bluuebunny/crossref_metadata_2025 huggingface dataset:
Invasion Signal
Authors: Foley John F.
Published: March 2008

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is implicated in many cancers; however, it is unclear specifically which EGFR pathways are involved in triggering tumor cell invasiveness. The abundance of the small guanosine triphosphatase Arf6, which regulates processes such as membrane trafficking and endocytosis, correlates with the invasiveness of malignant breast cancer cells. In this context, Morishige
et al
. used small interfering RNA (siRNA) to individually decrease the expression of the 10 genes that encode guanine nucleotide exchange factors (GEFs) that activate Arf family proteins in the invasive human breast cancer cell line MDA-MB-231. GEP100 was the only ArfGEF whose depletion resulted in the inhibition of MDA-MB-231 invasion activity, as measured by an in-gel invasiveness assay. Coimmunoprecipitation studies showed that EGF treatment of MDA-MB-231 cells triggered the association of GEP100 with EGFR. In transfected Cos-7 cells, the authors showed the EGF-dependent association of EGFR, GEP100, and Arf6. Further studies showed the usual involvement of the pleckstrin homology (PH) domain in the binding of GEP100 to phosphorylated Tyr
1068
and Tyr
1086
residues on EGF-activated EGFR. Immunohistochemical analyses of ductal carcinoma specimens revealed a correlation between the coexpression of EGFR and GEP100 and tumor severity. Transfection of noninvasive MCF7 cells with both GEP100 and Arf6 was sufficient to induce their invasiveness in response to EGF. Finally, lung metastasis in mice injected with mammary tumor cells that contained GEP100-specific siRNA was reduced compared with that in mice injected with control siRNA-transfected cells. As Valderrama and Ridley discuss in commentary, the specificity of the GEP100-Arf6 interaction in mediating tumor cell invasion may make it an attractive therapeutic target, especially in patients resistant to EGFR inhibitors.

M. Morishige, S. Hashimoto, E. Ogawa, Y. Toda, H. Kotani, M. Hirose, S. Wei, A. Hashimoto, A. Yamada, H. Yano, Y. Mazaki, H. Kodama, Y. Nio, T. Manabe, H. Wada, H. Kobayashi, H. Sabe, GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion.
Nat. Cell Biol.
10
, 85-92 (2008).
[PubMed]

F. Valderrama, A. J. Ridley, Getting invasive with GEP100 and Arf6.
Nat. Cell Biol
.
10
, 16-18 (2008).
[PubMed]
Protozoan Parasite Shares a Hormone with Plants
Authors: Adler Elizabeth M.
Published: March 2008

Research by Nagamune
et al
. has revealed that the plant hormone abscisic acid (ABA) acts in the human parasite
Toxoplasma gondii
. In the protozoan parasite
T. gondii
, calcium release from intracellular stores (which has been implicated in various processes that involve calcium-mediated protein secretion) is regulated by the second messenger cyclic ADP ribose (cADPR). Noting that, in plants, ABA regulates calcium release from intracellular stores by means of cADPR, Nagamune
et al
. exposed
T. gondii
to ABA and found that it increased both their cADPR content and the calcium-dependent secretion of the adhesin MIC2 (microneme protein 2). Biochemical analyses indicated that ABA was present in
T. gondii
lysates, and enzyme-linked immunosorbent assay indicated that its concentration was decreased by treatment with the herbicide fluridone (which inhibits ABA synthesis in plants). There was a spike in the intracellular concentration of ABA before parasite egress from infected cells, and, whereas fluridone failed to prevent invasion of host cells, it inhibited
T. gondii
exit from infected human foreskin fibroblasts. Instead, it promoted differentiation of the parasites into tissue cysts. Exogenous ABA stimulated early egress of parasites from host cells and enabled egress in the presence of fluridone. Fluridone also decreased parasite burden in infected mice and markedly improved survival of mice inoculated with
T. gondii
. The authors thus conclude that a plantlike ABA-mediated calcium-signaling pathway is present in the protozoan
T. gondii
and that disruption of ABA signaling may provide a therapeutic approach against toxoplasmosis.

K. Nagamune, L. M. Hicks, B. Fux, F. Brossier, E. N. Chini, L. D. Sibley, Abscisic acid controls calcium-dependent egress and development in
Toxoplasma gondii
.
Nature
451
, 207-210 (2008).
[PubMed]
NGF Targets Synaptotagmin 4
Authors: Foley John F.
Published: March 2008

After neurons have received a depolarizing signal, the resulting influx of Ca
2+
triggers the fusion of secretory vesicles with the plasma membrane and the release of neurotransmitters. Neurotrophic factors such as nerve growth factor (NGF) enhance this process, although the mechanism responsible is not known. Mori
et al
. expressed a fusion protein of neuropeptide Y and a fluorescent protein (NPY-Venus) in PC12 neuroendocrine cells and then monitored the release of NPY-Venus after applying a depolarizing signal to the cells. The authors confirmed that pretreatment of the cells with NGF enhanced the depolarization-induced secretion of NPY-Venus. Inhibition of c-Jun N-terminal kinase (JNK) blocked the enhancing effect of NGF on NPY-Venus secretion. Western blotting analyses showed that treatment of PC12 cells with NGF resulted in the phosphorylation of JNK and its substrate c-Jun. Yeast two-hybrid studies identified synaptotagmin 4 (Syt 4), a membrane-trafficking protein that regulates exocytosis, as a binding partner for JNK, and this interaction was confirmed in immunoprecipitation experiments. In vitro kinase assays showed that JNK phosphorylated Syt 4 at Ser
135
, and NGF treatment of PC12 cells resulted in JNK-mediated phosphorylation of Syt 4 at Ser
135
. Knockdown of Syt 4 in PC12 cells by short hairpin RNA blocked the enhancement of depolarization-induced secretion by NGF, compared with that in control cells, but had no effect on basal secretion. Finally, immunofluorescence studies showed that NGF stimulated the JNK-dependent localization of Syt 4 to mature secretory vesicles. Together, these data suggest a mechanism by which neurotrophins might enhance basal secretion of neurotransmitters.

Y. Mori, M. Higuchi, Y. Hirabayashi, M. Fukuda, Y. Gotoh, JNK phosphorylates synaptotagmin-4 and enhances Ca
2+
-evoked release.
EMBO J.
27
, 76-87 (2008).
[PubMed]
Where the PS Is
Authors: Hurtley Stella M.
Published: March 2008

The surface charge of the plasma membrane is a crucial determinant of the targeting of signaling proteins. Yeung
et al
. have developed a probe that can reveal the location of the major negatively charged phospholipid, phosphatidylserine (PS), within cell membranes. PS was found in the cytosolic leaflet of the plasma membrane and on endosomes and lysosomes. It appeared to play a role in the targeting of a variety of positively charged proteins within these membranes.

T. Yeung, G. E. Gilbert, J. Shi, J. Silvius, A. Kapus, S. Grinstein, Membrane phosphatidylserine regulates surface charge and protein localization.
Science
319
, 210-213 (2008).
[Abstract]
[Full Text]
Angiopoietin-1 Uncouples the VEGF Receptor from Src
Authors: Gough Nancy R.
Published: March 2008

Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) play complementary roles in vascular development during embryogenesis. In the adult, VEGF signaling increases vascular permeability and Ang1 blocks this VEGF-mediated response. Gavard
et al
. provide evidence that this function of Ang1 is mediated by its actions on the endothelial cells, preventing them from responding to VEGF signaling that triggers disruption of adherens junctions. When injected subcutaneously in mice, Ang1 prevented dermal vascular permeability in response to VEGF injection (acute in vivo vascular permeability model). Experiments with cultured mouse endothelial cells revealed that Ang1 did not prevent VEGF from stimulating downstream effectors involved in proliferation (phosphorylation of Akt, extracellular signal-regulated kinases 1 and 2, and focal adhesion kinase) but did prevent disruption of endothelial barrier function by VEGF. Preexposure of endothelial cell cultures to Ang1 inhibited the ability of VEGF to stimulate the phosphorylation on residue Ser
665
and subsequent endocytosis of the adhesion molecule VE-cadherin, an event associated with disruption of adherens junctions that connect cells to each other. A similar effect on phosphorylation and endocytosis was observed in the acute in vivo vascular permeability model. In cultured endothelial cells, Ang1 prevented the VEGF-mediated activation of Src but did not affect VEGF-mediated activation of two other Src family members, Fyn and Yes. Ang1 stimulated RhoA activation, and the ability of Ang1 to prevent VEGF-mediated endothelial barrier disruption was lost in cells in which RhoA activity was decreased (by RNA interference techniques, or expression of a dominant-negative mutant, or pharmacological inhibition). A downstream target of RhoA is mDia, and knockdown of either mDia1 or mDia2 prevented Ang1 from inhibiting VEGF-stimulated endothelial permeability, whereas overexpression of a constitutively active form of mDia1 blocked VEGF-mediated endothelial permeability. Ang1 stimulated the association of Src with mDia1 and prevented the accumulation of VEGF receptor-Src complexes in response to VEGF. Thus, by sequestering Src away from the VEGF receptor, Ang1 prevents VEGF-mediated vascular permeability without compromising VEGF-mediated proangiogenic signaling.

J. Gavard, V. Patel. J. S. Gutkind, Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia.
Dev. Cell
14
, 25-36 (2008).
[PubMed]
Organizing Brain Development
Authors: Hines Pamela J.
Published: March 2008

In early development, the cortex of the mammalian brain is built layer by layer, from the inside out. But before that occurs, the neural epithelium consists of only a single layer of stem cells. Mangale
et al
. (see the Perspective by Grove) have now determined that in mice the gene
Lhx2
determines cortical identity for cells at these earliest stages. Lhx2 establishes whether cells give rise to the cortical hem, which is now seen to function as an organizer of hippocampal development.

V. S. Mangale, K. E. Hirokawa, P. R. V. Satyaki, N. Gokulchandran, S. Chikbire, L. Subramanian, A. S. Shetty, B. Martynoga, J. Paul, M. V. Mai, Y. Li, L. A. Flanagan, S. Tole, E. S. Monuki, Lhx2 selector activity specifies cortical identity and suppresses hippocampal organizer fate.
Science
319
, 304-309 (2008).
[Abstract]
[Full Text]

E. A. Grove, Organizing the source of memory.
Science
319
, 288-289 (2008).
[Summary]
[Full Text]
FAK Goes Nuclear
Authors: Foley John F.
Published: March 2008

Under normal growth conditions, the tumor suppressor protein p53 is ubiquitinated and degraded, and cells proliferate. However, when cells are under stress, p53 accumulates and induces the expression of the gene encoding p21, which triggers cell cycle arrest. Signals from integrins, transmembrane receptors involved in cell adhesion, can counteract the activation of p53, but the mechanism involved is unclear. Lim
et
al
. investigated whether focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase stimulated by integrin activation, might regulate p53 activity. They generated
fak
–/–
mice, which were embryonic lethal due to a block in mesodermal cell proliferation. Western blotting analyses showed that the abundance of p53 was higher in
fak
–/–
embryos than in wild-type embryos. Whereas cells taken from
fak
–/–
embryos did not proliferate in culture, those taken from
fak
–/–
p53
–/–
or
fak
–/–
p21
–/–
embryos did. Expression of FAK, a kinase-inactive FAK, or the FAK FERM domain (which regulates kinase activity) in
fak
–/–
p21
–/–
fibroblasts reduced the abundance of p53 compared to that in control
fak
–/–
p21
–/–
fibroblasts. The effects of FAK and FAK FERM were blocked by a proteasomal inhibitor. Immunofluorescence and Western blotting studies demonstrated that FAK was localized to the nucleus (an effect dependent on FERM) and that it mediated the ubiquitination and degradation of p53. Coimmunoprecipitation studies showed the formation of a complex between FAK, p53, and the E3 ubiquitin ligase Mdm2, which was also FERM-dependent. Depletion of FAK from human fibroblasts resulted in increased abundance of p53 and p21 and an increased susceptibility to cisplatin-induced apoptosis. Together, these data suggest a kinase-independent, FERM-dependent role for FAK as a nuclear scaffold protein that targets p53 for degradation and enhances cell proliferation.

S.-T. Lim, X. L. Chen, Y. Lim, D. A. Hanson, T.-T. Vo, K. Howerton, N. Larocque, S. J. Fisher, D. D. Schlaepfer, D. Ilic, Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation.
Mol
.
Cell
29
, 9-22 (2008).
[PubMed]
How to Specify a Leg
Authors: Ray L. Bryan
Published: March 2008

During development, relatively simple signaling mechanisms have been elucidated that explain complicated patterns of distinct cell fates. The anteroposterior (AP) and dorsoventral (DV) axes in particular are derived from gradients in the concentration of morphogens that supply cells with positional information. Animals with limbs have another axis, the proximodistal (PD) axis, that must also be specified, and this results from activity of largely the same group of morphogens and transcriptional regulatory components that control the DV and AP axes, making things more complicated to explain. Estella and Mann use a series of experiments in which they manipulate spatiotemporal aspects of morphogen expression and are able to provide a model that explains how dorsal and ventral gradients of the signaling molecules Decapentaplegic (DPP) and Wingless (Wg) can be converted to circular and concentric domains of expression of two target genes,
Distalless
(
Dll
) and
dachshund
(
dac
), that control PD cell fate. Unlike in the wing, where the transcriptional repressor Brinker (Brk) is important for DV axis formation, Brk appears not to influence the DV axis in the leg but rather to control the PD axis. Further, the logical interpretation of Dpp signals is shown to be different in the DV and PD axes; that is, Dpp regulates cell fate through distinct transcriptional targets in each axis. In early stages, expression of
Dll
is dependent on the ratio of Brk activity to the strength of Wg signaling. Later on, the domains of dac and Dll expression become independent of Wg and Dpp. The authors interpret the results to explain formation of the PD axis and the generation of asymmetry in the DV axis of the leg.

C. Estella, R. S. Mann, Logic of Wg and Dpp induction of distal and medial fates in the
Drosophila
leg.
Development
135
, 627-636 (2008).
[Abstract]
[Full Text]
Specifying Gene Expression
Authors: Purnell Beverly A.
Published: March 2008

The efficiency of gene transcription by RNA polymerase II is modulated by the composition of promoter chromatin, including the presence of nucleosome-binding proteins. Using a genomic approach, Krishnakumar
et al
. show that poly(ADP-ribose) polymerase-1 (PARP-1) and the linker histone H1, two nucleosome-binding proteins that compete for binding to nucleosomes in vitro, exhibit a reciprocal pattern of binding at many promoters in vivo. At positively regulated promoters, PARP-1 is bound but H1 is not. Thus, promoter localization patterns of nucleosome-binding factors can specify global gene expression outcomes in vivo.

R. Krishnakumar, M. J. Gamble, K. M. Frizzell, J. G. Berrocal, M. Kininis, W. L. Kraus, Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes.
Science
319
, 819-821 (2008).
[Abstract]
[Full Text]
Integrin as Receptor for Neurotrophin
Authors: Gough Nancy R.
Published: March 2008

Integrins are dimeric receptors composed of an α and a β subunit and are well known for their ability to interact with the extracellular matrix (ECM), through which integrins contribute to cell adhesion and migration and cell survival. There are 18 α and 8 β subunits in mammals, and at least 24 heterodimers have been reported. Given this complexity, it may not be surprising that integrins have also been reported to interact with molecules other than those in the extracellular matrix. For example, α9β1, a widely distributed integrin, interacts with several classes of ligands, including tenascin, thrombospondin 1, and osteopontin (ECM constituents), VCAM1 (a cell adhesion molecule), ADAM12 and 15 (metalloproteinases), and VEGFs (growth factors). Staniszewska
et al
. now provide evidence that this promiscuous integrin α9β1 also binds to the neurotrophins NGF, NT3, and BDNF. Most of their experiments were performed with cells transfected to express α9β1 (α9SW480 cells). In a cell adhesion assay, α9SW480 cells adhered to microplate wells coated with purified mouse NGF, human recombinant NGF, BDNF, or NT3 at the same efficiency as those cells adhered to VCAM1, a known ligand for α9β1. Untransfected cells exhibited little adherence. Adherence to NGF-coated wells was blocked in the presence of an antibody specific for α9β1 (Y9A2), snake venom protein selective for α9β1 or α4β1 (VLO5), or an antibody selective for β1 (Lia1/2) but was not affected by antibodies or venom proteins specific for other integrins. Using an enzyme-linked immunosorbent assay (ELISA) with α9β1 coating the plate, human recombinant NGF bound in a dose-dependent manner with a Kd of ~4.5 nM. Although the α9SW480 cells did appear to have some high-affinity neurotrophin receptors (likely composed of TrkA and p75
NTR
), the responses detected appeared to be mediated by the integrin, because VLO5 or Y9A2 blocked the responses. The responses detected when the α9SW480 cells bound to NGF included proliferation (likely involving extracellular signal-regulated kinases 1 and 2, which were phosphorylated) and chemotaxis and migration (likely involving paxillin, which was phosphorylated). Neutrophils were used in a transmigration assay to show that a cell that endogenously expresses α9β1 could be stimulated to migrate by NGF and this migration was blocked by Y9A2.

I. Staniszewska, I. K. Sariyer, S. Lecht, M. C. Brown, E. M. Walsh, G. P. Tuszynski, M. Safak, P. Lazarovici, C. Marcinkiewicz, Integrin α9β1 is a receptor for nerve growth factor and other neurotrophins.
J. Cell Sci.
121
, 504-513 (2008).
[Abstract]
[Full Text]
Decisions, Decisions
Authors: Ray L. Bryan
Published: March 2008

By connecting cellular response systems to external and internal stimuli, complex signaling networks appear to provide not only a communication system but also an information processing system. Such decision-making on the basis of sophisticated pattern recognition of noisy inputs is not unlike human face recognition--a task relatively easy for the human brain but not trivial for a computer. Helikar
et al
. provide evidence that cellular signaling systems do indeed have the necessary characteristics for such complex information processing. The authors constructed a Boolean model of signal transduction by receptor tyrosine kinases, G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors, and integrins. They derived the logical instruction sets (the rules that govern activity of a particular protein or protein state, given the states of its interacting regulators) for 130 protein nodes in the model network from a set of 800 papers and then sampled the response of the system to 10,000 random inputs. They found that noisy random inputs were partitioned into a limited number of global cellular responses that appeared to be biologically relevant. Thus, the system demonstrated a basic characteristic of pattern recognition machines. The observations support the reasoning that characteristics of complex cellular signaling systems are not an accident of evolution but rather reflect the necessary structure of a pattern recognition system that allows cells to sample random inputs from hormones and growth factors and the like and then to decide on an appropriate response that ensures or enhances organismal survival.

T. Helikar, J. Konvalina, J. Heidel, J. A. Rogers, Emergent decision-making in biological signal transduction networks.
Proc. Natl. Acad. Sci. U.S.A.
105
, 1913-1918 (2008).
[Abstract]
[Full Text]
Looks Like a Protease, Walks Like a Protease…
Authors: Ray L. Bryan
Published: March 2008

The protein MALT1 (for mucosa-associated lymphoid tissue lymphoma translocation 1) has sequence similarity to proteases, but its best-characterized signaling role is not derived from protease activity. MALT1 is a component of the signalosome protein complex that transmits signals from the T cell and B cell antigen receptors to the transcriptional regulator NF-κB. In the signalosome, MALT1’s presumed protease activity appears not to be essential to activation of NF-κB, and MALT1 instead contributes to ubiquitination of IκB kinase γ (IKKγ). Two papers now show that the protease catalytic domain is indeed put to good use during T cell activation. Rebeaud
et al
. show that the adaptor protein Bcl-10 (also part of the signalosome) is a target for MALT1-mediated proteolysis. They went on to develop an inhibitor of MALT1 protease activity and showed that although NF-κB through the T cell receptor did not require MALT1 activity, the protease was necessary for optimal activation of NF-κB. Furthermore, depletion of MALT1 or Bcl-10 with siRNA showed that cleavage of Bcl-10 was required for optimal adhesion of activated T cells to fibronectin. Coornaert
et al
. discovered another target of MALT1-mediated proteolysis--the A20 protein, an inhibitor of NF-κB signaling pathway that they found was also recruited to the signalosome. A20 has deubiquitinase activity, and one of its targets is IKKγ. The MALT-1-mediated cleavage of A20 may inhibit deubiquitination of IKKγ by lopping off a binding domain of A20 that interacts with IKKγ. Thus, MALT1’s protease activity apparently has at least two effects on immune responses to antigen--inactivation of an inhibitor of the NF-κB signaling pathway and facilitation of integrin-mediated adhesion. McAllister-Lucas and Lucas provide background and commentary.

F. Rebeaud, S. Hailfinger, A. Posevitz-Fejfar, M. Tapernoux, R. Moser, D. Rueda, O. Gaide, M. Guzzardi, E. M. Iancu, N. Rufer, N. Fasel, M. Thome, The proteolytic activity of the paracaspase MALT1 is key in T cell activation.
Nat. Immunol
.
9
, 272-281 (2008).
[PubMed]

B. Coornaert, M. Baens, K. Heyninck, T. Bekaert, M. Haegman, J. Staal, L. Sun, Z. J. Chen, P. Marynen, R. Beyaert, T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-κB inhibitor A20.
Nat. Immunol.
9
, 263-271 (2008).
[PubMed]

L. M. McAllister-Lucas, P. C. Lucas, Finally, MALT1 is a protease!
Nat. Immunol.
9
, 231-233 (2008).
[PubMed]
Connecting Wnt to the Cytoskeleton
Authors: Gough Nancy R.
Published: March 2008

The planar cell polarity pathway is important for the proper orientation of cells in various tissues. Wnt is an extracellular signal that triggers this process by stimulating rearrangement of the actin cytoskeleton through the actions of the guanine triphosphatases (GTPases) Rho and Rac. However, the guanine nucleotide exchange factors (GEFs) that connect Wnt signaling to these GTPases are unknown. While screening
Xenopus
embryos for genes expressed in the notochord--a tissue that undergoes convergent extension, which is a process that mediates the anterior-posterior extension of the body axis during development--Tanegashima
et al
. identified a gene homologous to human
WGEF
(encoding weak-similarity GEF).
XWGEF
was expressed at the early gastula stage, became restricted to the notochord at the end of gastrulation, and then in the tail-bud stages expression was detected in the notochord and head region. When a tagged version of XWGEF was overexpressed, the protein was found colocalized with actin and appeared to be associated with the cell membrane. When expressed in human 293 cells, XWGEF coimmunoprecipitated and increased the abundance of GTP-bound RhoA but did not interact with and had no effect on Rac or Cdc42. When mRNA for hWGEF or XWGEF was injected into
Xenopus
embryos, RhoA activation was observed and the embryos exhibited phenotypes similar to those observed in which a constitutively active RhoA mRNA was injected. Injection of mRNA encoding a dominant-negative RhoA or a dominant-negative Rok (a downstream effector of RhoA), but not mRNA encoding a dominant-negative Rac1, rescued the body axis phenotype produced by overexpression of XWGEF. Using morpholino oligonucleotides to reduce XWGEF, the authors found that the embryos exhibited neural tube closure defects and a shortened axis, consistent with a defect in convergent extension. Epistasis experiments placed XWGEF downstream of Wnt and upstream of Daam-1 (a formin-homology protein). Tagged proteins expressed in mammalian cultured cells showed that WGEF interacted with both Dishevelled (Dsh, a component downstream of Wnt) and Daam-1. The authors propose a model whereby activation of the Frizzled receptor by Wnt leads to the formation of a complex of Dsh, Daam-1, and WGEF at the membrane, which allows WGEF to stimulate RhoA and regulate the actin cytoskeleton.

K. Tanegashima, H. Zhao, I. B. Dawid, WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in
Xenopus
gastrulation.
EMBO J.
27
, 606-617 (2008).
[PubMed]
Pulling on the TCR
Authors: Gough Nancy R.
Published: March 2008

The exact mechanism by which peptide-loaded major histocompatibility complex (pMHC) activates the T cell receptor (TCR) has been controversial. Various models have been proposed to account for the requirement of presentation by antigen-presenting cells (APCs) for effective TCR activation and the lack of activation by soluble pMHC, the ability of very low concentrations of agonist pMHC in the presence of endogenous pMHC to activate TCR signaling, and the apparent necessity of costimulatory interactions for effective activation. Ma
et al
. anchored pMHCs to either a fluid lipid bilayer or plastic surfaces and found that in the absence of nonagonist pMHC a minimum of 1 to 10 monomeric agonist pMHC (MCC-loaded pMHC) per T cell was sufficient to stimulate TCR signaling in T cells interacting with the artificial substrates. Furthermore, the T cell response (calcium signal) was not enhanced by the addition of nonagonist pMHC with the agonist pMHC or under conditions where the nonagonist pMHC and agonist pMHC were close enough to function as a dimer (by coupling to the same streptavidin molecule before covalent attachment to the artificial surface). To confirm that endogenous nonagonist pMHC did not enhance T cell activation in a more physiological situation, the authors loaded murine B cell lymphoma cells with various endogenous peptides and the MCC peptide and found that the production of interleukin-2 by the T cells was the same whether a previously reported costimulatory endogenous pMHC was present or not. The authors used another lipid bilayer system to which T cells would not bind unless the adhesion molecule ICAM was incorporated and found that adhesion was necessary for TCR triggering by MCC-loaded pMHC. When the T cells were treated with cytochalasin to depolymerize actin, the T cells failed to respond to MCC-loaded lymphoma cells or MCC-loaded pMHC bilayers or plastic surfaces, suggesting that the actin cytoskeleton is necessary for TCR triggering. The authors discuss how their results can be reconciled with the reports, suggesting that endogenous pMHC enhance T cell response to low-abundance agonist pMHC and propose a receptor deformation model for TCR activation. In their model, the agonist pMHC-TCR interaction leads to receptor deformation and activation when the cytoskeleton provides a pulling force on the complex. Endogenous pMHC-TCR interactions would be insufficiently strong to provide the necessary activation signal, and the complex would dissociate when "pulled on" by the cytoskeleton.

Z. Ma, K. A. Sharp, P. A. Janmey, T. H. Finkel, Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity.
PLoS Biol.
6
, e43 (2008).
[PubMed]
Extracellular Calcium Matters, Too
Authors: Foley John F.
Published: March 2008

During the development of the nervous system, neurons extend processes that navigate the microenvironment by responding to various guidance molecules; many of the signaling pathways involved depend on intracellular Ca
2+
. The extracellular calcium-sensing receptor (CaSR) is found in organs known to regulate Ca
2+
homeostasis; however, CaSR is also found in areas of the brain, such as the hippocampus, and on sympathetic neurons in the peripheral nervous system. This led Vizard
et
al
. to investigate whether CaSR might play a role in neuronal development. The authors used real-time polymerase chain reaction assays and immunofluorescence microscopy to show that the abundance of CaSR in the superior cervical ganglion (SCG) of the mouse peaks on embryonic day 18 (E18) before declining within a few days of birth. This coincides with a transient increase in the concentration of free extracellular Ca
2+
([Ca
2+
]
o
) in the embryo and with the period during which sympathetic axons innervate their targets. E18 SCG neurons cultured with nerve growth factor developed longer and more branched neurites when exposed to 2.3 mM [Ca
2+
]
o
than did those cultured in 0.7 mM [Ca
2+
]
o
. Expression in neurons of a dominant-negative CaSR inhibited the effect of [Ca
2+
]
o
on neurite growth and branching in vitro. The retinas of
Casr
−/−
mice showed decreased innervation by sympathetic axons compared with those of wild-type mice. Expression of dominant-negative CaSR in hippocampal pyramidal neurons in organ cultures resulted in reduced dendrite outgrowth and branching compared with that in control cultures. As Spitzer discusses in commentary, these data suggest that CaSR controls neurite outgrowth and branching in response to [Ca
2+
]
o
, which acts here as a first rather than second messenger.

T. N. Vizard, G. W. O’Keeffe, H. Gutierrez, C. H. Kos, D. Riccardi, A. M. Davies, Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor.
Nat
.
Neurosci
.
11
, 285-291 (2008).
[PubMed]

N. C. Spitzer, Calcium: First messenger.
Nat
.
Neurosci
.
11
, 243-244 (2008).
[PubMed]
Dissecting Function in the Living Brain
Authors: Kelner Katrina L.
Published: March 2008

Because the brain is composed of many interconnected cell types in close proximity, it is not easy to determine the precise function of any one class. Existing methods such as lesions or pharmacological inhibition are relatively crude, and their effects cannot reliably be used to eliminate the contribution of one cell type. Even genetic approaches, which can be targeted to certain cells, have only been used to inhibit one receptor subtype, and the deficits are often present throughout development, confounding interpretation. Nakashiba
et al
.

constructed a tetanus-toxin-based triple transgenic mouse that allows reversible inhibition of all the synaptic activity of one type of cell in the hippocampus, the CA3 pyramidal cell. When the CA3 hippocampal cells were silenced, the rats could still learn a spatial task but could not recall certain memories or perform rapid learning tasks.

T. Nakashiba, J. Z. Young, T. J. McHugh, D. L. Buhl, S. Tonegawa, Transgenic inhibition of synaptic transmission reveals role of CA3 output in hippocampal learning.
Science
319
, 1260-1264 (2008).
[Abstract]
[Full Text]
Meeting Report: cGMP MattersA report on the 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications, Dresden, Germany, 15 to 17 June 2007.
Authors: Kemp-Harper Barbara , Feil Robert
Published: March 2008

The second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) controls many cellular functions ranging from growth to contractility. Generated from guanylyl cyclases in response to natriuretic peptides or nitric oxide, cGMP transduces its effects through a number of cGMP effectors, including cGMP-regulated phosphodiesterases and protein kinases. Drugs that modulate cGMP levels are emerging as promising therapies, particularly for cardiovascular disorders. This report summarizes new data on the molecular mechanisms, (patho)physiological relevance, and therapeutic potential of the cGMP signaling system that were presented at the 3rd cGMP meeting held in June 2007 in Dresden, Germany.
Characterization and Growth of Polymorphic
Rickettsia felis
in a Tick Cell Line
Authors: Sunyakumthorn Piyanate , Bourchookarn Apichai , Pornwiroon Walairat , David Connie , Barker Steven A. , Macaluso Kevin R.
Published: March 2008

Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine
Rickettsia felis
morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form
R. felis
. Without exchange of host cell growth medium, polymorphic
R. felis
was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form
R. felis
organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form
R. felis
entered into and replicated in host cells faster than long-form
R. felis
. However, when both short- and long-form
R. felis
organisms were maintained in cell-free medium for 12 days, the infectivity of short-form
R. felis
was decreased compared to long-form
R. felis
organisms, which were capable of entering host cells, suggesting that long-form
R. felis
is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of
R. felis
may utilize metabolically and morphologically distinct forms for successful transmission.
Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS
Authors: Behrens Sebastian , Lösekann Tina , Pett-Ridge Jennifer , Weber Peter K. , Ng Wing-On , Stevenson Bradley S. , Hutcheon Ian D. , Relman David A. , Spormann Alfred M.
Published: March 2008

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic
Escherichia coli
and
Vibrio cholerae
cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with
13
C-carbon and
15
N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.
Concentration of Enteroviruses, Adenoviruses, and Noroviruses from Drinking Water by Use of Glass Wool Filters
Authors: Lambertini Elisabetta , Spencer Susan K. , Bertz Phillip D. , Loge Frank J. , Kieke Burney A. , Borchardt Mark A.
Published: March 2008

Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min
−1
), and its low cost makes it advantageous for studies requiring large numbers of samples.
Assessment of the Genetic Diversity of
Xanthomonas axonopodis
pv. phaseoli and
Xanthomonas fuscans
subsp.
fuscans
as a Basis To Identify Putative Pathogenicity Genes and a Type III Secretion System of the SPI-1 Family by Multiple Suppression Subtractive Hybridizations
Authors: Alavi Seyed Mehdi , Sanjari Saeideh , Durand Fabien , Brin Chrystelle , Manceau Charles , Poussier Stéphane
Published: March 2008

Fluorescent amplified fragment length polymorphism revealed that strains of
Xanthomonas axonopodis
pv. phaseoli and
Xanthomonas fuscans
subsp.
fuscans
are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.
Recovery, Purification, and Cloning of High-Molecular-Weight DNA from Soil Microorganisms
Authors: Liles Mark R. , Williamson Lynn L. , Rodbumrer Jitsupang , Torsvik Vigdis , Goodman Robert M. , Handelsman Jo
Published: March 2008

We describe here an improved method for isolating, purifying, and cloning DNA from diverse soil microbiota. Soil microorganisms were extracted from soils and embedded and lysed within an agarose plug. Nucleases that copurified with the metagenomic DNA were removed by incubating plugs with a high-salt and -formamide solution. This method was used to construct large-insert soil metagenomic libraries.
Isolation of the Exoelectrogenic Bacterium
Ochrobactrum anthropi
YZ-1 by Using a U-Tube Microbial Fuel Cell
Authors: Zuo Yi , Xing Defeng , Regan John M. , Logan Bruce E.
Published: March 2008

Exoelectrogenic bacteria have potential for many different biotechnology applications due to their ability to transfer electrons outside the cell to insoluble electron acceptors, such as metal oxides or the anodes of microbial fuel cells (MFCs). Very few exoelectrogens have been directly isolated from MFCs, and all of these organisms have been obtained by techniques that potentially restrict the diversity of exoelectrogenic bacteria. A special U-tube-shaped MFC was therefore developed to enrich exoelectrogenic bacteria with isolation based on dilution-to-extinction methods. Using this device, we obtained a pure culture identified as
Ochrobactrum anthropi
YZ-1 based on 16S rRNA gene sequencing and physiological and biochemical characterization. Strain YZ-1 was unable to respire using hydrous Fe(III) oxide but produced 89 mW/m
2
using acetate as the electron donor in the U-tube MFC. Strain YZ-1 produced current using a wide range of substrates, including acetate, lactate, propionate, butyrate, glucose, sucrose, cellobiose, glycerol, and ethanol. Like another exoelectrogenic bacterium (
Pseudomonas aeruginosa
),
O. anthropi
is an opportunistic pathogen, suggesting that electrogenesis should be explored as a characteristic that confers advantages to these types of pathogenic bacteria. Further applications of this new U-tube MFC system should provide a method for obtaining additional exoelectrogenic microorganisms that do not necessarily require metal oxides for cell respiration.
Regulation of Thermotolerance by Stress-Induced Transcription Factors in
Saccharomyces cerevisiae
Authors: Yamamoto Noritaka , Maeda Yuka , Ikeda Aya , Sakurai Hiroshi
Published: March 2008

The heat shock transcription factor Hsf1 and the general stress transcription factors Msn2 and Msn4 (Msn2/4) are major regulators of the heat shock response in
Saccharomyces cerevisiae
. Here, we show that transcriptional activation of their target genes, including
HSP104
, an antistress chaperone gene, is obligatory for thermotolerance. Although Hsf1 activity might be necessary before the exposure of cells to high temperature, severe heat shock induced the binding of hyperphosphorylated Hsf1 to its target promoters. However, promoter-bound, phosphorylated Hsf1 was inactive for transcription because RNA polymerase II was inactive at high temperatures. Rather, our results suggest that Hsf1 activates the transcription of most of its target genes during the recovery period following severe heat shock. This delayed upregulation by Hsf1, which would be induced by misfolded proteins that accumulate in severely heat-shocked cells, is required for the resumption of normal cell growth. In contrast, the factors Msn2/4 were not involved in the delayed upregulation of genes and were dispensable for cell growth during the recovery period, suggesting that they play a role before the exposure to high temperature. These results show that Hsf1 and Msn2/4 act differentially before and after exposure to extreme temperatures to ensure cell survival and growth.
Regulation of Gene Expression in a Mixed-Genus Community: Stabilized Arginine Biosynthesis inStreptococcus gordoniiby Coaggregation withActinomyces naeslundii
Authors: Jakubovics Nicholas S. , Gill Steven R. , Iobst Stacey E. , Vickerman M. M. , Kolenbrander Paul E.
Published: March 2008

Interactions involving genetically distinct bacteria, for example, between oral streptococci and actinomyces, are central to dental plaque development. A DNA microarray identifiedStreptococcus gordoniigenes regulated in response to coaggregation withActinomyces naeslundii. The expression of 23 genes changed >3-fold in coaggregates, including that of 9 genes involved in arginine biosynthesis and transport. The capacity ofS. gordoniito synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically at low arginine concentrations. In dual-species cultures containing coaggregates, however,S. gordoniigrew to high cell density at low arginine concentrations. Equivalent cocultures without coaggregates showed no growth until coaggregation was evident (9 h). AnargHmutant was unable to grow at low arginine concentrations with or withoutA. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative reverse transcriptase PCR, the expression ofargC,argG, andpyrAbwas strongly (10- to 100-fold) up-regulated inS. gordoniimonocultures after 3 h of growth when exogenous arginine was depleted. Cocultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation withA. naeslundii, the expression ofargC,argG, andpyrAbinS. gordoniiwas partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus,A. naeslundiistabilizesS. gordoniiexpression of arginine biosynthesis genes in coaggregates but not cocultures and enables aerobic growth when exogenous arginine is limited.
Phylogenetic Analysis of the Incidence ofluxGene Horizontal Transfer inVibrionaceae
Authors: Urbanczyk Henryk , Ast Jennifer C. , Kaeding Allison J. , Oliver James D. , Dunlap Paul V.
Published: March 2008

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT inVibrionaceaeand its possible impact on speciation, we assessed the incidence of interspecies transfer of theluxgenes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11Aliivibrio,Photobacterium, andVibriospecies, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrBorpyrH) and a representativeluxgene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB,recA, andpyrH) and multipleluxgenes (luxCDABEG). In nearly all cases, housekeeping gene andluxgene phylogenies were congruent, and there was no instance in which theluxgenes of one luminous species had replaced theluxgenes of another luminous species. Therefore, theluxgenes are predominantly vertically inherited inVibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) theluxgenes of the only known luminous strain ofVibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to theluxgenes ofVibrio harveyi; (ii) theluxgenes of two luminous strains ofVibrio chagasii, 21N-12 and SB-52, were closely related to those ofV. harveyiandVibrio splendidus, respectively; (iii) theluxgenes of a luminous strain ofPhotobacterium damselae, BT-6, were closely related to theluxgenes of thelux-rib2operon ofPhotobacterium leiognathi; and (iv) a strain of the luminous bacteriumPhotobacterium mandapamensiswas found to be merodiploid for theluxgenes, and the second set ofluxgenes was closely related to theluxgenes of thelux-rib2operon ofP. leiognathi. In none of these cases of apparent HGT, however, did acquisition of theluxgenes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of theluxgenes in nature is rare and that horizontal acquisition of theluxgenes apparently has not contributed to speciation in recipient taxa.