bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibiting Akt
Authors: Gough Nancy R.
Published: December 2007

The Akt family of protein kinases is involved in mediating cell survival signals, and cancers are often associated with mutations in genes encoding negative regulators of Akt activity, such as the lipid phosphatase PTEN. Activation of Akt involves recruitment to the plasma membrane, followed by phosphorylation by activating kinases. Cinar
et al
. report that the kinases Mst1 and Mst2 inhibit Akt activity in a mechanism that is independent of the kinase activity of Mst. Fractionation of lipid rafts (Triton X-100-insoluble membranes) showed that ~10% of Akt1 was present in this compartment in the prostate cancer cell line LNCaP (a PTEN-deficient cell line in which Akt1 is constitutively active), and coimmunoprecipitation followed by mass spectrometry analysis revealed that Mst1 (also known as STK4) was in a complex with Akt1 in this compartment. The interaction appeared to be direct in that purified proteins interacted. Mst1 is cleaved by caspase into a C-terminal domain containing the regulatory region (Mst1-C) and a N-terminal domain containing the kinase domain (Mst1-N). Full-length Mst1, Mst1-C, and Mst1-N interacted with a tagged form of Akt1 when overexpressed in HEK 293T cells. A kinase-inactive form of the Mst1-N also interacted with Akt1, suggesting that kinase activity was not required for the interaction. All three forms of Mst1 interacted with the C-terminal hydrophobic domain of Akt1 based on experiments with Akt1 mutants. Proteins expressed in transfected cells [Mst1, Mst1-N, and Mst1-C and GFP-Myr-Akt1 (myristoylated and tagged with green fluorescent protein)] and in LNCaP cells (endogenous phosphorylated Akt1 and transfected Mst1-N and Mst1-C) showed that although the full-length Mst1 and Mst1-C colocalized with Akt1 at the plasma membrane and cytoplasm, Mst1-N did not colocalize with Akt1 at the membrane; instead, in the LNCaP cells it colocalized with Akt1 in the nucleus. RNA interference experiments with LNCaP cells showed that depletion of either Mst1 or the related protein Mst2 enhanced Akt1 activity, whereas overexpression of the Mst proteins inhibited Akt1 activity. Expression of Mst1, Mst1-C, Mst1-N, or the kinase-inactive Mst1-N reverted a gastrulation defect induced by expression of constitutively active Akt (Myr-Akt1) in zebrafish embryos, further confirming the negatively regulatory role of Mst. Finally, immunohistochemical analysis of a human prostate cancer tissue array showed that tissues with the highest levels of activated Akt had the lowest levels of Mst1, suggesting that loss of this inhibitory input may be clinically relevant during cancer progression.

B. Cinar, P.-K. Fang, M. Lutchman, D. Di Vizio, R. M. Adam, N. Pavlova, M. A. Rubin, P. C. Yelick, M. R. Freeman, The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1.
EMBO J
.
26
, 4523-4534 (2007).
[PubMed]
Reversing to the Nucleus
Authors: Foley John F.
Published: December 2007

Membrane-bound ephrinB proteins bind to and activate EphB receptor tyrosine kinases on adjacent cells after cell-to-cell contact and regulate many processes, including cell adhesion. The activation of EphB receptors is considered a "forward" signal of ephrinB. In the cell expressing ephrinB, "reverse signaling" occurs and involves phosphorylation of the C terminus of ephrinB leading to the recruitment of the adaptor protein Grb4 (growth factor receptor-bound protein 4) and the activation of focal adhesion kinase (FAK). Sequence analysis of the ephrinB C terminus revealed the presence of the conserved YXXQ motif that is important for the binding and activation of signal transducer and activator of transcription 3 (STAT3), which led Bong
et al
. to investigate whether ephrinB might also activate STAT3. The authors performed a series of immunoprecipitation and Western blotting experiments in
Xenopus
oocytes (and mammalian cell lines) transfected with wild-type and mutant forms of ephrinB and STAT3. ephrinB was activated either by the addition of the EphB2 ectodomain fused to the Fc portion of human immunoglobulin or in response to fibroblast growth factor (FGF) receptor activation, which is known to lead to ephrinB phosphorylation. ephrinB that was phosphorylated at two specific tyrosine residues recruited STAT3, which resulted in STAT3 phosphorylation that was Janus kinase 2 (Jak2)-dependent. Reporter assays and gel-shift experiments showed that ephrinB activation also increased both the DNA-binding and transcriptional activity of STAT3. Immunofluorescence microscopy in mouse embryonic neuroepithelial cells showed that ephrinB activation resulted in the phosphorylation and nuclear localization of STAT3. Together, these data suggest that STAT3-mediated regulation of gene transcription may represent another "reverse" signaling pathway for ephrinB.

Y.-S. Bong, H.-S. Lee, L. Carim-Todd, K. Mood, T. G. Nishanian, L. Tessarollo, I. O. Daar, ephrinB1 signals from the cell surface to the nucleus by recruitment of STAT3.
Proc. Natl. Acad. Sci. U.S.A.
104
, 17305-17310 (2007).
[Abstract]
[Full Text]
The Sound of Silence
Authors: Foley John F.
Published: December 2007

The spontaneous firing of auditory neurons triggered by the release of the neurotransmitter glutamate from the inner hair cells (IHCs) of the cochlea is critical for neuronal survival during development of the auditory system. However, how these responses are triggered before the auditory canal has opened (that is, without sound as a stimulus) is unknown. Tritsch
et al
. removed cochlea from postnatal rats with closed auditory canals, performed whole-cell voltage clamp experiments, and measured low-frequency spontaneous currents and extracellular potentials in the supporting cells of a structure called Kölliker’s organ, which resides close to the IHCs. Antagonists of the P2 purinergic receptors P2Y and P2X inhibited these spontaneous potentials, as did the ATP-hydrolyzing enzyme, apyrase. The supporting cells contain connexin 26 and are connected to each other by gap junctions, and ATP-dependent spontaneous potentials were blocked by gap junction inhibitors. The authors then simultaneously measured light scattering and electrical activity in the Kölliker’s organ and nearby IHCs and demonstrated ATP-dependent spontaneous currents in the IHCs that were coincident with waves of cell-shape changes and currents in the supporting cells of the Kölliker’s organ. Whole-cell recordings of afferent neurons at the base of IHCs showed that both endogenous (from the supporting cells) and exogenous ATP elicited glutamate-dependent synaptic currents. Finally, cell-shape changes, extracellular potentials, and spontaneous IHC currents were almost completely absent in the cochlea of rats whose auditory canals had opened. The lack of spontaneous action potentials in the IHCs of animals that can hear prevents interference with the accurate detection of sound. Forsythe discusses the implications of these findings in accompanying commentary.

N. X. Tritsch, E. Yi, J. E. Gale, E. Glowatzki, D. E. Bergles, The origin of spontaneous activity in the developing auditory system.
Nature
450
, 50-55 (2007).
[PubMed]

I. D. Forsythe, A fantasia on Kölliker’s organ.
Nature
450
, 43-44 (2007).
[PubMed]
Commonalities Between pDCs and B Cells
Authors: Gough Nancy R.
Published: December 2007

Plasmacytoid dendritic cells (pDCs) are a subpopulation of dendritic cells that secrete large amounts of interferons (IFNs) in response to stimuli that activate the Toll-like receptors TLR9 or TLR7. The C-type lectin blood dendritic cell antigen 2 (BDCA2) is uniquely expressed by pDCs, and antibodies that bind BDCA2 inhibit IFN production in response to TLR signaling. Cao
et al
. found that the abundance of BDCA2 at the cell surface was increased, and signaling was reconstituted in Jurkat cells or a mouse T cell hybridoma cell line when the cells were also transfected to express the γ subunit of the Fcε receptor (FcεR1γ) but not when BDCA2 was coexpressed with the immunoreceptor tyrosine-based activation motif (ITAM) adaptor DAP12 or the related protein DAP10. Analysis of the mRNA and protein abundance of signaling proteins associated with either B cell receptor (BCR) or T cell receptor (TCR) signaling revealed that the signaling cascade of isolated human pDCs most resembled that downstream of BCRs and included the B cell-specific adaptor B cell linker (BLNK, also known as SLP-65 or BASH). Studies with stably transfected human Burkitt lymphoma Namalwa cells (expressing BDCA2, FcεR1γ, and IRF7, a necessary downstream factor) showed that antibody-mediated cross-linking of BDCA2 inhibited IFN production in response to TLR9, TLR7, or TLR8 activation. Thus, BDCA2 appears to use the FcεR1γ as the ITAM adaptor to signal through a BCR-like signaling pathway to mediate inhibition of TLR signaling in pDCs.

W. Cao, L. Zhang, D. B. Rosen, L. Bover, G. Watanabe, M. Bao, L. L. Lanier, Y.-J. Liu, BDCA2/FCεRIγ complex signals through a novel BCR-like pathway in human plasmacytoid dendritic cells.
PLoS Biol.
5
, e248 (2007).
[PubMed]
Molecular Culprit in Gestational Diabetes
Authors: Kiberstis Paula A.
Published: December 2007

During pregnancy, maternal pancreatic islet β-cells expand to accommodate the increasing physiological demands placed on the mother by the growing fetus. The molecular mechanisms controlling this physiological response, which helps prevent the development of "gestational diabetes" in the mother, have been unclear. Studying mouse models, Karnik
et al
. now show that menin, a protein previously identified as an endocrine tumor suppressor and transcriptional regulator, inhibits the growth of islet β-cells during pregnancy. Transgenic expression of menin in maternal β-cells prevented islet expansion and caused the mice to develop several hallmark features of gestational diabetes. Menin appears to be maintained at low levels in islets through the actions of the pregnancy-associated hormone prolactin.

S. K. Karnik, H. Chen, G. W. McLean, J. J. Heit, X. Gu, A. Y. Zhang, M. Fontaine, M. H. Yen, S. K. Kim, Menin controls growth of pancreatic β-cells in pregnant mice and promotes gestational diabetes mellitus.
Science
318
, 806-809 (2007).
[Abstract]
[Full Text]
Dissecting a Core Oscillator
Authors: Purnell Beverly A.
Published: December 2007

The core circadian oscillator of the cyanobacterium
Synechococcus elongatus
can be reconstituted in vitro by mixing the proteins KaiA, KaiB, and KaiC, resulting in oscillations in KaiC phosphorylation. The underlying mechanism of this biochemical oscillator has been elusive. Rust
et al
. (see the Perspective by Poon and Ferrell) now show that phosphorylation of KaiC at two residues is cyclically ordered and that the abundance of each phosphorylated form determines the phase of the oscillator. This sequential phosphorylation of KaiC, combined with negative feedback caused by one of the phosphorylation states, is sufficient to explain stable oscillation.

M. J. Rust, J. S. Markson, W. S. Lane, D. S. Fisher, E. K. O'Shea, Ordered phosphorylation governs oscillation of a three-protein circadian clock.
Science
318
, 809-812 (2007).
[Abstract]
[Full Text]

A. C. Poon, J. E. Ferrell Jr., A clock with a flip switch.
Science
318
, 757-758 (2007).
[Summary]
[Full Text]
The Role of Intracellular Calcium and RhoA in Neuronal Migration
Authors: Faux Clare H. , Parnavelas John G.
Published: December 2007

The successive steps involved in cell migration include extension of the leading process, followed by translocation of the soma and retraction of the trailing process. These events require the coordinated activity of various intracellular signaling mechanisms. Recent evidence suggests that the intracellular distribution of calcium ions and of the Rho guanosine triphosphatase (RhoGTPase), RhoA, are important components of these mechanisms. During migration, the growth cone of the leading process senses guidance cues present in the extracellular environment. These cues, acting through appropriate receptors on the growth cone, induce changes in the concentration of calcium, both in the growth cone and in the soma. These changes in the distribution of calcium cause a redistribution of intracellular RhoA and the eventual translocation of the soma. The trailing process is retracted as the cell moves forward.
Tumbling, an Interactive Way to Move Forward
Authors: Sano Hiroko , Ricardo Sara , Lehmann Ruth
Published: December 2007

The migration of
Drosophila
border cells has become a powerful model with which to genetically identify guidance cues that control the directed migration of a group of interconnected cells. During oogenesis, border cells delaminate from an epithelial layer and move collectively toward the oocyte. In vivo observation has been added to the impressive experimental toolkit available to study border cell migration. These studies reveal two previously unknown migratory behaviors: one in which cells within the border cell cluster constantly change their position, and another called "tumbling," by which the entire border cell cluster rotates forward. Unexpectedly, the same receptor tyrosine kinases control these different modes of migration through separate downstream pathways. An early mode is mediated by the actin regulatory proteins ELMO and Mbc and resembles cellular polarization during individual cell migration; whereas during a later phase, communication between cells, facilitated by mitogen-activated protein kinase and phospholipase C–γ, organizes the polarity of the entire cluster.
Dual Regulation of Endothelial Junctional Permeability
Authors: Komarova Yulia A. , Mehta Dolly , Malik Asrar B.
Published: December 2007

G protein–coupled receptors (GPCRs) of endothelial cells transmit diverse intracellular signals that regulate adherens junction (AJ) permeability. Increased endothelial permeability contributes to pathological processes such as inflammation, atherogenesis, and acute lung injury. Thrombin, a serine protease, and sphingosine-1-phosphate (S1P), a bioactive lipid, regulate endothelial barrier function by activating their respective GPCRs—the protease-activated receptor PAR
1
and the S1P receptor S1P
1
—which initiate intracellular signals that regulate AJ integrity and cytoskeleton organization. The distinct patterns of PAR
1
and S1P
1
signal transduction underlie the functional antagonism between thrombin and S1P. Evidence points to a role for activation of the S1P
1
receptor that is induced by PAR
1
-mediated signaling in the mechanism of AJ reannealing and endothelial barrier repair. Understanding the molecular basis of AJ integrity in the context of inflammation is important in developing novel anti-inflammatory therapeutics. This Review provides a working model for molecular mechanisms for the dual regulation of endothelial barrier function.
PINK Participates in Parkinson’s Pathway
Authors: Ray L. Bryan
Published: December 2007

A neurodegenerative disease in mice with similarities to Parkinson’s disease in humans is caused by missense mutations in a serine protease known as HtrA2 (also called Omi). Plun-Favreau
et al
. describe a connection between this protein and PINK1, a mitochondrial protein kinase that is mutated in certain forms of Parkinson’s disease. HtrA2 functions to detect damaged or misfolded proteins caused by stress in the mitochondria. Misfolded proteins bind to a regulatory domain and activate the protease. The new work indicates that HtrA2 may also be regulated by phosphorylation in response to the stress-activated p38 MAP (mitogen-activated protein) kinase pathway. Although PINK1 binds to HtrA2 and was required for p38-activated phosphorylation and activation of HtrA2, PINK appeared not to directly phosphorylate HtrA2. Consistent with a role of this putative regulatory system in Parkinson’s disease, phosphorylation of HtrA2 was increased in samples of brain from patients with idiopathic Parkinson’s disease, as might be expected if mitochondrial stress in the diseased cells caused activation of the p38 pathway and consequent phosphorylation and activation of HtrA2. Commentary by Alnemri provides further context for the regulatory mechanism and its possible relevance to disease.

H. Plun-Favreau, K. Klupsch, N. Moisoi, S. Gandhi, S. Kjaer, D. Frith, K. Harvey, E. Deas, R. J. Harvey, N. McDonald, N. W. Wood, L. M. Martins, J. Downward, The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-associated kinase PINK1.
Nat. Cell Biol.
9
, 1243-1252 (2007).
[PubMed]

E. S. Alnemri, HtrA2 and Parkinson’s disease: Think PINK?
Nat. Cell Biol.
9
, 1227-1229 (2007).
[PubMed]
Turning Smo On
Authors: Gough Nancy R.
Published: December 2007

Morphogens of the Hedgehog (Hh) family bind to the receptor Patched, and this alleviates inhibition of the signal-transducing membrane protein Smoothened. This activation of Smoothened involves increased cell surface abundance and phosphorylation of the cytoplasmic tail of Smoothened by protein kinase A and casein kinase I. By expressing Smoothened proteins with deletions or mutations in the cytoplasmic region, Zhao
et al
. found that multiple Arg clusters appeared responsible for limiting the cell surface accumulation of Smoothened expressed in S2 cells. Furthermore, the response was graded such that as more Arg clusters were mutated, the abundance of Smoothened at the surface progressively increased, and when these mutant Smoothened proteins were expressed in fly wing discs, the activity of Smoothened also was graded, with mutation of more Arg clusters leading to greater activity. The Arg clusters are adjacent to the phosphorylation sites, and mutations of the Arg clusters in combination with either mutation of the phosphorylation sites or introduction of phosphomimetic residues suggested that the negative charge created by phosphorylation activates Smoothened by neutralizing the positive charge of the Arg clusters. Fluorescence resonance energy transfer (FRET) experiments to detect intermolecular interactions suggested that Smoothened existed as a dimer constitutively (N-terminally tagged proteins produced a basal FRET signal) but that the cytoplasmic domains of the dimers did not interact (FRET produced by the cytoplasmic domains of C-terminally tagged proteins was low). Instead, the interaction between adjacent Smoothened cytoplasmic domains was increased by Hh. Proteins tagged to detect intramolecular interactions revealed that the C-terminal cytoplasmic domain was in close proximity to the third intracellular loop and that Hh decreased FRET between these. FRET analysis of the Smoothened mutants was consistent with phosphorylation adding a negative charge to mediate a change in the conformation of Smoothened. The affinity of the interaction between the Smoothened autoinhibitory domain (SAID) containing the Arg clusters and the phosphorylation sites and a minimal interacting fragment was progressively decreased by increasing phosphorylation or the introduction of increasing numbers of charge-neutralizing mutations. The authors propose that in the absence of Hh, Smoothened is a dimer and the cytoplasmic tail loops to form an inactive conformation mediated by the Arg clusters. Phosphorylation in response to Hh disrupts the cytoplasmic intramolecular interaction, allowing the cytoplasmic regions of the Smoothened dimer to interact. Whether increasing the number of sites phosphorylated triggers multiple active states or whether it increases the probability that the protein will adopt the active conformation remains unknown.

Y. Zhao, C. Tong, J. Jiang, Hedgehog regulates smoothened activity by inducing a conformational switch.
Nature
450
, 252-258 (2007).
[PubMed]
Regulating the Regulators
Authors: Ray L. Bryan
Published: December 2007

Growth factors and nutrients, particularly amino acids, regulate cell growth and proliferation in part through the serine-threonine protein kinase mTOR (mammalian target of rapamycin). Activity of mTOR is regulated through the small guanosine triphosphatase Rheb (Ras homolog enriched in brain). Bai
et al
. (see the Perspective by Proud) reveal a mechanism by which Rheb could control activity of mTOR. The protein FKBP38 binds to mTOR and inhibits its kinase activity. Active GTP-bound Rheb can interact with FKBP38 and thereby free mTOR from inhibition. This process may represent a critical control mechanism whose altered function is associated with human diseases, including cancer.

X. Bai, D. Ma, A. Liu, X. Shen, Q. J. Wang, Y. Liu, Y. Jiang, Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38.
Science
318
, 977-980 (2007).
[Abstract]
[Full Text]

C. G. Proud, mTOR, unleashed.
Science
318
, 926-927 (2007).
[Summary]
[Full Text]
Adenylyl Cyclase 5: A New Clue in the Search for the "Fountain of Youth"?
Authors: Chester Julia A. , Watts Val J.
Published: December 2007

The adenylyl cyclase (AC)­–cyclic adenosine monophosphate (cAMP) signaling pathway is involved in a number of important physiological functions in both the peripheral and central nervous systems. A report now indicates that genetic disruption of AC5 increases mouse life span and confers resistance to aging-related conditions, including bone loss and cardiomyopathies. It is proposed that these beneficial effects may be the result of the increased activity of second messenger signaling proteins such as mitogen-activated or extracellular signal–regulated protein kinase kinase (MAPKK, also known as MEK) and extracellular signal–regulated kinase (ERK), or of enzymes such as manganese superoxide dismutase (MnSOD) that promote cell survival through protection against oxidative stress and apoptosis. These intriguing findings should stimulate additional research aimed at dissecting the complex cellular mechanisms regulated by AC isoforms and may lead to novel genetic and pharmacological approaches to delay aging-related conditions and to extend life span.
Entrainment of the
Drosophila
Circadian Clock: More Heat Than Light
Authors: Fan Jin-Yuan , Muskus Michael J. , Price Jeffrey L.
Published: December 2007

Circadian rhythms are produced by a biological clock that is synchronized (or entrained) by cycles of light and temperature. In
Drosophila
, light triggers the interaction of the photoreceptor cryptochrome (CRY) with the circadian clock protein timeless (TIM). The absence of this interaction in

cry
b

mutants eliminates this entrainment mechanism. The abundance of TIM and period (PER) oscillate throughout the day, and they form a complex that moves to the nucleus to rhythmically repress transcription of the
per
and
tim
genes. Because the CRY:TIM interaction triggers rapid degradation of TIM, the phase of these molecular oscillations is reset by light, which thereby entrains the circadian clock. A study now shows that heat pulses trigger an association between CRY and PER:TIM, which suggests that CRY:PER:TIM also contributes to entrainment by temperature. In wild-type flies, CRY:PER:TIM formation requires high temperatures and is only triggered by heat pulses in the early night, but in

per
L

mutants, which exhibit a temperature-sensitive lengthening of circadian periods, CRY:PER
L
:TIM formation is triggered by lower temperatures and throughout the night. Because CRY:PER:TIM is formed under the same conditions that entrain circadian behavior, formation of the complex is likely to mediate entrainment by heat pulses. Whereas

per
L

flies exhibit longer periods at higher temperatures,

per
L
;cry
b

flies exhibit similar periods at different temperatures, which suggests that an altered interaction between CRY and PER
L
:TIM contributes to a lack of temperature compensation. Future work should determine how the interaction between CRY and PER:TIM entrains rhythms to temperature and affects temperature compensation.
Vasorelaxation with Garlic? It’s a Gas
Authors: Adler Elizabeth M.
Published: December 2007

Garlic is reputed to have various beneficial effects on health, and its consumption has been correlated with a decrease in several risk factors for cardiovascular disease. Noting that garlic is rich in organosulfur compounds and that hydrogen sulfide (H
2
S), like nitric oxide and carbon monoxide, may act as a gaseous signaling molecule in the cardiovascular system (see Lefer), Benavides
et al.
investigated the possibility that polysulfide compounds in garlic might provide a source of vascular H
2
S. Using a polarographic H
2
S sensor, they showed that addition of garlic juice to human red blood cells (RBCs) elicited H
2
S production. Glucose, which enabled the maintenance of the pool of reduced RBC glutathione, promoted sustained production of H
2
S in the presence of garlic; moreover, glutathione plus garlic produced H
2
S even in the absence of RBCs. Experiments with membrane-permeant and -impermeant thiol-blocking reagents indicated that H
2
S production involved exofacial RBC membrane thiols, as well as intracellular thiols. H
2
S produced in the presence of either garlic or garlic-derived polysulfides elicited relaxation of precontracted rat aortic rings, an effect that did not require a functional endothelium. Thus, the authors propose that garlic produces beneficial vasoactive effects by promoting the production of H
2
S.

G. A. Benavides, G. L. Squadrito, R. W. Mills, H. D. Patel, T. S. Isbell, R. P. Patel, V. M. Darley-Usmar, J. E. Doeller, D. W. Kraus, Hydrogen sulfide mediates the vasoactivity of garlic.
Proc. Natl. Acad. Sci. U.S.A.
104
, 17977-17982 (2007).
[Abstract]
[Full Text]

D. J. Lefer, A new gaseous signaling molecule emerges: Cardioprotective role of hydrogen sulfide.
Proc. Natl. Acad. Sci. U.S.A.
104
, 17907-17908 (2007).
[Full Text]
Vitamin C as Neuronal Modulator
Authors: Gough Nancy R.
Published: December 2007

T-type calcium channels contribute to neuronal excitability and can be regulated by signals that produce reactive oxygen species (ROSs). Nelson
et al
. found that ascorbic acid (vitamin C), which is abundant in neurons, inhibited T-type calcium channels in dissociated dorsal root ganglion (DRG) cells and in brain slices containing thalamic nuclei. Only brain slices that contained the Ca
v
3.2 subtype of channel exhibited inhibition, and this subtype selectivity was confirmed with channels expressed in HEK293 cells, a nonneuronal cell line. Domain swapping between Ca
v
3.2 and Ca
v
3.1 indicated that domain 1 of Ca
v
3.2 was required for ascorbate inhibition. Furthermore, mutation of His191 to Gln in Ca
v
3.2 abolished ascorbate-mediated inhibition, and introduction of a His at position 172 in Ca
v
3.1 conferred ascorbate-mediated inhibition. Histidines bind divalent transition metals, which can contribute to metal-catalyzed oxidation. Metal chelation enhanced basal T-currents and prevented ascorbate-mediated inhibition of DRG neurons. Catalase, which degrades peroxide, also prevented ascorbate-mediated inhibition, as did a ROS scavenger. Cu
2+
, but not Zn
2+
, participates in metal-catalyzed oxidation, and increasing the Cu
2+
/Zn
2+
ratio in the extracellular solution increased ascorbate-mediated inhibition, which is consistent with a metal-catalyzed oxidation mechanism for inhibition. Ascorbate applied at physiological concentrations inhibited firing of reticular thalamic neurons in response to low-threshold Ca
2+
spikes and burst firing. Thus, ascorbate may be an endogenous modulator of Ca
v
3.2-mediated neuronal activity.

M. T. Nelson, P. M. Joksovic, P. Su, H.-W. Kang, A. Van Deusen, J. P. Baumgart, L. S. David, T. P. Snutch, P. Q. Barrett, J.-H. Lee, C. F. Zorumski, E. Perez-Reyes, S. M. Todorovic, Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate.
J. Neurosci.
27
, 12577-12583 (2007).
[Abstract]
[Full Text]
Regeneration in Liver and Pancreas: Time to Cut the Umbilical Cord?
Authors: Dor Yuval , Stanger Ben Z.
Published: December 2007

Organisms that are capable of robust tissue regeneration, including the urodele amphibians, use mechanisms that recapitulate embryonic development to regrow organs. Although mammals are not so adept at regeneration, several adult tissues exhibit partial or complete regrowth after injury. An ability to influence growth in mammalian tissues has become more imperative with the emergence of "regenerative medicine" as a discipline. For this field to fulfill its promise of providing functional tissues for clinical use, a more detailed picture will be required of how adult human tissues maintain mass during normal homeostasis and after injury. Studies of developing and regenerating liver and pancreas now suggest that mammals use distinct programs to regulate tissue growth during embryogenesis and adulthood.
New Insights into the Mechanisms of SOS Activation
Authors: Quilliam Lawrence A.
Published: December 2007

The activation of the small guanosine triphosphatase Ras is critical for many biological events. It is therefore not surprising that the ubiquitously expressed Ras guanine nucleotide exchange factor (GEF) SOS (Son of Sevenless), which couples protein tyrosine kinases to Ras activation, is under tight autoinhibitory control. Several studies have revealed how multiple regulatory domains might affect SOS activity. Most notably, a second Ras-binding site on SOS allosterically regulates the duration and amplitude of Ras activation. This allosteric Ras-GTP is produced by another GEF, Ras guanine nucleotide–releasing protein 1 (RasGRP1). SOS and RasGRP1 are both activated downstream of phospholipase D
2
, and gain-of-function mutants of SOS contribute to inherited diseases. These studies not only enable us to better appreciate the complexity of the regulation of GEFs but also prompt us to reevaluate our current understanding of pathways that lead to Ras activation.
Substrate-Bound Protein Gradients for Cell Culture Fabricated by Microfluidic Networks and Microcontact Printing
Authors: von Philipsborn Anne C. , Lang Susanne , Jiang Zhongxiang , Bonhoeffer Friedrich , Bastmeyer Martin
Published: December 2007

Graded distributions of proteins are pivotal for many signaling processes during development, such as morphogenesis, cell migration, and axon guidance. Here, we describe a technique to fabricate substrate-bound stepwise protein gradients by means of a microfluidic network etched into a silicon wafer with an array of parallel 14-micrometer–wide channels, which can be filled with a series of arbitrarily chosen protein solutions. In a subsequent microcontact printing step, the protein pattern is transferred onto a surface and is used as a substrate for cell culture. Cellular responses to a defined microscopic pattern of a protein, such as guided axonal outgrowth and directed migration, cell polarization, changes in morphology, and signaling, can be thus studied in a controlled in vitro environment.
Starved into Submission?
Authors: Adler Elizabeth M.
Published: December 2007

CD4
+
CD25
+
thymus-derived regulatory T cells (T
reg
cells) suppress the activity of naïve and effector T cells, limiting the immune response and helping guard against autoimmune activity. The mechanisms whereby they inhibit the activity of target cells, however, have been controversial and unclear (see Scheffold
et al.
). Pandiyan
et al
. found that T
reg
cells elicited the apoptosis of activated "responder" CD4
+
T cells with which they were cocultured, without blocking their proliferation or the early production of interleukin-2 (IL-2). Apoptosis of responder T cells occurred in the presence of T
reg
cells from perforin-deficient or Fas ligand mutant mice, occurred over several days (peaking 3 to 4 days after activation), depended on the proapoptotic protein Bim, and was blocked by cytokines that signal through the common γ chain. Pharmacological analysis of the cytokine-dependent Akt pathway indicated that the ability of IL-7 to rescue responder T cell survival depended on phosphatidylinositol-3-OH kinase (which activates Akt), whereas Akt phosphorylation in responder T cells was decreased in the presence of T
reg
cells, as was phosphorylation (and thereby deactivation) of the proapoptotic protein Bad. Bad phosphorylation was enhanced during IL-7 rescue. In a mouse model of inflammatory bowel disease involving the transfer of colitogenic CD4
+
CD45RB
hi
T cells, T
reg
cells protected against colitis and stimulated the Bim-dependent apoptosis of the transferred T cells. The authors thus conclude that T
reg
cells can inhibit effector T cell responses both in vitro and in vivo by depriving them of cytokines and eliciting their apoptotic death.

P. Pandiyan, L. Zheng, S. Ishihara, J. Reed, M. J. Lenardo, CD4
+
CD25
+
Foxp3
+
regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4
+
T cells.
Nat. Immunol
.
8
, 1353-1362 (2007).
[PubMed]

A. Scheffold, K. M. Murphy, T. Höfer, Competition for cytokines: T
reg
cells take all.
Nat. Immunol
.
8
, 1285-1287 (2007).
[PubMed]
Domesticated Mobile Elements Modulate Light Responses
Authors: Purnell Beverly A.
Published: December 2007

Plants constantly gauge their environments and adjust their growth and development in order to survive. Plants can "see" their surroundings using a battery of photoreceptors to determine the color, intensity, direction, and duration of light around them. Phytochrome A (phyA) mediates various far-red light-induced responses through a cascade of signaling events. Lin
et al.
report that two proteins co-opted from ancestral
Mutator
-like transposable element(s) act as transcription factors essential for phyA-controlled gene expression. These results provide insight into the mechanisms and evolutionary history of phytochrome signaling in higher plants and lend support for a role of mobile elements as driving forces for host evolution.

R. Lin, L. Ding, C. Casola, D. R. Ripoll, C. Feschotte, H. Wang, Transposase-derived transcription factors regulate light signaling in
Arabidopsis
.
Science
318
, 1302-1305 (2007).
[Abstract]
[Full Text]
β
2
-Adrenergic Receptor Structures
Authors: Vinson Valda , Gough Nancy
Published: December 2007

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are transmembrane proteins that respond to diverse extracellular stimuli to trigger many different cellular responses. They are important pharmacological targets, but drug design has been hampered because structural information is only available for the rather distinctive member of this family, rhodopsin (see the Perspective by Ranganathan). Three studies now provide structural insights into another GPCR, the human β
2
-adrenergic receptor. All three groups crystallized the protein in the presence of an inverse agonist that stabilizes the inactive conformation of the receptor. Rosenbaum
et al
. stabilized the protein for crystallization by protein engineering and analyzed mutagenesis data in the context of the structure to provide insight into how ligand binding is coupled. Cherezov
et al
. describe in detail the 2.4 Å structure of the fusion protein and how it compares to the rhodopsin structure. Rasmussen
et al
. crystallized the protein in a lipid environment in the presence of a monoclonal antibody fragment to obtain a 3.4 Å structure. It appears that conserved helices provide a common core in class A GPCRs, whereas variable helices provide binding-site plasticity.

V. Cherezov, D. M. Rosenbaum, M. A. Hanson, S. G. F. Rasmussen, F. S. Thian, T. S. Kobilka, H.-J. Choi, P. Kuhn, W. I. Weis, B. K. Kobilka, R. C. Stevens, High-resolution crystal structure of an engineered human β
2
-adrenergic G protein-coupled receptor.
Science
318
, 1258-1265 (2007).
[Abstract]
[Full Text]

D. M. Rosenbaum, V. Cherezov, M. A. Hanson, S. G. F. Rasmussen, F. S. Thian, T. S. Kobilka, H.-J. Choi, X.-J. Yao, W. I. Weis, R. C. Stevens, B. K. Kobilka, GPCR engineering yields high-resolution structural insights into β
2
-adrenergic receptor function.
Science
318
, 1266-1273 (2007).
[Abstract]
[Full Text]

S. G. F. Rasmussen, H.-J. Choi, D. M. Rosenbaum, T. S. Kobilka, F. S. Thian, P. C. Edwards, M. Burghammer, V. R. P. Ratnala, R. Sanishvili, R. F. Fischetti, G. F. X. Schertler, W. I. Weis, B. K. Kobilka, Crystal structure of the human β
2
adrenergic G-protein-coupled receptor.
Nature
450
, 383-387 (2007).
[PubMed]

R. Ranganathan, Signaling across the cell membrane.
Science
318
, 1253-1254 (2007).
[Abstract]
[Full Text]
Genomic Maintenance: The p53 Poly(ADP-ribosyl)ation Connection
Authors: Alvarez-Gonzalez Rafael
Published: December 2007

The integrity of the genome in higher eukaryotes, as well as the modulation of its complex structure and functions, is exquisitely regulated. This genomic regulation occurs as a function of time in a very sophisticated and elaborate biological process called cell cycle progression, resulting in cell division, and is also controlled by a highly coordinated and intricate network of molecular signaling pathways, which in turn orchestrate very specific macromolecular interactions among nuclear proteins and DNA at the biochemical level. Among the latter, a prominent enzymatic cycle that is involved in maintaining the integrity of mammalian chromosomes is covalent protein-poly[adenosine diphosphate (ADP)–ribosyl]ation. The importance of this posttranslational modification is illustrated by the close cooperation between two "guardian angels" of the genome, one constitutive and one inducible protein, namely poly(ADP-ribose) polymerase–1 (PARP-1) and p53, and the integration of these pivotal signaling processes with genomic maintenance.
Two’s Company, Three’s a Complex
Authors: Foley John F.
Published: December 2007

Activation of gene transcription by members of the nuclear factor κB (NF-κB) family of transcription factors is crucial to such processes as proliferation and inflammation. Perhaps the best-characterized NF-κB is a heterodimer composed of p50 and p65; however, some aspects of its regulation remain poorly understood. Differences between the properties of NF-κB from nuclear extracts and those of recombinant NF-κB led Wan
et al
. to search for other proteins that might form part of endogenous NF-κB complexes. They affinity-purified and analyzed by mass spectrometry those proteins from 293T cells that bound to a tagged form of p65 and identified ribosomal protein S3 (RPS3) as a major binding partner. Reporter assays showed that knockdown of RPS3 by small inhibitory (si) RNAs impaired T cell receptor (TCR)-stimulated NF-κB activation in Jurkat T cells. Electrophoretic mobility shift assays (EMSAs) demonstrated the TCR-stimulated formation of complexes containing p50, p65, and RPS3. Chromatin immunoprecipitation (ChIP) assays showed that recruitment of p65 to target genes was greatly diminished in RPS3-knockdown cells compared with that in control cells. TCR-stimulated activation of these genes was also lower in RPS3-knockdown cells than in control cells, as assessed by real-time reverse transcription polymerase chain reaction assays. Comparison of RPS3-knockdown with p65-knockdown T cells by microarray assays showed that RPS3 was necessary for the expression of a subset of p65 target genes, one of which encodes the cytokine interleukin-2 (IL-2). Consistent with this, TCR-stimulated proliferation, which is dependent on IL-2, was blocked in RPS3-knockdown T cells. These results challenge the current dimer-based model of NF-κB activity and suggest that RPS3 is a required subunit of NF-κB that provides target specificity.

F. Wan, D. E. Anderson, R. A. Barnitz, A. Snow, N. Bidere, L. Zheng, V. Hegde, L. T. Lam, L. M. Staudt, D. Levens, W. A. Deutsch, M. J. Lenardo, Ribosomal protein S3: A KH domain subunit in NF-κB complexes that mediates selective gene regulation.
Cell
131
, 927-939 (2007).
[PubMed]
Looking After Polysialic Acid
Authors: Adler Elizabeth M.
Published: December 2007

Many aspects of brain function are modulated by neuronal activity; in some cases, the effects of activity are restricted to particular developmental stages, or critical periods. For instance, loss of visual input from one eye early in life leads to a shift in the responsiveness of neurons in the visual cortex toward the functional eye. The onset and time course of this critical period for ocular dominance plasticity is influenced by the maturation of GABAergic innervation, which is itself regulated by visual input and neuronal activity. But how? Di Cristo
et al
. found that polysialic acid [PSA, a homopolymer of sialic acid that attaches to neural cell adhesion molecules (NCAMs) and modulates intercellular bonds] and NCAMs were abundant in the neonatal mouse visual cortex but that PSA underwent a steep decline after eye opening. Pups reared in the dark showed increased PSA compared with pups reared under conventional light/dark cycles, and pharmacological suppression of visual signals suppressed the decline. In organotypic cortical cultures, enzymatic removal of PSA with endoneuramidase led to premature maturation of the innervation of pyramidal cell somata by GABAergic basket interneurons. Similarly, endoneuramidase injection into the visual cortex promoted both perisomatic GABAergic innervation and the frequency of miniature inhibitory postsynaptic currents. Moreover, it stimulated the early onset of the critical period for ocular dominance plasticity. Thus, the authors conclude that maturation of GABAergic inhibition--and thereby the initiation of ocular dominance plasticity--is regulated by an activity-dependent decrease in PSA.

G. Di Cristo, B. Chattopadhyaya, S. J. Kuhlman, Y. Fu, M.-C. Bélanger, C. Z. Wu, U. Rutishauser, L. Maffei, Z. J. Huang, Activity-dependent PSA expression regulates inhibitory maturation and onset of critical period plasticity.
Nat. Neurosci
.
10
, 1569-1577 (2007).
[PubMed]
Old Enough to Remember?
Authors: Ray L. Bryan
Published: December 2007

Long-term potentiation (LTP) is a form of synaptic plasticity thought to mediate learning and memory in hippocampal neurons. At a molecular level, neuronal function is altered during LTP when AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) neurotransmitter receptors accumulate in postsynaptic sites. This movement of receptors is promoted by PKA (cyclic AMP-dependent protein kinase). Lu
et al.
explored the role of a scaffolding protein, AKAP150, that positions PKA appropriately so that it can phosphorylate AMPA receptors. When the authors examined mice expressing a mutant form of AKAP150 that doesn’t bind PKA, they found that its effects were strongly dependent on the age of the mice examined. LTP induced by a single tetanic electrical stimulus was normal in animals 4 weeks old but was not detected in animals 7 to 12 weeks old. Inhibitors of PKA or of AMPA receptors that lack the GluR2 subunit also blocked LTP in 8-week-old mice but not in 4-week-old mice. (AMPA receptors that lack GluR2 contain the GluR1 subunit that is a substrate for PKA.) Thus, the authors propose that mice undergo two developmental transitions: They require PKA for LTP for the first few weeks of life, don’t need it around 4 weeks of age, and then again need PKA’s actions for LTP at around 8 weeks of age. This might reflect changes in function of the developing brain. In the immature brain, neurons may not be ready to form permanent synaptic connections, so LTP may be restrained by additional requirements--activation of PKA and GluR2-lacking AMPA receptors. The older brain may have a period of increased LTP (during which requirements are relaxed, i.e., PKA is not required) and then undergo restriction as the brain becomes more mature and connections are stabilized. (The authors carefully note other studies that provide contrasting results regarding the precise ages at which PKA signaling is required and whether GluR2-lacking receptors are the main ones used in 8-week-old animals.)

Y. Lu, M. Allen, A. R. Halt, M. Weisenhaus, R. F. Dallapiazza, D. D. Hall, Y. M. Usachev, G. S. McKnight, J. W. Hell, Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP.
EMBO J.
26
, 4879-4890 (2007).
[PubMed]
Die Another Way
Authors: Foley John F.
Published: December 2007

Epithelial cells depend for their survival on integrin-mediated signals generated by attachment to the extracellular matrix (ECM). Epithelial cells also form adherens junctions that are dependent on interactions between cadherin proteins on adjacent cells. Detachment of epithelial cells from the ECM triggers their apoptotic cell death in a process known as anoikis. Overholtzer
et al
. were studying small aggregates of human mammary epithelial cells (MCF10A) in suspension when they noticed that some cells were contained within large vacuoles in other cells. By labeling populations of cells with different fluorescent dyes and mixing the cells, the authors found that cell invasion occurred within a few hours of detachment from the ECM. Whereas cell invasion was not prevented by inhibitors of apoptosis, its occurrence was reduced by inhibitors of actin polymerization or myosin contraction. Inhibition of the guanosine triphosphatase Rho, which regulates actin and myosin, or of its downstream target Rho-associated kinase (ROCK) prevented epithelial cell internalization. Further experiments with differentially labeled and differentially treated cells showed that Rho and ROCK activity in the invading cells were necessary for cell invasion, which the authors refer to as entosis. An antibody against E-cadherin inhibited both the formation of MCF10A aggregates and entosis. Fluorescent microscopy showed that, whereas the invading cells did not contain activated caspase-3, a marker of apoptosis, many of the invading cells colocalized with host cell lysosomal markers and the protease cathepsin B. Finally, immunohistochemical analyses showed evidence of entosis in human primary and metastatic breast tumors. As White points out in commentary, entosis may be a mechanism by which epithelial cancers, characterized by unregulated proliferation of detached epithelial cells, may be suppressed.

M. Overholtzer, A. A. Mailleux, G. Mouneimne, G. Normand, S. J. Schnitt, R. W. King, E. S. Cibas, J. S. Brugge, A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion.
Cell
131
, 966-979 (2007).
[PubMed]

E. White, Entosis: It’s a cell-eat-cell world.
Cell
131
, 840-842 (2007).
[PubMed]
Tacking Myelin Seams
Authors: Hines Pamela J.
Published: December 2007

Schwann cells wrap layers of myelin around nerve axons to provide the electrical insulation that speeds conduction of the action potential. At the gaps between the Schwann cells are nodes that the electrical impulse must jump. At these nodes, the myelin layers are held down particularly tightly to provide an insulating seal. Scheiermann
et al.
have now identified JAM-C, a junctional adhesion protein known for its role in adhesion between endothelial cells and intestinal cells, as one of the proteins responsible for sealing these edges of the myelin sheath.

C. Scheiermann, P. Meda, M. Aurrand-Lions, R. Madani, Y. Yiangou, P. Coffey, T. E. Salt, D. Ducrest-Gay, D. Caille, O. Howell, R. Reynolds, A. Lobrinus, R. H. Adams, A. S. L. Yu, P. Anand, B. A. Imhof, S. Nourshargh, Expression and function of junctional adhesion molecule-C in myelinated peripheral nerves.
Science
318
, 1472-1475 (2007).
[Abstract]
[Full Text]
Type I Interferons as Anti-Inflammatory Mediators
Authors: Benveniste Etty N. , Qin Hongwei
Published: December 2007

The type I interferons (IFNs), IFN-α and IFN-β, are cytokines that have antiviral, antiproliferative, and immunomodulatory activities. Data are now emerging that suggest that type I IFNs are also important mediators of anti-inflammatory responses. These findings, largely driven by studies to explain the beneficial effects of IFN-β in the treatment of multiple sclerosis, an autoimmune disease of the central nervous system, offer a number of mechanisms for the anti-inflammatory properties of type I IFNs. Type I IFNs, through their ability to induce the immunosuppressive cytokine interleukin-10 (IL-10), mediate the inhibition of proinflammatory gene products. In addition, type I IFNs induce other immunosuppressive mediators such as suppressor of cytokine signaling–1 (SOCS-1) and tristetrapolin (TTP), which act by divergent mechanisms to restore homeostasis to the immune system. Furthermore, type I IFNs mediate anti-inflammatory and protective effects in a variety of autoimmune disease models such as experimental colitis, experimental allergic encephalomyelitis, experimental arthritis, and neonatal inflammation. Here, we discuss the molecular basis for the anti-inflammatory properties of type I IFNs and their therapeutic potential in autoimmune and inflammatory diseases.