bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysophosphatidic acid possesses dual action in cellproliferation.
Authors: Tigyi G , Dyer D L , Miledi R
Published: May 2006

Lysophosphatidic acid (LPA) induces mitogenic
responses in cultured fibroblasts through a pertussis toxin-sensitive signaling
pathway. In contrast, we have shown that LPA inhibits the proliferation of
Sp2/0-Ag14 myeloma cells. To resolve this apparent controversy, LPA-elicited
responses in cell proliferation and the underlying second messenger mechanisms
were compared in Sp2/0-Ag14 myeloma and NIH 3T3 fibroblast cells. The
antimitogenic response was not elicited by micromolar concentrations of
phosphatidic acid, phosphatidylglycerol, or diacylglycerol. In NIH 3T3 and Sp2
cells, LPA elicited an increase in inositol trisphosphate and a subsequent
transient increase in free cytoplasmic Ca2+. Unlike the mitogenic response in
NIH 3T3 cells, the antimitogenic effect was not affected by pertussis toxin; on
the contrary, it was accompanied by an increase in cAMP. In Sp2 cells, cAMP
analogs, forskolin, and isobutylmethylxanthine inhibited cell proliferation and
enhanced LPA action in an additive manner, suggesting that an LPA-elicited
increase in cAMP-mediated signaling was responsible for the antimitogenic
response. In addition to the mitogenic response in fibroblasts and the
antimitogenic response in tumor cell lines, there are some cell types (Jurkat
T-cell lymphoma and primary astrocytes) in which LPA is ineffective in altering
cell proliferation. The cell-type-specific dual action of LPA suggests that this
endogenous lipid mediator when released from activated cells might play an
important role as a regulator, rather than a ubiquitous inducer, of cell
proliferation.
Temperature acclimation and competitive fitness: anexperimental test of the beneficial acclimation assumption.
Authors: Leroi A M , Bennett A F , Lenski R E
Published: May 2006

Phenotypic acclimation is generally assumed to
confer an advantage in the environment that stimulates the response. To test
this beneficial acclimation assumption explicitly, we investigated the
consequences of temperature acclimation for the fitness of Escherichia coli at
two temperatures, 32 degrees C and 41.5 degrees C. Both temperatures permit
growth and long-term persistence of the genotypes in serial culture. We found
that prior acclimation to 32 degrees C, relative to acclimation to 41.5 degrees
C, enhanced fitness at 32 degrees C, consistent with the assumption. But prior
acclimation to 41.5 degrees C actually reduced fitness at 41.5 degrees C,
relative to acclimation to 32 degrees C. Hence, the assumption that acclimation
always confers an advantage is demonstrated to be false. Acclimation to 41.5
degrees C did, however, improve survival at 50 degrees C, a lethal temperature.
This protective response has been shown to be associated with the induction of
stress proteins. The reduced competitive fitness caused by acclimation at 41.5
degrees C may reflect a physiological burden associated with expression of
stress proteins when they are not needed to prevent lethal damage. Whatever the
cause, acclimation to the higher temperature decreased competitive fitness at
that temperature.
An SRY-related sequence on the marsupial X chromosome:implications for the evolution of the mammalian testis-determininggene.
Authors: Foster J W , Graves J A
Published: May 2006

The SRY gene on the human, mouse, and marsupial Y
chromosomes is the testis-determining gene that initiates male development in
mammals. The SRY protein has a DNA-binding domain (high mobility group or HMG
box) similar to those found in the high-mobility-group proteins. SRY is specific
for the Y chromosome, but many autosomal genes have been identified that possess
a similar HMG box region; those with the most closely SRY-related box regions
form a gene family now referred to as SOX genes. We have identified a sequence
on the marsupial X chromosome that shares homology with SRY. Sequence
comparisons show near-identity with the mouse and human SOX3 gene (formerly
called a3), the SOX gene which is the most closely related to SRY. We suggest
here that the highly conserved X chromosome-linked SOX3 represents the ancestral
SOX gene from which the sex-determining gene SRY was derived. In this model
SOX3/SRY divergence and the acquisition of a testis-determining role by SRY
might have preceded (and initiated) sex chromosome differentiation or,
alternatively, might have been a consequence of X chromosome-Y chromosome
differentiation initiated at the locus of an original sex-determining gene(s),
later superseded by SRY.
A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.
Authors: Garcia E P , Gatti E , Butler M , Burton J , De Camilli P
Published: May 2006

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.
Hemispheric encoding/retrieval asymmetry in episodic memory: positron emission tomography findings.
Authors: Tulving E , Kapur S , Craik F I , Moscovitch M , Houle S
Published: May 2006

Data are reviewed from positron emission tomography studies of encoding and retrieval processes in episodic memory. These data suggest a hemispheric encoding/retrieval asymmetry model of prefrontal involvement in encoding and retrieval of episodic memory. According to this model, the left and right prefrontal lobes are part of an extensive neuronal network that subserves episodic remembering, but the two prefrontal hemispheres play different roles. Left prefrontal cortical regions are differentially more involved in retrieval of information from semantic memory and in simultaneously encoding novel aspects of the retrieved information into episodic memory. Right prefrontal cortical regions, on the other hand, are differentially more involved in episodic memory retrieval.
Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.
Authors: Staehelin C , Granado J , Müller J , Wiemken A , Mellor R B , Felix G , Regenass M , Broughton W J , Boller T
Published: May 2006

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.
A role for histones H2A/H2B in chromatin folding and transcriptional repression.
Authors: Hansen J C , Wolffe A P
Published: May 2006

Histone octamers or histone H3/H4 tetramers were reconstituted onto either closed circular plasmids containing a single Xenopus 5S rRNA gene or a reiterated array of Lytechinus 5S rRNA genes. All "reconstitutes" were found to undergo both Na(+)-dependent and Mg(2+)-dependent compaction. However, in each case, the compaction of nucleosomal templates containing H2A/H2B was much more extensive than compaction of templates containing only H3/H4 tetramers. Inclusion of 5 mM MgCl2 in the transcription buffer increased the level of compaction of nucleosomal templates and led to a marked inhibition of both transcription initiation and elongation by RNA polymerase III. The inhibitory effect of Mg2+ was reduced significantly when DNA templates contained only H3/H4 tetramers, consistent with their lesser extent of Mg(2+)-dependent compaction. Thus, the removal of histones H2A/H2B from nucleosomal arrays enhances gene activity, in part because of decreased levels of chromatin folding.
Evolution of drug-resistant tuberculosis: a tale of two species.
Authors: Iseman M D
Published: May 2006

The history of the disease tuberculosis is briefly discussed. Now human societal failures have potentiated the evolution of drug-resistant strains of the tubercle bacillus in the United States and around the world. Until recently, this evolutionary change largely posed a threat to the health and survival of the individual in whom inadequate therapy promoted the drug resistance. However, the human immunodeficiency virus epidemic threatens to promote wholesale transmission of multidrug-resistant tuberculosis with the potential for immense morbidity and mortality. Reinforced treatment and control programs for tuberculosis are vital.
Reaction of germinal centers in the T-cell-independent response to the bacterial polysaccharide alpha(1-->6)dextran.
Authors: Wang D , Wells S M , Stall A M , Kabat E A
Published: May 2006

Primary immunization of BALB/c mice with alpha(1-->6)dextran (DEX), a native bacterial polysaccharide, induces an unexpected pattern of splenic B-cell responses. After a peak of antibody-secreting B-cell response at day 4, deposition of dextran-anti-dextran immune complexes, as revealed by staining with both dextran and antibodies to dextran, occurs and persists in splenic follicles until at least the fourth week after immunization. Antigen-specific B cells appear and proliferate in such follicles, leading by day 11 to development of DEX-specific germinal centers as characterized by the presence of distinct regions of DEX+ peanut agglutinin-positive (PNA+) cells. At this time, fluorescence-activated cell sorter analysis also reveals the appearance of a distinct population of DEX+ PNA+ splenic B cells. In contrast, DEX+ PNA- cells, characterized by intense cytoplasmic staining, are present outside of splenic follicles, peak at day 4 to day 5, and persist until at least day 28. The frequency of these cells correlates with DEX-specific antibody-secreting cells, as detected by the ELISA-spot assay. Thus, in addition to the expected plasma cellular response, the typical T-cell-independent type II antigen, DEX, surprisingly also elicits the formation of antigen-specific germinal centers. These observations raise fundamental questions about the roles of germinal centers in T-cell-independent immune responses.
Recombinational basis of serovar diversity in Salmonella enterica.
Authors: Li J , Nelson K , McWhorter A C , Whittam T S , Selander R K
Published: May 2006

The fliC gene, which encodes phase 1 flagellin, was sequenced in strains of 15 Salmonella enterica serovars expressing flagellar antigenic factors of the g series. The occurrence of each of the flagellin serotypes g,m, m,t, and g,z51 in distantly related strains is the result of horizontal exchange of DNA, as indicated by identity or close similarity in nucleotide sequence of all or parts of the antigenic factor-determining central region of fliC. The flagellin genes of some serovars are complex mosaic structures composed of diverse segments derived through multiple recombination events. Thus, recombination of horizontally transferred segments (intragenic) or entire genes (assortative) within and among subspecies is identified as a major evolutionary mechanism generating both allelic variation at the fliC locus and serovar diversity in natural populations. Evidence that flagellar serological diversity is promoted by diversifying selection in adaptation to host immune defense system or flagellotropic phage is discussed.
Light and nutritional regulation of transcripts encoding a wheat protein kinase homolog is mediated by cytokinins.
Authors: Sano H , Youssefian S
Published: May 2006

Many metabolic processes in plants are regulated by phosphorylation of proteins by kinases, but little is known of the roles that specific protein kinase play in the various signal transduction pathways or the mechanisms by which these kinases themselves are regulated. We report here the isolation of a gene, wpk4, encoding a putative protein kinase from wheat that appears to belong to the SNF1 kinase subfamily and that shows increased transcript levels in response to multiple stimuli: light, nutrient deprivation, and cytokinin application. Although wpk4 mRNA is undetectable in etiolated seedlings, it rapidly accumulates within 1 hr of illumination. General nutrient deprivation also increases wpk4 mRNA levels, but only under light conditions. In addition, of the various phytohormones tested, cytokinin (N6-benzylaminopurine) specifically increases wpk4 mRNA levels regardless of the light conditions, whereas in the presence of a cytokinin antagonist the level of wpk4 mRNA is not increased by either light or nutrient deprivation. These results suggest that the light and nutrient signals that induce wpk4 mRNA accumulation may be mediated through cytokinins and provide a strong basis for examining the coordinated regulation of protein phosphorylation by light, cytokinins, and nutritional cues in a single transduction pathway.
Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities.
Authors: Varghese J N , Garrett T P , Colman P M , Chen L , Høj P B , Fincher G B
Published: May 2006

The three-dimensional structures of (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII and (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII from barley have been determined by x-ray crystallography at 2.2- to 2.3-A resolution. The two classes of polysaccharide endohydrolase differ in their substrate specificity and function. Thus, the (1-->3)-beta-glucanases, which are classified amongst the plant "pathogenesis-related proteins," can hydrolyze (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls and may therefore contribute to plant defense strategies, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall hydrolysis during mobilization of the endosperm in germinating grain or during the growth of vegetative tissues. Both enzymes are alpha/beta-barrel structures. The catalytic amino acid residues are located within deep grooves which extend across the enzymes and which probably bind the substrates. Because the polypeptide backbones of the two enzymes are structurally very similar, the differences in their substrate specificities, and hence their widely divergent functions, have been acquired primarily by amino acid substitutions within the groove.
Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition.
Authors: Myatt E A , Westholm F A , Weiss D T , Solomon A , Schiffer M , Stevens F J
Published: May 2006

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.
ATP-dependent phosphorylation of serine-46 in the phosphocarrier protein HPr regulates lactose/H+ symport in Lactobacillus brevis.
Authors: Ye J J , Reizer J , Cui X , Saier M H
Published: May 2006

Lactobacillus brevis takes up lactose and the nonmetabolizable lactose analogue thiomethyl beta-galactoside (TMG) by a permease-catalyzed lactose/H+ symport mechanism. Earlier studies have shown that TMG, previously accumulated in L. brevis cells, rapidly effluxes from the cells upon addition of glucose, and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeant metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular ATP or extravesicular arginine. TMG uptake into these vesicles was not inhibited, and preaccumulated TMG did not efflux from them upon addition of glucose. Intravesicular but not extravesicular wild-type phosphocarrier protein HPr of Bacillus subtilis restored regulation. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-bisphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular serine-46-->alanine mutant HPr cold not promote regulation of lactose permease activity when electroporated into the vesicles with or without glucose or the various phosphorylated metabolites, but the serine-46-->aspartate mutant HPr promoted regulation, even in the absence of glucose or a metabolite. HPr(Ser-P) appears to convert the lactose/H+ symporter into a sugar uniporter. These results establish that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates lactose permease activity in L. brevis. A direct allosteric mechanism is proposed.
Isolation and analysis of the breakpoint sequences of chromosome inversion In(3L)Payne in Drosophila melanogaster.
Authors: Wesley C S , Eanes W F
Published: May 2006

Chromosomal rearrangements constitute a significant feature of genome evolution, and inversion polymorphisms in Drosophila have been studied intensely for decades. Population geneticists have long recognized that the sequence features associated with inversion breakpoints would reveal much about the mutational origin, uniqueness, and genealogical history of individual inversion polymorphisms, but the cloning of breakpoint sequences is not trivial. With the aid of a method for rapid recovery of DNA clones spanning rearrangement breakpoints, we recover and examine the DNA sequences spanning the breakpoints of the cosmopolitan inversion In(3L)Payne in Drosophila melanogaster. By examining the sequence diversity associated with six standard and seven inverted chromosomes from natural populations, we find that the inversion is monophyletic in origin, the sequences are genetically isolated from recombination at the breakpoints, and there is no association with features such as transposable elements. The inverted sequences show 17-fold less nucleotide polymorphism, but there are eight fixed differences in the region spanning both breakpoints. This suggests that this inversion is not recently derived. Finally, Northern analysis and transcript mapping find that the distal breakpoint has disrupted three transcripts that are normally expressed in the standard arrangement. Incidentally, the method introduced here can be used to isolate breakpoint sequences of arrangements associated with many human diseases.
Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha.
Authors: Gietl C , Faber K N , van der Klei I J , Veenhuis M
Published: May 2006

We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of various genetically altered watermelon MDH genes, whose protein products were localized by immunocytochemical techniques. It is shown that the presequence of gMDH is essential and sufficient for peroxisomal targeting; it can target the mature part of the mitochondrial MDH to microbodies, whereas deletion of the presequence results in accumulation of the mature form of gMDH in the cytosol. Alignment of the N termini of several peroxisomal proteins that are assumed to contain a peroxisomal targeting signal at the N terminus (PTS2) suggested the consensus seqence RL-X5-HL. A similar motif is present in the presequence of watermelon gMDH--namely, 10RI-X5-17HL. Mutational analysis revealed that substitutions of 10RI into DD or 17HL into DE destroyed the topogenic information, whereas substitutions of 25M into I and 26EE into LV did not. By combining our data with recent analyses of others on the presequences of mammalian thiolases, it is concluded that the peroxisomal targeting information of PTS2 is contained in the consensus sequence RL/I-X5-HL. In contrast to the higher plant and mammals, the Hansenula yeast peroxisomes seem to lack an enzyme capable of removing microbody presequences of higher eukaryotes.
Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.
Authors: Hall S D , Kolodner R D
Published: May 2006

RecT protein of Escherichia coli promotes the formation of joint molecules between homologous linear double-stranded M13mp19 replicative-form bacteriophage DNA and circular single-stranded M13mp19 DNA in the presence of exonuclease VIII, the recE gene product. The joint molecules were formed by a mechanism involving the pairing of the complementary strand of the linear double-stranded DNA substrate with the circular single-stranded DNA substrate coupled with the displacement of the noncomplementary strand. When the homologous linear double-stranded DNA substrate had homologous 3' or 5' single-stranded tails, then RecT promoted homologous pairing and strand exchange in the absence of exonuclease VIII. Histone H1 could substitute for RecT protein; however, joint molecules formed in the presence of histone H1 did not undergo strand exchange. These results indicate that under the reaction conditions used, the observed strand exchange reaction is promoted by RecT and is not the result of spontaneous branch migration. These results are consistent with the observation that expression of RecE (exonuclease VIII) and RecT substitutes for RecA in some recombination reactions in E. coli.
Glutamate regulates intracellular calcium and gene expression in oligodendrocyte progenitors through the activation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.
Authors: Pende M , Holtzclaw L A , Curtis J L , Russell J T , Gallo V
Published: May 2006

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.
Cooperative interactions between the interleukin 2 receptor alpha and beta chains alter the interleukin 2-binding affinity of the receptor subunits.
Authors: Roessler E , Grant A , Ju G , Tsudo M , Sugamura K , Waldmann T A
Published: May 2006

The interleukin 2 (IL-2) receptor (IL-2R) is a multisubunit receptor that includes three major IL-2 binding subunits, the IL-2R alpha, beta, and gamma chains. We have detected and analyzed cooperative interactions between the IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively) in COS cells transfected with cDNAs encoding the IL-2R alpha, the IL-2R beta, or both cDNAs. We demonstrated that IL-2 F42A, an analog that fails to bind to the isolated IL-2R alpha subunit and would be predicted by the hierarchical affinity-conversion model to have impaired binding to cells expressing both chains, instead readily binds to the IL-2R alpha/beta heterodimer in COS cells. Furthermore, this binding is abolished by the antibody HIEI that separates the two IL-2R subunits. The monoclonal antibodies anti-Tac and Mik-beta 1 directed at the IL-2-binding sites on IL-2R alpha and IL-2R beta, respectively, block ligand binding to the heterodimer. This binding pattern is inconsistent with the strict hierarchical affinity-conversion model that mandates an initial binding of IL-2 to IL-2R alpha followed by binding of the IL-2/IL-2R alpha complex to IL-2R beta. Instead, our results support an alternative model of preformed complexes of IL-2R beta with other IL-2R subunits. In this alternative model, IL-2R alpha and -beta exist in part as preformed complexes in which the affinity of IL-2R beta for IL-2 is altered by the proximity of IL-2R alpha, through mechanisms that do not require the prior binding of IL-2 to IL-2R alpha.
Truncation mutants define and locate cytoplasmic barriers to lateral mobility of membrane glycoproteins.
Authors: Edidin M , Zúñiga M C , Sheetz M P
Published: May 2006

The lateral mobility of cell membrane glycoproteins is often restricted by dynamic barriers. These barriers have been detected by measurements of fluorescence photobleaching and recovery (FPR) and barrier-free path (BFP). To define the location and properties of the barriers, we compared the lateral mobility, measured by FPR and BFP, of wild-type class I major histocompatibility complex (MHC) membrane glycoproteins with the lateral mobility of mutant class I MHC glycoproteins truncated in their cytoplasmic domains. Mutants with 0 or 4 residues in the cytoplasmic domain were as mobile as lipid-anchored class I MHC molecules, molecules whose lateral mobility is relatively unrestricted by barriers. In contrast, mobility of class I MHC molecules with 7-residue cytoplasmic domains was as restricted as mobility of class I molecules with full-length, 31-residue cytoplasmic domains. Though some of the difference between the mobilities of mutants with 4- or 0-residue domains and the other class I molecules may be due to differences in the net charge of the cytoplasmic domain, FPR measurements of the mobility of molecules with 7-residue domains show that length of the cytoplasmic domain has an important influence on the lateral mobility. Model calculations suggest that the barriers to lateral mobility are 2-3 nm below the membrane bilayer.
C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins.
Authors: Tanaka S , Morishita T , Hashimoto Y , Hattori S , Nakamura S , Shibuya M , Matuoka K , Takenawa T , Kurata T , Nagashima K
Published: May 2006

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa. The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae. The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins. Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence. The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues. The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.
Triterpene derivatives that block entry of human immunodeficiency virus type 1 into cells.
Authors: Mayaux J F , Bousseau A , Pauwels R , Huet T , Hénin Y , Dereu N , Evers M , Soler F , Poujade C , De Clercq E
Published: May 2006

A series of triterpene compounds characterized by a stringent structure-activity relationship were identified as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. Currently studied botulinic derivatives have 50% inhibitory concentrations (IC50) against HIV-1 strain IIIB/LAI in the 10 nM range in several cellular infection assays but are inactive against HIV-2. These compounds did not significantly inhibit the in vitro activities of several purified HIV-1 enzymes. Rather, they appeared to block virus infection at a postbinding, envelope-dependent step involved in the fusion of the virus to the cell membrane.
Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers.
Authors: Oswald E , Sugai M , Labigne A , Wu H C , Fiorentini C , Boquet P , O'Brien A D
Published: May 2006

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.
Expression of TrkA confers neuron-like responsiveness to nerve growth factor on an immortalized hypothalamic cell line.
Authors: Zhou J , Holtzman D M , Weiner R I , Mobley W C
Published: May 2006

The result of nerve growth factor (NGF) actions depends upon the cells in which it signals. To define how signaling is influenced by cellular context, it would be useful to examine cells committed to different fates or cells of a single type at different developmental stages. Interest in NGF actions on neurons of the central nervous system led us to examine GT1-1 cells, an immortalized hypothalamic cell line. GT1-1 cells demonstrated neuronal properties but were unresponsive to NGF and other neurotrophins. Through transfection, trkA expression conferred NGF signaling leading to enhanced neuronal differentiation, including dose-dependent induction of neurite outgrowth and a rapid transient increase in c-fos and NGFI-A mRNA. Under serum-free culture conditions, NGF also delayed cell death. These findings suggest that trkA transfection of neurons and neuronal precursors can be used to better define NGF signaling.
Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).
Authors: Ishiai M , Wada C , Kawasaki Y , Yura T
Published: May 2006

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.
Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A.
Authors: Price E R , Jin M , Lim D , Pati S , Walsh C T , McKeon F D
Published: May 2006

Cyclophilin B is targeted to the secretory pathway via an endoplasmic reticulum signal sequence. We analyzed the localization and trafficking of endogenous and transfected cyclophilin B in mammalian cells. Cyclophilin B accumulates both in the endoplasmic reticulum and in complexes on the plasma membrane. The immunosuppressant cyclosporin A specifically mobilizes cyclophilin B from the endoplasmic reticulum, and promotes the secretion of cyclophilin B into the medium. We suggest that cyclosporin A competes with endogenous plasma membrane proteins for association with cyclophilin B in the secretory pathway. These findings argue in favor of a role for cyclophilin B as a chaperone to proteins destined for the plasma membrane, rather than solely as a proline isomerase functioning within the endoplasmic reticulum.
The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity.
Authors: Skinner H B , McGee T P , McMaster C R , Fry M R , Bell R M , Bankaitis V A
Published: May 2006

The Saccharomyces cerevisiae protein SEC14p is required for Golgi function and cell viability in vivo. This requirement is obviated by mutations that specifically inactivate the CDP-choline pathway for phosphatidylcholine biosynthesis. The biochemical basis for the in vivo relationship between SEC14p function and the CDP-choline pathway has remained obscure. We now report that SEC14p effects an in vivo depression of CDP-choline pathway activity by inhibiting choline-phosphate cytidylyltransferase (CCTase; EC 2.7.7.15), the rate-determining enzyme of the CDP-choline pathway. Moreover, this SEC14p-mediated inhibition of CCTase was recapitulated in vitro and was saturable. Finally, whereas the SEC14p-dependent inhibition of CCTase in vitro was markedly reduced under assay conditions that were expected to increase levels of phosphatidylinositol-bound SEC14p, assay conditions expected to increase levels of phosphatidylcholine-bound SEC14p resulted in significant potentiation of CCTase inhibition. The collective data suggest that the phosphatidylcholine-bound form of SEC14p effects an essential repression of CDP-choline pathway activity in Golgi membranes by inhibiting CCTase and that the phospholipid-binding/exchange activity of SEC14p represents a mechanism by which the regulatory activity of SEC14p is itself controlled.
Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.
Authors: Wang Y K , Huff E J , Schwartz D C
Published: May 2006

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.
The chemistry of eavesdropping, alarm, and deceit.
Authors: Stowe M K , Turlings T C , Loughrin J H , Lewis W J , Tumlinson J H
Published: May 2006

Arthropods that prey on or parasitize other arthropods frequently employ those chemical cues that reliably indicate the presence of their prey or hosts. Eavesdropping on the sex pheromone signals emitted to attract mates allows many predators and parasitoids to find and attack adult insects. The sex pheromones are also useful signals for egg parasitoids since eggs are frequently deposited on nearby plants soon after mating. When the larval stages of insects or other arthropods are the targets, a different foraging strategy is employed. The larvae are often chemically inconspicuous, but when they feed on plants the injured plants respond by producing and releasing defensive chemicals. These plant chemicals may also serve as "alarm signals" that are exploited by predators and parasitoids to locate their victims. There is considerable evidence that the volatile "alarm signals" are induced by interactions of substances from the herbivore with the damaged plant tissue. A very different strategy is employed by several groups of spiders that remain stationary and send out chemical signals that attract prey. Some of these spiders prey exclusively on male moths. They attract the males by emitting chemicals identical to the sex pheromones emitted by female moths. These few examples indicate the diversity of foraging strategies of arthropod predators and parasitoids. It is likely that many other interesting chemically mediated interactions between arthropod hunters and their victims remain to be discovered. Increased understanding of these systems will enable us to capitalize on natural interactions to develop more ecologically sound, environmentally safe methods for biological control of insect pests of agriculture.
Chemistry of sex attraction.
Authors: Roelofs W L
Published: May 2006

The chemical communication system used to attract mates involves not only the overt chemical signals but also indirectly a great deal of chemistry in the emitter and receiver. As an example, in emitting female moths, this includes enzymes (and cofactors, mRNA, genes) of the pheromone biosynthetic pathways, hormones (and genes) involved in controlling pheromone production, receptors and second messengers for the hormones, and host plant cues that control release of the hormone. In receiving male moths, this includes the chemistry of pheromone transportation in antennal olfactory hairs (binding proteins and sensillar esterases) and the chemistry of signal transduction, which includes specific dendritic pheromone receptors and a rapid inositol triphosphate second messenger signal. A fluctuating plume structure is an integral part of the signal since the antennal receptors need intermittent stimulation to sustain upwind flight. Input from the hundreds of thousands of sensory cells is processed and integrated with other modalities in the central nervous system, but many unknown factors modulate the information before it is fed to motor neurons for behavioral responses. An unknown brain control center for pheromone perception is discussed relative to data from behavioral-threshold studies showing modulation by biogenic amines, such as octopamine and serotonin, from genetic studies on pheromone discrimination, and from behavioral and electrophysiological studies with behavioral antagonists.
Chemical signals in the marine environment: dispersal, detection, and temporal signal analysis.
Authors: Atema J
Published: May 2006

Chemical signals connect most of life's processes, including interorganismal relationships. Detection of chemical signals involves not only recognition of a spectrum of unique compounds or mixtures of compounds but also their spatial and temporal distribution. Both spectral and temporal signal processing determine what is a signal and what is background noise. Each animal extracts its unique information from the chemical world and uniquely contributes to it. Lobsters have provided important information on temporal signal processing. Marine chemical signals can be measured with high spatio-temporal resolution giving us a novel view of the lobster's environment. Lobster chemoreceptor cells have flicker fusion frequencies of 4 Hz and can integrate stimuli over 200 ms, closely corresponding to odor sampling behavior with 4-Hz "sniffs." Using this information, spatial odor gradients can be determined from temporal analysis of odor patches typical of turbulent dispersal. Lobsters appear to use this information to locate odor sources. Lobster social behavior depends greatly on chemical signals. Urine carries important information for courtship, dominance, and individual recognition. A novel gland in the nephropore is strategically located to release its products into the urine. Urine, in turn, is injected into the gill current, which jets water 1-2 m ahead of the animal. Lobsters control three different currents that carry chemical signals to and from them. The study of odor dynamics has only just begun. It will be exciting to see how signal dispersal, receptor temporal tuning, neural processing, and animal behavior interact to enhance signals for communication and detection and to reduce signals for chemical camouflage.
Induced oleoresin biosynthesis in grand fir as a defense against bark beetles.
Authors: Steele C L , Lewinsohn E , Croteau R
Published: May 2006

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated.