bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Sphingosine 1-Phosphate, Ceramides, and Other Bioactive Sphingolipids by High-Performance Liquid Chromatography-Tandem Mass Spectrometry
Authors: Sullards M. Cameron , Merrill Alfred H.
Published: May 2003

The lipid backbones of sphingolipids and their metabolites are highly bioactive compounds that affect diverse cellular functions. The metabolites that have been most extensively studied with respect to their effects on cell behavior are ceramides, sphingosine (and other sphingoid bases), and sphingosine 1-phosphate. Additionally, there is interest in other naturally occurring species such as lysosphingolipids (sphingosine, phosphorylcholine, and psychosines) and N-methyl (di- and tri-methyl)-sphingosines. In many cases, studies of cell signaling mediated by these compounds have focused on a single category (such as ceramides or sphingosine 1-phosphate) because of the technical difficulty of more comprehensive analyses. One obstacle in such studies is that most of these compounds are metabolically interconvertable, so it is difficult to assign a conclusive relationship. In this article, we describe the analytical methods for extraction, identification, and quantitation of sphingolipids using state-of-the-art tandem mass spectrometry (MS/MS). Precursor ion scans are used to distinguish various species of sphingolipids in crude extracts by their unique molecular decomposition products. Specific headgroup, sphingoid base, and fatty acid chain combinations can be readily determined. Quantitation is achieved by multiple reaction monitoring (MRM) in conjunction with high-pressure liquid chromatography (HPLC). Compared to precursor ion scans alone, MRM experiments yield greater sensitivity and lower limits of detection by monitoring a specific precursor and product ion pair. This sensitivity facilitates detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each species, eliminating any ambiguity that may arise from differences in the kinetics of dissociation.
Ras GTPases: Singing in TuneA review of the meeting "The Ras Superfamily of Small GTP-Binding Proteins," FASEB Summer Research Conference, Snowmass, Colorado, 15 through 20 July 2000
Authors: Symons Marc , Takai Yoshimi
Published: May 2003

The molecular cloning of the human proto-oncogene encoding Ras was reported nearly 20 years ago. Since then, Ras has become the prototypical member of a superfamily of small guanosine triphosphatase proteins. Despite the maturity of this field of research, the discovery of new functions and interactions between the superfamily members continues unabated. Symons and Takai have written a meeting report on the latest findings on the Ras superfamily.
Arrestins as Signaling Molecules Involved in Apoptotic Pathways: A Real Eye Opener
Authors: Miller William E. , Lefkowitz Robert J.
Published: May 2003

Recent data suggest that internalized receptor and arrestin complexes are actively involved in signal transduction. Miller and Lefkowitz discuss evidence from the
Drosophila
visual system that suggests that intracellular rhodopsin and arrestin2 complexes induce apoptosis. Experiments with activated mammalian G protein-coupled receptor and arrestin complexes point to a mechanism by which proliferative or proapoptotic signals can be mediated largely independent from G protein activation.
The Ethylene Pathway: A Paradigm for Plant Hormone Signaling and Interaction
Authors: Alonso Jose M. , Ecker Joseph R.
Published: May 2003

To dissect the web of signals that control plant growth, it is important to understand how the individual components of the pathway are modulated. Ethylene is a plant hormone involved in a large number of developmental processes. Biochemical and genetic approaches have provided a detailed view of the biosynthetic and signal transduction pathways of this hormone in the reference plantArabidopsis thaliana. The effects of several hormones and of developmental changes on the regulation of the key enzymes of ethylene biosynthesis, ACC synthase and ACC oxidase, serve as a clear example of interaction between signals in the generation of complex responses. We now have a picture of how ethylene is sensed by the ethylene receptors and how the signal is further transduced to the nucleus. Although some of the ethylene receptors show a tissue-specific pattern of expression, little is known about the regulation of the components of the ethylene transduction cascade by other hormones or developmental factors. Once the ethylene signal reaches the nucleus, it activates a transcriptional cascade that results in changes in the expression of a number of genes. We describe some of the results that suggest an interaction at the transcriptional level between ethylene, other hormones, and stress signals.
A Progress Report on Translational Control in Eukaryotes
Authors: Kozak Marilyn
Published: May 2003

An extraordinary amount of regulation goes into making sure that protein expression is controlled correctly. Several layers of regulation function to achieve the proper levels and proper timing of protein expression. Much is known about the protein machinery involved in translation, but we are lagging behind in understanding the mechanisms of control in eukaryotes. Kozak reviews the new second edition of
Translational Control of Gene Expression
, which attempts to catalog the mechanisms used by prokaryotes and eukaryotes, and the viruses that infect them and subvert their translational machinery.

Review and commentary on
Translational Control of Gene Expression
Edited by Nahum Sonenberg, John W. B. Hershey, and Michael B. Mathews Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000, 1020 pp. $115
ISBN: 0-87969-568-4
Immunoisolation of TCR Signaling Complexes from Jurkat T Leukemic Cells
Authors: Harder Thomas , Kuhn Marina
Published: May 2003

The formation of multimolecular assemblies of signaling molecules at the plasma membrane is key to triggering signaling cascades following activation of transmembrane receptors at the plasma membrane. We have developed a method to immunoisolate activated T cell receptors (TCRs) and associated signaling molecules in plasma membrane subdomains from Jurkat T leukemic cells. The immunoisolation procedure for the signaling complexes uses magnetic beads, which are coupled to TCR-activating antibodies. Following mechanical cellular disruption using nitrogen cavitation, conjugates of these beads with Jurkat cells are formed and isolated. TCR-signaling complexes within plasma membrane fragments associate with the antibody-coupled beads and are retrieved along with the beads using a magnet. We found that the immunoisolated plasma membrane fragments are highly enriched for activated TCRs and associated signaling proteins. This isolation procedure allows a detailed and precise biochemical analysis of the assembly of signaling proteins in plasma membrane subdomains.
A Signal Transduction PrimerReview and commentary on
Principles of Molecular Regulation
, edited by P. Michael Conn and Anthony R. Means. Humana Press, Totowa, NJ, 2000, 474 pp. $135.00 ISBN: 0-896-03630-8
Authors: Blumenthal Donald K.
Published: May 2003

Because of the complexity and the rapid pace of research developments in the field of signal transduction, there are few textbooks devoted to this subject specifically. Blumenthal provides an overview of the text
Principles of Molecular Regulation
. The review emphasizes the strong points of the text and makes suggestions for improving later editions.
Two Mammalian Mitotic Aurora Kinases: Who's Who?
Authors: Descamps Simon , Prigent Claude
Published: May 2003

Several serine-threonine kinases related to the Ipl1p kinase in budding yeast, termed aurora kinases, have been cloned recently. Their characterization revealed them to be important regulators of mitotic functions, including (i) the separation of the centrosome, (ii) assembly of the spindles, and (iii) segregation of the chromosomes. The Perspective by Descamps and Prigent delves into the latest observations on aurora kinases in humans and the specific roles of each kinase within the process of mitosis.
Targeted Gene Repair in Mammalian Cells Using Chimeric RNA/DNA Oligonucleotides and Modified Single-Stranded Vectors
Authors: Parekh-Olmedo Hetal , Czymmek Kirk , Kmiec Eric B.
Published: May 2003

Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.
Intracellular Signaling by the Killer Immunoglobulin-Like Receptors and Ly49
Authors: McVicar Daniel W. , Burshtyn Deborah N.
Published: May 2003

Once thought to be promiscuous killers, it is now known that natural killer (NK) cells possess an elaborate array of receptors that regulate NK cytotoxic and secretory functions upon interaction with target cell MHC class I proteins. These receptors, known as killer cell immunoglobulin-like receptors (KIRs) in humans, and Ly49 receptors in the mouse, have become the focus of intense study in an effort to discern the underlying biology of these large receptor families. These receptor families include both inhibitory and activating receptors. Interrogation of a target expressing KIR ligands leads to coengagement of the inhibitory receptor with as-yet poorly defined activation receptors. Kinases activated during engagement mediate the phosphorylation of the KIR or Ly49 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The phosphorylated ITIMs serve as efficient recruitment points for the cytosolic protein tyrosine phosphatases, SHP-1 and SHP-2, resulting in the dephosphorylation of substrates critical for cellular activation. In contrast, some KIRs and Ly49s lack the ITIM and possess a charged residue in their transmembrane domains that mediates interaction with the DAP12 signal transduction chain. DAP12 uses its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) to mediate cellular activation. Engagement of a DAP12 coupled KIR or Ly49 results in phosphorylation of DAP12, and other key substrates, including the Syk tryosine kinase, phospholipase C, and c-Cbl. DAP12 activation then leads to the Mapk cascade and ultimately to enhanced degranulation, and production of cytokines and chemokines. Although the context in which inhibitory and activating KIR and Ly49s function is not yet known, the dissection of the activating and inhibitory signal transduction pathways should shed light on their method of integration into the activation sequela of NK cells. Ultimately, this work will lead to concrete understanding of the immunobiology of these seemingly antagonistic receptor systems.
Signal Transduction Pathways as Targets for TherapeuticsMeeting information: AAAS 2001 Annual Meeting and Science Innovation Exposition, San Francisco, California, February 15 through 20, 2001.
Authors: Gough Nancy R.
Published: May 2003

Science's STKE
sponsored a symposium at the AAAS Annual Meeting in February 2001. Five speakers addressed the signaling pathways that are modified in wide-ranging pathologies including inflammation, impotence, diabetes, obesity, and cancer. The molecular targets of signaling pathways included cell surface molecules, such as the G protein-coupled receptors (GPCRs) and receptor tyrosine kinases, and intracellular signaling components, such as phosphodiesterases (PDEs) and components of the small guanosine triphosphatase (GTPase) Ras signaling pathway. Analysis of the therapeutic strategies to impinge on these various pathways provides insight into both the potential of signaling pathways as relevant drug targets and the possible pitfalls that make complex signaling networks unpredictably difficult targets for such manipulation.
Terminal Transferase-Dependent PCR (TDPCR) for In Vivo UV Photofootprinting of Vertebrate Cells
Authors: Chen Hsiu-Hua , Kontaraki Joanna , Bonifer Constanze , Riggs Arthur D.
Published: May 2003

Terminal transferase-dependent PCR (TDPCR) is a versatile, sensitive method for detecting DNA lesions such as those generated by the footprinting agents commonly used to detect in vivo protein-DNA interactions. Data similar to those obtained by ligation-mediated PCR (LMPCR) are obtained, but one advantage of TDPCR is that no special enzymes are needed other than terminal deoxynucleotide transferase, T4 DNA ligase, and thermostable DNA polymerases. A detailed TDPCR protocol is given for using UV photofootprinting to detect in vivo footprints and chromatin fine structure in vertebrate cells. One version of the protocol makes use of nonradioactive labeling by near-infrared fluorochromes and detection by a LI-COR DNA sequencing instrument. Sensitivity similar to that of
32
P-labeling is obtained, but with superior band resolution and quantitation.
A New Role for Protein Methylation: Switching Partners at the Phosphatase Ball
Authors: Mumby Marc
Published: May 2003

Reversible protein methylation may be another posttranslational modification that serves an important role in modulating signal transduction pathways. Not only does protein phosphatase 2A (PP2A), a key regulator of many signal transduction cascades, undergo this modification, but the methylation process itself may be regulated by various cellular stimuli or states. Mumby describes how PP2A is regulated and how methylation may alter its subcellular targeting and substrate specificity by influencing its interaction with regulatory subunits.
New Functions for DNA Binding Domains
Authors: Rao Anjana
Published: May 2003

How the same transcription factor can be both an activator and a repressor of transcription is a biological mystery. Rao describes work that shows that the structure of the transcription factor may actually change depending on the DNA sequence bound by the factor. This change in structure may influence the binding of activators or repressors of transcription, thus allowing a single transcription factor to have two different activities.
CREB Signaling-Timing Is Everything
Authors: Impey Soren , Goodman Richard H.
Published: May 2003

Depolarization of neurons can lead to changes in gene expression that are important for such processes as synaptic plasticity, neuronal differentiation, and apoptosis. Impey and Goodman discuss some of the opposing models for how gene transcription in response to neuronal activity and elevations in intracellular calcium concentration is regulated. The pathways appear to converge on cyclic AMP response element-binding (CREB) protein, with the mitogen-activated protein kinase pathways playing an important role. The continuing debate about the involvement of calmodulin kinase IV is also described.
Targeting Silence: The Use of Site-Specific Recombination to Introduce In Vitro Methylated DNA into the Genome
Authors: Schübeler Dirk , Lorincz Matthew C. , Groudine Mark
Published: May 2003

DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.
Neurotrophins: Which Way Did They Go?
Authors: Segal Rosalind
Published: May 2003

The classic view of the neurotrophin as a messenger produced by the postsynaptic cell that signals survival to the presynaptic cell has become expanded. Segal discusses the evidence that the neurotrophin brain-derived nerve growth factor (BDNF) can be also released from the presynaptic cell onto the dendritic spine of a postsynaptic cell to regulate the neuronal circuit in two directions: forward (from presynaptic cell to postsynaptic dendrite) and the classic backward direction (from postsynaptic cell to the presynaptic axon). These results remind us that in the brain, neurons exist in a complex circuit and one neuron's presynaptic cell is another neuron's postsynaptic cell. The evidence that a single growth factor may have different actions based on the localization of the receipt of the signal adds more complexity to the brain's neuronal circuitry.
Lnk Adaptor: Novel Negative Regulator of B Cell Lymphopoiesis
Authors: Rudd Christopher E.
Published: May 2003

Originally thought to have the functions now ascribed to the linker for activation of T cells protein (LAT), Lnk is coming into its own as an adaptor protein that mediates signaling through several receptor pathways. An essential role for Lnk in B cell development and maturation was recently uncovered by Perlmutter and colleagues. Rudd discusses the role of Lnk in B cells and hypothesizes a mechanism whereby Lnk, and its closely related protein family members, the adaptor molecules containing pleckstrin homology (PH) and Src-homology 2 (SH2) domains (APS), and Src-homology 2-B protein (SH2-B), may mediate signal promotion or attenuation.
The Biotin Switch Method for the Detection of
S
-Nitrosylated Proteins
Authors: Jaffrey Samie R. , Snyder Solomon H.
Published: May 2003

Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. Here, we describe a novel method for detecting proteins that contain nitrosothiols. In this three-step procedure, nitrosylated cysteines are converted to biotinylated cysteines. Biotinylated proteins can then be detected by immunoblotting or can be purified by avidin-affinity chromatography. We include examples of the detection of
S
-nitrosylated proteins in brain lysates after in vitro
S
-nitrosylation, as well as the detection of endogenous
S
-nitrosothiols in selected neuronal proteins.
S
-Nitrosylation Is Emerging as a Specific and Fundamental Posttranslational Protein Modification: Head-to-Head Comparison with
O
-Phosphorylation
Authors: Lane Paul , Hao Gang , Gross Steven S.
Published: May 2003

Nitric oxide (NO) is a free-radical product of mammalian cell metabolism that plays diverse and important roles in the regulation of cell function. Biological actions of NO arise as a direct consequence of chemical reactions between NO or NO-derived species and protein targets. Reactions of NO with transition metals in target proteins have garnered the most attention to date as the principal mechanism of NO signaling; nonetheless,
S
-nitrosylation of protein Cys residues is rapidly moving to center stage in importance. In general, however, there has been a delay in adequate appreciation of the role of
S
-nitrosylation in biological signaling by NO. This lag is attributed to a poor understanding of the basis for selective targeting of NO to particular thiols, and methodological limitations in accurately quantifying this modification--recent breakthroughs in concepts and methods diminish these barriers. Here, we consider the wheres and whys of protein
S
-nitrosylation and its basis for specificity. Protein
S
-nitrosylation potentially represents a ubiquitous and fundamental mechanism for posttranslational control of protein activity on a par with that of
O
-phosphorylation.
Manipulating Programmed Cell Death for Better Living!

Review and commentary on
Apoptosis in Health and Disease
,  edited by Robert R. Ruffolo, Jr. and Frank Walsh,   Harwood Academic Publishers, Amsterdam, Netherlands, 2000, 249 pp. $80.00, ISBN: 90-5823-086-4
Authors: Manolagas Stavros C.
Published: May 2003

Manolagas provides a succinct review of the book
Apoptosis in Health and Disease
. He praises the book for the clarity with which it covers the topics, but he also points out two missing health issues for which regulating apoptosis may prove clinically important: One is the health and regulation of the female reproductive system and the other is the treatment of osteoporosis.
TGF-β Flips the Myc Switch
Authors: Orian Amir , Eisenman Robert N.
Published: May 2003

Although transforming growth factor-β (TGF-β) can affect cell cycle arrest, not much molecular detail is known about how TGF-β-dependent arrest is mediated. Two recent papers shed some light on how this is accomplished. Orian and Eisenman discuss how Myc interacts with Miz-1 to block the expression of a cell cycle inhibitory protein, p15
INK4b
, and how TGF-β is able to unblock Myc-dependent repression of Miz-1.
Cycling of Synaptic Vesicles: How Far? How Fast!
Authors: Galli Thierry , Haucke Volker
Published: May 2003

Synaptic transmission is based on the regulated exocytotic fusion of synaptic vesicles filled with neurotransmitter. In order to sustain neurotransmitter release, these vesicles need to be recycled locally. Recent data suggest that two tracks for the cycling of synaptic vesicles coexist: a slow track in which vesicles fuse completely with the presynaptic plasma membrane, followed by clathrin-mediated recycling of the vesicular components, and a fast track that may correspond to the transient opening and closing of a fusion pore. In this review, we attempt to provide an overview of the components involved in both tracks of vesicle cycling, as well as to identify possible mechanistic links between these two pathways.
PACT and PKR: Turning on NF-κB in the Absence of Virus
Authors: D'Acquisto Fulvio , Ghosh Sankar
Published: May 2003

Double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) has been generally thought to be solely regulated by dsRNA, an intermediate in the replication of many viruses. However, the notion that PKR acts solely as a sensor for viral infection has been challenged by recent findings that alteration of PKR activity has effects on cellular growth and by the discovery of a virus-independent activator of PKR, a cellular protein called PACT (PKR-activating protein). The activation of the transcription factor nuclear factor kappa B (NF-κB) by PKR has been shown to account for the host antiviral response. We summarize the most recent findings on the molecular mechanisms leading to the activation of NF-κB by PKR and discuss three major unanswered questions. First, is PACT an alternative to dsRNA as a direct activator of the PKR-NF-κB pathway? Second, how is PACT itself activated and targeted to PKR? And third, what are the biological functions of PKR in the absence of viral infection?
Signal Integration via PKR
Authors: Williams Bryan R. G.
Published: May 2003

The vital role of interferons (IFNs) as mediators of innate immunity is well established. It has recently become apparent that one of the pivotal proteins in mediating the antiviral activity of IFNs, the double-stranded RNA (dsRNA)-activated protein kinase (PKR), also functions as a signal transducer in the proinflammatory response to different agents. PKR is a member of a small family of kinases that are activated by extracellular stresses and that phosphorylate the α subunit of protein synthesis initiation factor eIF-2, thereby inhibiting protein synthesis. The activation of PKR during infection by viral dsRNA intermediates results in the inhibition of viral replication. PKR also mediates the activation of signal transduction pathways by proinflammatory stimuli, including bacterial lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α), and interleukin 1 (IL-1). PKR is a component of the inhibitor of κB (IκB) kinase complex and plays either a catalytic or structural role in the activation of IκB kinase, depending on the stimulus. The activities of the stress-activated protein kinases p38 and c-Jun NH
2
-terminal kinase (JNK) are also regulated by PKR in a pathway that leads to the production of proinflammatory cytokines. This review will focus on the role of PKR in nuclear factor κ B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, because these have been the subjects of a series of publications over the past year that have reported conflicting findings. Although the conflicts may not be resolved in this review, suggestions are made for experiments that could lead to a clearer understanding of the mechanisms involved.
Physiology, Phylogeny, and Functions of the TRP Superfamily of Cation Channels
Authors: Montell Craig
Published: May 2003

The transient receptor potential (TRP) protein superfamily consists of a diverse group of Ca
2+
permeable nonselective cation channels that bear structural similarities to Drosophila TRP. TRP-related proteins play important roles in nonexcitable cells, as demonstrated by the recent finding that a mammalian TRPC protein is expressed in endothelial cells and functions in vasorelaxation. However, an emerging theme is that many TRP-related proteins are expressed predominantly in the nervous system and function in sensory physiology. The TRP superfamily can be divided into six subfamilies, the first of which is composed of the "classical TRPs" (TRPC subfamily). These proteins all share the common features of three to four ankryin repeats, ≥30% amino acid homology over ≥750 amino acids, and a gating mechanism that operates through phospholipase C. Some classical TRPs may be store-operated channels (SOCs), which are activated by release of Ca
2+
from internal stores. The mammalian TRPC proteins are also expressed in the central nervous system, and several are highly enriched in the brain. One TRPC protein has been implicated in the pheromone response. The archetypal TRP, Drosophila TRP, is predominantly expressed in the visual system and is required for phototransduction. Many members of a second subfamily (TRPV) function in sensory physiology. These include VR1 and OSM-9, which respond to heat, osmolarity, odorants, and mechanical stimuli. A third subfamily, TRPN, includes proteins with many ankyrin repeats, one of which, NOMPC, participates in mechanotransduction. Among the members of a fourth subfamily, TRPM, is a putative tumor suppressor termed melastatin, and a bifunctional protein, TRP-PLIK, consisting of a TRPM channel fused to a protein kinase. PKD2 and mucolipidin are the founding members of the TRPP and TRPML subfamilies, respectively. Mutations in PKD2 are responsible for polycystic kidney disease, and mutations in mucolipidin result in a severe neurodegenerative disorder. Recent studies suggest that alterations in the activities of SOC and TRP channels may be at the heart of several additional neurodegenerative diseases. Thus, TRP channels may prove to be important new targets for drug discovery.
Slit: A Roadblock for Chemotaxis
Authors: Fernandis Aaron Z. , Ganju Ramesh K.
Published: May 2003

The immune system and nervous system display striking similarities. Fernandis and Ganju discuss yet another example where a protein (Slit) originally identified for its role in modulating axon pathfinding is able to regulate immune cell migration. Slit isoforms expressed in the nervous system interact with members of the Robo receptor family to modify movement stimulated by the secreted attractants netrins and their receptors. In leukocytes, Slit 2 interacting with Robo receptors inhibits movement stimulated by the chemokine receptor (CXCR4). Fernandis and Ganju discuss the therapeutic potential of Slit as an antiviral agent and in the treatment of inflammatory diseases, autoimmune disorders, and cancer.
A Call to Arms: The Cytokine Selection Service
Authors: Nelson John W.
Published: May 2003

The process by which naïve T helper (T
H
) cells differentiate into the T
H
1 and T
H
2 subtypes has been well studied. However, there remain some unresolved issues pertaining to the requirements for the initial step of T
H
cell differentiation. Much debate exists about whether the roles of cytokines include the forcing of the initial steps of differentiation on naïve T
H
cells, termed "instruction," or whether cytokines act in a supportive role, termed "selection," whereby newly differentiating T
H
cells are given the proper signals for survival and proliferation. A recent paper by Mullen
et al
., which helps delineate the role of cytokines in T
H
1 cell development, is addressed by Nelson; it appears that cytokines act in the selection stage of T
H
cell maturation.
RNA Localization Meets Wingless Signaling
Authors: Manseau Lynn J.
Published: May 2003

In many tissues in many organisms, messenger RNA (mRNA) is not randomly distributed throughout a cell, but is targeted and accumulated in specific subcellular locations. Signal transduction pathways can be extremely sensitive to gradients of signaling proteins and represent a cellular phenomenon where RNA localization may be important. Manseau discusses how targeting of the RNA for a secreted ligand, Wingless (Wg), is important in promoting proper Wg signaling in early Drosophila embryos and the mechanism for achieving this subcellular targeting. The possible mechanistic models for how restricted wg mRNA distribution influences Wg signaling are also detailed.
HOG on the Promoter: Regulation of the Osmotic Stress Response
Authors: Chellappan Srikumar P.
Published: May 2003

Members of the mitogen-activated protein (MAP) kinase family regulate transcription through phosphorylation of specific transcription factors. New studies indicate that this process may be more complex than previously anticipated. The yeast Hog1 protein kinase (a homolog of the mammalian p38 MAP kinase) interacts with transcription factors and perhaps with the general transcription machinery at target promoters. Chellappan discusses the recent results and their implications for understanding control of transcription by stress-activated MAP kinases.
Analysis of Protein Arginine Methylation and Protein Arginine-Methyltransferase Activity
Authors: Mowen Kerri A. , David Michael
Published: May 2003

Posttranslational modification of proteins allows cells to adapt and react quickly to their environment beyond the boundaries set forth by genetic code. Arginine methylation, a protein modification discovered almost 30 years ago, has recently experienced a renewed interest as several new arginine methyltransferases have been identified and numerous proteins were found to be regulated by methylation on arginine residues. Until recently, the detection of arginine methylation required the use of chromatography and mass-spectrometrical analysis. The following protocol provides guidelines for the straightforward identification of arginine-methylated proteins, made possible by the availability of novel, commercially available reagents.
Stressed to Death: Regulation of Apoptotic Signaling Pathways by the Heat Shock Proteins
Authors: Beere Helen M.
Published: May 2003

Cellular damage can engage two fundamental cellular responses: apoptosis, a precisely regulated form of cell death; and the heat shock protein (Hsp), or stress response, which functions to protect cells and to mediate an accelerated recovery following damage. The coordinated balance between these two opposing pathways governs the ultimate fate of the cell--whether it lives or dies. The self-destruction of a cell is mediated by one of many signaling pathways culminating in the activation of the caspase proteases. The Hsps regulate the activity of multiple intracellular signaling intermediates, many of which are intimately involved in the execution of the apoptotic signaling pathways. This review addresses whether the antiapoptotic activities of several Hsps, including Hsp70, Hsp90, and Hsp27, can be attributed to their collective ability to regulate the activities, expression, or both of apoptotic signaling molecules. In summary, the functional interface between the ancient heat shock or stress protein response and the highly conserved biochemical pathways leading to the activation of apoptosis governs the susceptibility of a cell to damaging stimuli.
New Roles for Introns: Sites of Combinatorial Regulation of Ca
2+
- and Cyclic AMP-Dependent Gene Transcription
Authors: Finkbeiner Steven
Published: May 2003

Because some proteins can facilitate cell division or the expression of many genes simultaneously, it comes as no surprise that the expression of very important gene products is a tightly controlled process. Although gene expression is often thought of in terms of complexes of transcription factors binding to promoter elements, some studies indicate that intronic DNA sequences may also regulate gene expression. Finkbeiner examines recent work by Schlegel and colleagues demonstrating that sequences within the first intron of the c-
fos
gene help to regulate Fos expression under different conditions.
Neuronal Signaling through Alternative Splicing: Some Exons CaRRE...
Authors: O'Donovan Kevin J. , Darnell Robert B.
Published: May 2003

Alternative splicing represents a mechanism by which a single gene can be used to create proteins with different functions. Neurons use alternative splicing to produce channels with different sequences and biophysical or regulatory properties. O'Donovan and Darnell discuss a mechanism by which neurons can alter channel splicing in response to neuronal activity through a signal generated by calcium and calcium/calmodulin-dependent kinase activity.
GABA May Act as a Self-Limiting Trophic Factor at Developing Synapses
Authors: Kriegstein Arnold R. , Owens David F.
Published: May 2003

Early in development, synapses with glycine or γ-aminobutyric acid (GABA)-gated chloride channels exhibit the ability to depolarize postsynaptic cells. As the synapses mature and the gradient of chloride ions across the cell membrane is altered, these neurotransmitters signal an inhibitory response, hyperpolarizing the membrane and decreasing neuronal excitability. Kriegstein and Owens discuss how GABA-stimulated up-regulation of the expression of the potassium chloride cotransporter KCC2 may be the mechanism underlying this synaptic switch.
RNA Interference of Gene Expression (RNAi) in Cultured
Drosophila
Cells
Authors: Worby Carolyn A. , Simonson-Leff Nancy , Dixon Jack E.
Published: May 2003

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on
Drosophila
-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of
Drosophila
, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all
Drosophila
cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene.
Drosophila
cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.
PODs in the Nuclear Spot: Enigmas in the Magician's Pot
Authors: Hatta Mitsutoki , Fukamizu Akiyoshi
Published: May 2003

The promyelocytic leukemia (PML) nuclear body, also known as the PML oncogenic domain (POD), is implicated in the pathophysiology of PML. These nuclear subcompartments are dynamic structures. The PML protein, which undergoes a fusion event in patients with promyelocytic leukemia, is normally found in PODs. The PML protein may be a major regulator of the constituents of PODs, controlling POD organization and function. Hatta and Fukamizu describe the functions of PML and discuss how the POD structure and organization may be regulated and affect apoptosis, gene expression, and cellular transformation.
Ras Isoform-Specific Signaling: Location, Location, Location
Authors: Wolfman Alan
Published: May 2003

The proteins that compose the Ras family of small guanosine triphosphatases share a remarkably high degree of sequence similarity, yet recent evidence indicates that they may have unique biological properties. How is it that similar proteins carry out different jobs in the cell? Wolfman addresses this question by surveying recent reports that indicate that different biological roles may be born out of distinct subcellular localizations of the Ras proteins. It appears that the small differences in their amino acid sequences and their different posttranslational modifications may be all that is necessary to direct various Ras proteins to different sites.
IKKα on Center Stage
Authors: DiDonato Joseph A.
Published: May 2003

Inhibitor of κB kinase α (IKKα) was originally identified as a component of a multiprotein kinase complex that regulates the activity of the transcription factor nuclear factor-κB (NF-κB) through phosphorylation of its inhibitor proteins, the IκBs. DiDonato discusses new roles that have been discovered for IKKα, focusing especially on its role in epidermal differentiation and on a new function of IKKα in B cell maturation. In epidermal differentiation, IKKα regulates the production of a secreted differentiation factor through a pathway that is independent of its role in activation of NF-κB. In B cell maturation, conventional NF-κB signal-induced activation of IKKα results in phosphorylation of p100 precursor proteins and increased proteolytic processing and constitutive NF-κB activation.
A Hitchhiker's Guide to the Proteasome
Authors: von Arnim Albrecht G.
Published: May 2003

Regulated degradation of proteins is essential for viability and is involved in the control of many signal transduction pathways. von Arnim discusses a new model for how substrates destined for degradation by the 26S proteasome may be presented to the proteasome through a physical interaction between the proteasome and a complex consisting of the substrate and a ubiquitin-ligase. The new model suggests that the SCF (Skp1/cullin/F-box) protein complex may physically associate with the proteasome and that this interaction may be regulated by posttranslational modifications, such as phosphorylation or the covalent attachment of the Nedd8 protein, called neddylation. Additionally, other proteins may compete with the SCF complexes for binding to the proteasome and thus present another layer of regulation for controlling substrate targeting for ubiquitin-mediated degradation.
Unzipping Ion Channels
Authors: MacFarlane Stacey Nee , Levitan Irwin B.
Published: May 2003

The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.
The Torso Ligand, Unmasked?
Authors: Stein David , Stevens Leslie M.
Published: May 2003

When a transmembrane receptor protein tyrosine kinase (RTK) is expressed throughout the plasma membrane, yet only a specific handful of them must be activated, what's a ligand to do? During the development of the anterior and posterior termini of the
Drosophila
embryo, uniformly secreted ligand precursors are activated by proteolysis near the location of the receptors that must be activated. Stein and Stevens discuss the recent publication by Casali and Casanova that describes the mechanism of activation of the
Drosophila
RTK called Torso. In addition, Casali and Casanova may have identified a physiologically relevant ligand for Torso called Trunk. Proteolytic cleavage of the Trunk precursor can activate Torso-dependent signaling, but the existence of cleaved Trunk has not yet been demonstrated in vivo for
Drosophila
. Stein and Stevens discuss the ramifications of such a highly regulated process of ligand activation, and also proffer alternative scenarios for Torso activation.
Fungal Histidine Kinases
Authors: Santos Jennifer L. , Shiozaki Kazuhiro
Published: May 2003

Eukaryotic cells predominantly use serine, threonine, and tyrosine phosphorylation in various intracellular signal transduction pathways. In contrast, prokaryotic organisms employ numerous "two-component" systems, in which signaling is achieved by transferring a phosphoryl group from phosphohistidine in the "sensor kinase" component to aspartate in the "response regulator" component. In the last several years, genetic screens and genome projects have identified sensor kinases and response regulators in lower eukaryotes and plants, revealing that eukaryotic organisms also make use of His-Asp phosphotransfer in a limited number of signaling pathways. Extensive studies in yeasts have demonstrated that a variation of the two-component system, a multistep "phosphorelay," is the prevailing mechanism among distantly related yeast species. In the budding yeast
Saccharomyces cerevisiae
, a His-Asp-His-Asp phosphorelay transmits osmotic stress signals to a mitogen-activated protein kinase (MAPK) cascade to induce adaptive responses. A phosphorelay in the fission yeast
Schizosaccharomyces pombe
, analogous to the
S. cerevisiae
phosphorelay, is responsible for MAPK activation in response to peroxide stress. Mammalian cells do not have any two-component or phosphorelay systems, although protein histidine kinases unrelated to the sensor kinase may be involved in cellular signaling. Because some phosphorelay proteins are essential for virulence of microbial pathogens, including the yeast fungus
Candida albicans
, novel antibiotics targeted to phosphorelays may be effective against eukaryotic pathogens without causing host cell damage.
Raf-1 Without MEK?
Authors: Murakami Monica S. , Morrison Deborah K.
Published: May 2003

The Ras-Raf-MEK [(mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase]-MAPK signaling pathway controls the activation of many cellular functions. Recent reports of Raf-1-deficient mice have indicated that MEK may not be an important downstream substrate for Raf-1 and that, in fact, Raf-1 is important for blocking apoptosis rather than for cell proliferation. Murakami and Morrison examine these recent findings and discuss their implications, as well as other possible conclusions that may be drawn from the published data.
IL-23: A Cytokine That Acts on Memory T Cells
Authors: Frucht David M.
Published: May 2003

The newly discovered cytokine interleukin (IL)-23 shares some in vivo functions with IL-12, including the activation of the transcription factor STAT4 (signal tranducer and activator of transcription-4). Indeed, the receptors for each appear to share one subunit, but also have at least one distinct subunit. Frucht discusses the similarities of IL-12 and IL-23 and the effects that distinguish one from the other. In contrast to IL-12, IL-23 appears to participate in the proliferative signal in memory T cells. More functions that distinguish IL-23 from IL-12 are likely to be uncovered as soon as the other component(s) of the IL-23 receptor are molecularly cloned and characterized.
Unexpected Cross Talk: Small GTPase Regulation of Calcium Channel Trafficking
Authors: Trimmer James S.
Published: May 2003

Calcium ions can serve as both charge carriers and second messengers. Most cells have voltage-dependent calcium channels that control the membrane permeability to calcium. These channels at the membrane open in response to changes in membrane potential. Their activity is further modulated by phosphorylation by various kinases, such as protein kinase A and protein kinase C, and by changes in intracellular calcium concentration through the action of calcium calmodulin (Ca
2+
/CaM). Trimmer discusses a potential mechanism by which the expression of these channels can be regulated through an interaction with a small guanosine triphosphatase (GTPase), kir/Gem, that influences trafficking of the channels through effects on assembly with auxiliary subunits that occurs in the biosynthetic pathway.
Meeting at Mitosis: Cell Cycle-Specific Regulation of c-Src by RPTPα
Authors: Mustelin Tomas , Hunter Tony
Published: May 2003

Exquisite regulation is required for cells to properly enter and exit the phases of the cell cycle. The transmembrane receptor-like protein tyrosine phosphatase RPTPα, an important protein that participates in the transition of the cell cycle from G2 to mitosis activates the protein tyrosine kinase c-Src in vivo. Mustelin and Hunter discuss new findings that describe the highly regulated activation of RPTPα and c-Src that occurs just before entry into the mitotic phase. These findings also raise several questions that pertain to redistribution of RPTPα in the cell, and the role of phosphorylation and dimerization in regulating RPTPα activity.
A New Gun in Town: The U Box Is a Ubiquitin Ligase Domain
Authors: Patterson Cam
Published: May 2003

Ubiquitin ligases determine protein stability in a highly regulated manner by coordinating the addition of polyubiquitin chains to proteins that are then targeted to the proteasome for degradation. Ubiquitin ligases have generally been separated into two groups--those containing HECT domains and those with RING finger domains. Recently, a third group of ubiquitin ligases has emerged: those containing a U-box domain. Patterson discusses what is known about the few U-box-containing proteins that have been characterized, although the general properties of U-box proteins that distinguish them from other ubiquitin ligases are still a matter of speculation.
Phototropin Blue Light Receptors and Light-Induced Movement Responses in Plants
Authors: Lin Chentao
Published: May 2003

Two blue light receptors in
Arabidopsis
, termed phot1 and phot2, which contain flavin chromophores and have intrinsic protein kinase activity, participate in several physiological processes. Lin looks at the genetic evidence regarding how these photoreceptors mediate blue light-induced phototropism, chloroplast relocalization, and opening of the stomatal aperture.