cells were more efficiently reprogrammed than were the fibroblasts. Chimeric mice containing fibroblast-derived iPS cells had a high risk of developing cancer as adults, presumably because the iPS cells contained a retrovirally encoded copy of the oncogene
c-Myc
. The incidence of cancer was reduced in chimeras containing liver-derived iPS cells. Furthermore, it was possible to generate epithelium-derived iPS cells without retroviral expression of the oncogene
c-Myc
, a feature that could be used to further limit the propensity of these cells to cause cancer.

T. Aoi, K. Yae, M. Nakagawa, T. Ichisaka, K. Okita, K. Takahashi, T. Chiba, S. Yamanaka, Generation of pluripotent stem cells from adult mouse liver and stomach cells.
Science
321
, 699-702 (2008).
[Abstract]
[Full Text]
Science Signaling
Podcast: 05 August 2008
Authors: Gough Nancy R. , VanHook Annalisa M.
Published: September 2008

This conversation is about research highlighted in the Editors' Choice summary titled, "Anesthesia Increases Postoperative Pain." The highlighted article is J. A. Matta, P. M. Cornett, R. L. Miyares, K. Abe, N. Sahibzada, G. P. Ahern, General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.
Proc. Natl. Acad. Sci. U.S.A.
105
, 8784–8789 (2008). (Length: 11 min; file size: 5 MB; file format: mp3; location:
http://podcasts.aaas.org/science_signaling/ScienceSignaling_080805.mp3
)
Telling Polys Apart
Authors: Foley John F.
Published: September 2008

Polyubiquitination is a tightly regulated posttranslational modification of proteins that has important implications for their function. When individual ubiquitin molecules in a chain are linked to each other through Lys
48
(K48) residues, the ubiquitinated protein is often targeted for proteasomal degradation. However, if the linkages occur through Lys
63
(K63), the ubiquitinated protein is not degraded; instead, its activity is often enhanced. Distinguishing between these polyubiquitin chains has been technically challenging and often involves mass spectrometry or the use of mutant ubiquitin molecules. By screening a phage display library for low-affinity antibodies against specific polyubiquitin chains, solving the structures of some of the positive clones, and then using these structures to design changes to engineered antibodies, Newton
et al
. developed linkage-specific antibodies that recognized either K48- or K63-linked polyubiquitinated proteins. Immunofluorescent and immunohistochemical analyses performed with these antibodies showed, as expected, that K48-linked, but not K63-linked, polyubiquitinated proteins colocalized with proteasomes. Stimulation of the tumor necrosis factor receptor (TNFR) is known to lead to the recruitment and K63-linked polyubiquitination of the adaptor kinase RIP1, which enables RIP1 to activate nuclear factor κB (NF-κB) signaling. The authors first used their antibodies to confirm the K63-linked polyubiquitination of RIP1 immediately after TNFR stimulation and then found that RIP1 was later polyubiquitinated by K48-linked chains and degraded, coincident with the termination of NF-κB signaling. A similar pattern of polyubiquitin editing was found for interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1), an adaptor kinase recruited to activated Toll-like receptors. These antibodies will likely prove very useful in determining whether other adaptor kinases undergo polyubiquitin editing to modulate their function and in identifying other modified proteins.

K. Newton, M. L. Matsumoto, I. E. Wertz, D. S. Kirkpatrick, J. R. Lill, J. Tan, D. Dugger, N. Gordon, S. S. Sidhu, F. A. Fellouse, L. Komuves, D. M. French, R. E. Ferrando, C. Lam, D. Compaan, C. Yu, I. Bosanac, S. G. Hymowitz, R. F. Kelley, V. M. Dixit, Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.
Cell
134
, 668-678 (2008).
[Online Journal]
Sensing Alarm
Authors: Stern Peter
Published: September 2008

In 1973, Hans Grueneberg observed the presence of a structure at the tip of the rodent nose that, he thought, belonged to the Nervus terminalis. Recently, using transgenic techniques, several groups reported the rediscovery of this structure. They named this structure the Grueneberg ganglion in memory of the original work. However, the function of these cells remains a matter of controversy. Despite the lack of typical olfactory neuronal features, the ganglion was suggested to have some olfactory function, based on the expression of olfactory marker protein and on its neural connections to the olfactory bulb of the brain. Brechbühl
et al.
have now identified a function for the Grueneberg ganglion cells. A combination of anatomical, surgical, and behavioral techniques was used to suggest that the Grueneberg ganglion is involved in alarm pheromone detection.

J. Brechbühl, M. Klaey, M.-C. Broillet, Grueneberg ganglion cells mediate alarm pheromone detection in mice.
Science
321
, 1092-1095 (2008).
[Abstract]
[Full Text]
Fixed But Flexible
Authors: Kelner Katrina L.
Published: September 2008

To what extent is stochastic noise responsible for phenotypic variability in biological systems? Feinerman
et al.
combine computer modeling with measurements of expression levels of multiple proteins required for signaling in populations of T cells, along with the response of the same cells to antigenic stimulation. Together these revealed how stochastic variation in expression of different components of antigen-induced signaling pathways had distinct effects on the response potential of T cells. For a clone of T cells responding to a given antigen, this could help by optimizing flexibility within the responding cell population while maintaining the overall uniformity required for reliable antigen discrimination.

O. Feinerman, J. Veiga, J. R. Dorfman, R. N. Germain, G. Altan-Bonnet, Variability and robustness in T cell activation from regulated heterogeneity in protein levels.
Science
321
, 1081-1084 (2008).
[Abstract]
[Full Text]
Rainbow Signals
Authors: Ash Caroline
Published: September 2008

In addition to an antiseptic function, phenazines—pigmented antibiotics made by bacteria—organize colony structure by activating a superoxidative stress regulator.
Big Brother's a Bully
Authors: Gough Nancy R.
Published: September 2008

In
Caenorhabditis elegans
, a large noncoding RNA inhibits the complex that produces small RNAs.
New Ramifications of Strigolactone Signaling?
Authors: Adler Elizabeth M.
Published: September 2008

Strigolactones inhibit lateral branching in plants.
“SHP”-ed Out of the Nucleus
Authors: Ray L. Bryan
Published: September 2008

Cell cycle-dependent phosphorylation of the phosphatase SHP-1 results in proteolytic cleavage and movement out of the nucleus.
Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis
Authors: Kodiha Mohamed , Brown Claire M. , Stochaj Ursula
Published: September 2008

High-throughput screening and MetaXpress software modules can be adapted to quantify the subcellular localization of fluorescently labeled molecules.
Quality Counts
Authors: Foley John F.
Published: September 2008

Whether T cell receptors in the central region of the immunological synapse signal or are degraded depends on the strength of the antigenic stimulus.
Prospective Study of Fecal Colonization by Extended-Spectrum-β-Lactamase-Producing
Escherichia coli
in Neutropenic Patients with Cancer
Authors: Calatayud L. , Arnan M. , Liñares J. , Dominguez M. A. , Gudiol C. , Carratalà J. , Batlle M. , Ribera J. M. , Gudiol F.
Published: September 2008

Fecal colonization by extended-spectrum-β-lactamase-producing
Escherichia coli
in 912 stool samples collected from 154 neutropenic patients with cancer, hospitalized at two teaching institutions, was prospectively studied. Forty-nine (31.8%) patients were colonized, 22 of them at hospital admission. Most strains were clonally unrelated and carried a CTX-M-9 group enzyme.
Rationale for Reading Fluconazole MICs at 24 Hours Rather than 48 Hours When Testing
Candida
spp. by the CLSI M27-A2 Standard Method
Authors: Ostrosky-Zeichner Luis , Rex John H. , Pfaller Michael A. , Diekema Daniel J. , Alexander Barbara D. , Andes David , Brown Steven D. , Chaturvedi Vishnu , Ghannoum Mahmoud A. , Knapp Cindy C. , Sheehan Daniel J. , Walsh Thomas J.
Published: September 2008

We investigated if CLSI M27-A2
Candida
species breakpoints for fluconazole MIC are valid when read at 24 h. Analysis of a data set showed good correlation between 48- and 24-h MICs, as well as similar outcomes and pharmacodynamic efficacy parameters, except for isolates in the susceptible dose-dependent category, such as
Candida glabrata
.
In Vitro and In Vivo Validation of
ligA
and
tarI
as Essential Targets in
Staphylococcus aureus
Authors: Streker Karin , Schäfer Tina , Freiberg Christoph , Brötz-Oesterhelt Heike , Hacker Jörg , Labischinski Harald , Ohlsen Knut
Published: September 2008

A conditional expression system has been developed using the isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible P
spac
promoter to validate essential genes of
Staphylococcus aureus
in vivo. The system has been applied to prove the essentiality of
ligA
and to evaluate the function of
tarI
, which was found to be essential in vitro but not in vivo.
Population Pharmacokinetics of Fluconazole in Young Infants
Authors: Wade K. C. , Wu D. , Kaufman D. A. , Ward R. M. , Benjamin D. K. , Sullivan J. E. , Ramey N. , Jayaraman B. , Hoppu K. , Adamson P. C. , Gastonguay M. R. , Barrett J. S.
Published: September 2008

Fluconazole is being increasingly used to prevent and treat invasive candidiasis in neonates, yet dosing is largely empirical due to the lack of adequate pharmacokinetic (PK) data. We performed a multicenter population PK study of fluconazole in 23- to 40-week-gestation infants less than 120 days of age. We developed a population PK model using nonlinear mixed effect modeling (NONMEM) with the NONMEM algorithm. Covariate effects were predefined and evaluated based on estimation precision and clinical significance. We studied fluconazole PK in 55 infants who at enrollment had a median (range) weight of 1.02 (0.440 to 7.125) kg, a gestational age at birth (BGA) of 26 (23 to 40) weeks, and a postnatal age (PNA) of 2.3 (0.14 to 12.6) weeks. The final data set contained 357 samples; 217/357 (61%) were collected prospectively at prespecified time intervals, and 140/357 (39%) were scavenged from discarded clinical specimens. Fluconazole population PK was best described by a one-compartment model with covariates normalized to median values. The population mean clearance (CL) can be derived for this population by the equation CL (liter/h) equals 0.015 · (weight/1)
0.75
· (BGA/26)
1.739
· (PNA/2)
0.237
· serum creatinine (SCRT)
−4.896
(when SCRT is >1.0 mg/dl), and using a volume of distribution (
V
) (liter) of 1.024 · (weight/1). The relative standard error around the fixed effects point estimates ranged from 3 to 24%. CL doubles between birth and 28 days of age from 0.008 to 0.016 and from 0.010 to 0.022 liter/kg/h for typical 24- and 32-week-gestation infants, respectively. This population PK model of fluconazole discriminated the impact of BGA, PNA, and creatinine on drug CL. Our data suggest that dosing in young infants will require adjustment for BGA and PNA to achieve targeted systemic drug exposures.
Genetic Heterogeneity in
pbp
Genes among Clinically Isolated Group B Streptococci with Reduced Penicillin Susceptibility
Authors: Nagano Noriyuki , Nagano Yukiko , Kimura Kouji , Tamai Kiyoko , Yanagisawa Hideji , Arakawa Yoshichika
Published: September 2008

The recent emergence of group B streptococcal isolates exhibiting increased penicillin MICs at the Funabashi Municipal Medical Center and other hospitals in Japan prompted a comparative analysis of the penicillin-binding proteins (PBPs) from those strains with the PBPs from penicillin-susceptible strains comprising four neonatal invasive strains isolated from 1976 to 1988 and two recent isolates. The PBP sequences of the penicillin-susceptible strains were highly conserved, irrespective of their isolation date. Of six strains with reduced susceptibility to penicillin (penicillin MICs, 0.25 to 0.5 μg/ml), strains R1, R2, R5, and R6 shared a unique set of five amino acid substitutions, including V405A adjacent to the
402
SSN
404
motif in PBP 2X and one in PBP 2B. The remaining two strains, R3 and R4, shared several substitutions, including Q557E adjacent to the
552
KSG
554
motif in PBP 2X, in addition to the substitutions in PBP 2B, which are commonly found among penicillin-insusceptible strains. Strains R7 and R8, which had a penicillin MIC of 1 μg/ml, shared a unique set of eight amino acid substitutions (two in PBP 2X; two in PBP 2B, including G613R adjacent to the
614
KTG
616
motif; three in PBP 1A; and one in PBP 2A), and the Q557E substitution in PBP 2X was common to R3 and R4. The binding of Bocillin FL was reduced or not detected in some PBPs, including PBP 2X of penicillin-insusceptible strains, but no significant reduction in the level of
pbp2x
transcription was found in such strains. The results of phylogenetic comparative analyses imply the absence of epidemic penicillin-insusceptible strains, and several genetic lineages of penicillin-insusceptible strains have been independently emerging through the accumulation of mutations in their
pbp
genes, especially in
pbp2x
.
Inhibition of
Bacillus anthracis
Spore Outgrowth by Nisin
Authors: Gut Ian M. , Prouty Angela M. , Ballard Jimmy D. , van der Donk Wilfred A. , Blanke Steven R.
Published: September 2008

The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several
Bacillus
species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using
B. anthracis
Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC
50
) and an IC
90
of 0.57 μM and 0.90 μM, respectively. Viable
B. anthracis
organisms were not recoverable from cultures containing concentrations of nisin greater than the IC
90
. These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.
First Countrywide Survey of Acquired Metallo-β-Lactamases in Gram-Negative Pathogens in Italy
Authors: Rossolini Gian Maria , Luzzaro Francesco , Migliavacca Roberta , Mugnaioli Claudia , Pini Beatrice , De Luca Filomena , Perilli Mariagrazia , Pollini Simona , Spalla Melissa , Amicosante Gianfranco , Toniolo Antonio , Pagani Laura
Published: September 2008

Metallo-β-lactamases (MBLs) can confer resistance to most β-lactams, including carbapenems. Their emergence in gram-negative pathogens is a matter of major concern. Italy was the first European country to report the presence of acquired MBLs in gram-negative pathogens and is one of the countries where MBL producers have been detected repeatedly. Here, we present the results of the first Italian nationwide survey of acquired MBLs in gram-negative pathogens. Of 14,812 consecutive nonreplicate clinical isolates (12,245
Enterobacteriaceae
isolates and 2,567 gram-negative nonfermenters) screened for reduced carbapenem susceptibility during a 4-month period (September to December 2004), 30 isolates (28
Pseudomonas aeruginosa
isolates, 1
Pseudomonas putida
isolate, and 1
Enterobacter cloacae
isolate) carried acquired MBL determinants. MBL producers were detected in 10 of 12 cities, with a predominance of VIM-type enzymes over IMP-type enzymes (4:1). Although having an overall low prevalence (1.3%) and significant geographical differences, MBL-producing
P. aeruginosa
strains appeared to be widespread in Italy, with a notable diversity of clones, enzymes, and integrons carrying MBL gene cassettes.
Gain-of-Function Mutations in the Transcription Factor
MRR1
Are Responsible for Overexpression of the
MDR1
Efflux Pump in Fluconazole-Resistant
Candida dubliniensis
Strains
Authors: Schubert Sabrina , Rogers P. David , Morschhäuser Joachim
Published: September 2008

Candida dubliniensis
, a yeast that is closely related to
Candida albicans
, can rapidly develop resistance to the commonly used antifungal agent fluconazole in vitro and in vivo during antimycotic therapy. Fluconazole resistance in
C. dubliniensis
is usually caused by constitutive overexpression of the
MDR1
gene, which encodes a multidrug efflux pump of the major facilitator superfamily. The zinc cluster transcription factor Mrr1p has recently been shown to control
MDR1
expression in
C. albicans
in response to inducing stimuli, and gain-of-function mutations in the
MRR1
gene result in constitutive upregulation of the
MDR1
efflux pump. We identified a gene with a high degree of similarity to
C. albicans MRR1
(Ca
MRR1
) in the
C. dubliniensis
genome sequence. When
C. dubliniensis MRR1
(Cd
MRR1
) was expressed in
C. albicans mrr1
Δ mutants, it restored benomyl-inducible
MDR1
expression, demonstrating that Cd
MRR1
is the ortholog of Ca
MRR1
. To investigate whether
MDR1
overexpression in
C. dubliniensis
is caused by mutations in
MRR1
, we sequenced the
MRR1
alleles from a fluconazole-resistant, clinical
C. dubliniensis
isolate and a matched, fluconazole-susceptible isolate from the same patient as well as those from four in vitro-generated, fluconazole-resistant
C. dubliniensis
strains derived from two different
C. dubliniensis
isolates. We found that all five resistant strains contained single nucleotide substitutions or small in-frame deletions that resulted in amino acid changes in Mrr1p. Expression of these mutated alleles in
C. albicans
resulted in the constitutive activation of the
MDR1
promoter and multidrug resistance. Therefore, mutations in
MRR1
are the major cause of
MDR1
upregulation in both
C. albicans
and
C. dubliniensis
, demonstrating that the transcription factor Mrr1p plays a central role in the development of drug resistance in these human fungal pathogens.
High Prevalence of Plasmid-Mediated Quinolone Resistance Genes
qnr
and
aac(6′)-Ib-cr
in Clinical Isolates of
Enterobacteriaceae
from Nine Teaching Hospitals in China
Authors: Yang Hong , Chen Hongbin , Yang Qiwen , Chen Minjun , Wang Hui
Published: September 2008

Quinolone resistance is an emerging problem in China. To investigate the prevalence of the plasmid-mediated quinolone resistance genes
qnr
and
aac(6′)-Ib-cr
, a total of 265 clinical isolates of
Escherichia coli
,
Klebsiella pneumoniae
,
Citrobacter freundii
, and
Enterobacter cloacae
with ciprofloxacin MICs of ≥0.25 μg/ml were screened at nine teaching hospitals in China. The
qnrA
,
qnrB
,
qnrS
, and
aac(6′)-Ib
genes were detected by PCR. The
aac(6′)-Ib-cr
gene was further identified by digestion with BtsCI and/or direct sequencing. The
qnr
gene was present in significantly smaller numbers of isolates with cefotaxime MICs of <2 μg/ml than isolates with higher MICs (≥2.0 μg/ml) (20.6% and 42.1%, respectively;
P
< 0.05).
aac(6′)-Ib-cr
was present in 17.0% of the isolates tested, and 7.9% of the isolates carried both the
qnr
and the
aac(6′)-Ib-cr
genes. Among the isolates with cefotaxime MICs of ≥2.0 μg/ml,
qnr
and
aac(6
′
)-Ib-cr
were present in 65.7% and 8.6% of
E. cloacae
isolates, respectively; 65.5% and 21.8% of
K. pneumoniae
isolates, respectively; 63.3% and 26.7% of
C. freundii
isolates, respectively; and 6.5% and 16.9% of
E. coli
isolates, respectively. The 20 transconjugants showed 16- to 128-fold increases in ciprofloxacin MICs, 14 showed 16- to 2,000-fold increases in cefotaxime MICs, and 5 showed 8- to 32-fold increases in cefoxitin MICs relative to those of the recipient due to the cotransmission of
bla
CTX-M-14
,
bla
CTX-M-3
,
bla
DHA-1
,
bla
SHV-2
, and
bla
SHV-12
with the
qnr
and
aac(6
′
)-Ib-cr
genes. Southern hybridization analysis showed that these genes were located on large plasmids of different sizes (53 to 193 kb). These findings indicate the high prevalence of
qnr
and
aac(6′)-Ib-cr
in members of the family
Enterobacteriaceae
and the widespread dissemination of multidrug resistance in China.
Mechanisms of T-Lymphocyte Accumulation during Experimental Pleural Infection Induced byMycobacterium bovisBCG
Authors: Souza Mariana C. , Penido Carmen , Costa Maria F. S. , Henriques Maria Graças
Published: September 2008

Tuberculous pleurisy is a frequent extrapulmonary manifestation characterized by accumulation of fluid and inflammatory cells in the pleural space. Here, we investigated the mechanisms of T-lymphocyte accumulation in the pleural space by using a murine model of pleurisy induced byMycobacterium bovisBCG. Intrathoracic (i.t.) injection of BCG (4.5 × 105bacteria/cavity) induced accumulation of T lymphocytes in the pleural cavities of C57BL/6 mice. We observed the presence of CFU in pleural washes conducted 1, 2, 3, 7, and 15 days after pleurisy induction. Pretreatment with fucoidan inhibited T-lymphocyte accumulation at 1 day, but not at 15 days, after BCG-induced pleurisy. Accordingly, adoptive transfer of fluorescein isothiocyanate-labeled blood mononuclear cells to infected mice showed that T lymphocytes migrated into the pleural cavity 1 day (but not 15 days) after BCG injection. Cell-free pleural wash fluids recovered from mice 1 day after BCG i.t. stimulation (day 1 BCG-PW), but not day 7 or day 15 BCG-PW, induced in vitro T-cell transmigration, which was dependent on L-, P-, and E-selectins. In contrast, day 7 BCG-PW (but not day 1 BCG-PW) induced in vitro T-lymphocyte proliferation via interleukin-2 (IL-2) and gamma interferon (IFN-γ). Accordingly, in vivo IL-2 or IFN-γ neutralization abolished T-lymphocyte accumulation 7 days after pleurisy induction. Our results demonstrate that pleural infection induced by BCG leads to T-lymphocyte accumulation in two waves. The acute phase depends on selectin-mediated migration, while the second wave of T-lymphocyte accumulation seems to depend on a local proliferation induced by cytokines produced in situ.
Interleukin-4-Promoted T Helper 2 Responses EnhanceNippostrongylus brasiliensis-Induced Pulmonary Pathology
Authors: Mearns Helen , Horsnell William G. C. , Hoving J. Claire , Dewals Benjamin , Cutler Antony J. , Kirstein Frank , Myburgh Elmarie , Arendse Berenice , Brombacher Frank
Published: September 2008

The role of CD4+T-cell interleukin-4 (IL-4) receptor alpha (IL-4Rα) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4+T-cell IL-4Rα knockout (KO) mice with the parasitic nematodeNippostrongylus brasiliensis, which induces strong host TH2 responses. AlthoughN. brasiliensisexpulsion was not affected in CD4+T-cell IL-4Rα KO mice, the associated lung pathology was reduced. Infected CD4+T-cell IL-4Rα KO mice showed abrogation of airway mucus production. Furthermore, CD4+T-cell IL-4Rα KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4+T cells was reduced in infected CD4+T-cell IL-4Rα KO mice. Restimulation of tissue-derived CD4+T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4+T-cell IL-4Rα KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4+T-cell IL-4Rα KO mice. Restimulation withN. brasiliensisantigen of total lung cell populations and populations with CD4+T cells depleted showed that CD4+T cells were a key TH2 cytokine source. These data demonstrated that CD4+T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated withN. brasiliensisinfections.
Low Prevalence of Antibodies to Preerythrocytic but Not Blood-StagePlasmodium falciparumAntigens in an Area of Unstable Malaria Transmission Compared to Prevalence in an Area of Stable Malaria Transmission
Authors: Noland Gregory S. , Hendel-Paterson Brett , Min Xinan M. , Moormann Ann M. , Vulule John M. , Narum David L. , Lanar David E. , Kazura James W. , John Chandy C.
Published: September 2008

In areas where levels of transmission ofPlasmodium falciparumare high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.
Borrelia burgdorferi
Lacking DbpBA Exhibits an Early Survival Defect during Experimental Infection
Authors: Weening Eric H. , Parveen Nikhat , Trzeciakowski Jerome P. , Leong John M. , Höök Magnus , Skare Jonathan T.
Published: September 2008

Several
Borrelia burgdorferi
genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the
dbpBA
locus, whose products bind host decorin and glycosoaminoglycans. Recently, the
dbpBA
genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a Δ
dbpBA
::Gent
r
derivative of
B. burgdorferi
strain B31. The results indicate that the Δ
dbpBA
::Gent
r
mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of
B. burgdorferi
, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the
dbpBA
mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by
B. burgdorferi
and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.
Characterization of Two
Campylobacter jejuni
Strains for Use in Volunteer Experimental-Infection Studies
Authors: Poly Frédéric , Read Timothy D. , Chen Yu-Han , Monteiro Mario A. , Serichantalergs Oralak , Pootong Piyarat , Bodhidatta Ladaporn , Mason Carl J. , Rockabrand David , Baqar Shahida , Porter Chad K. , Tribble David , Darsley Michael , Guerry Patricia
Published: September 2008

The development of vaccines against
Campylobacter jejuni
would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most
C. jejuni
strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of
C. jejuni
that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing
N
-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-
ido
-heptose, and, in smaller amounts,
d
-
glycero
-
d
-
ido
-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-
altro
-heptose,
N
-acetyl-glucosamine, and minor amounts of 6-deoxy-3-
O
-Me-
altro
-heptose. Both CPSs were shown to carry
O
-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of
C. jejuni
RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.
Evidence that Membrane Rafts Are Not Required for the Action of
Clostridium perfringens
Enterotoxin
Authors: Caserta Justin A. , Hale Martha L. , Popoff Michel R. , Stiles Bradley G. , McClane Bruce A.
Published: September 2008

The action of bacterial pore-forming toxins typically involves membrane rafts for binding, oligomerization, and/or cytotoxicity.
Clostridium perfringens
enterotoxin (CPE) is a pore-forming toxin with a unique, multistep mechanism of action that involves the formation of complexes containing tight junction proteins that include claudins and, sometimes, occludin. Using sucrose density gradient centrifugation, this study evaluated whether the CPE complexes reside in membrane rafts and what role raft microdomains play in complex formation and CPE-induced cytotoxicity. Western blot analysis revealed that the small CPE complex and the CPE hexamer 1 (CH-1) complex, which is sufficient for CPE-induced cytotoxicity, both localize outside of rafts. The CH-2 complex was also found mainly in nonraft fractions, although a small pool of raft-associated CH-2 complex that was sensitive to cholesterol depletion with methyl-β-cyclodextrin (MβCD) was detected. Pretreatment of Caco-2 cells with MβCD had no appreciable effect on CPE-induced cytotoxicity. Claudin-4 was localized to Triton X-100-soluble gradient fractions of control or CPE-treated Caco-2 cells, indicating a raft-independent association for this CPE receptor. In contrast, occludin was present in raft fractions of control Caco-2 cells. Treatment with either MβCD or CPE caused most occludin molecules to shift out of lipid rafts, possibly due (at least in part) to the association of occludin with the CH-2 complex. Collectively, these results suggest that CPE is a unique pore-forming toxin for which membrane rafts are not required for binding, oligomerization/pore formation, or cytotoxicity.