bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.
Authors: Loukas S , Mercouris M , Panetsos F , Zioudrou C
Published: May 2006

Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.
Ras signaling in the activation of glucose transport by insulin.
Authors: Manchester J , Kong X , Lowry O H , Lawrence J C
Published: May 2006

An approach involving microinjection and microanalysis has been developed to investigate signal-transduction pathways involved in the hormonal control of metabolism. We have applied this strategy to investigate the role of Ras signaling in the acute activation of glucose transport by insulin in cardiac myocytes. Glucose transport activity was assessed by measuring the initial rate of accumulation of 2-deoxyglucose 6-phosphate (dGlc6P) in individual cells after incubation in 2-deoxyglucose. Insulin increased accumulation of dGlc6P by 3- to 4-fold, consistent with its stimulatory effect on glucose transport. Accumulation of dGlc6P was increased severalfold by microinjecting the nonhydrolyzable GTP analogue, guanosine 5'-[gamma-thio]triphosphate, which activates members of the Ras superfamily of GTP-binding proteins. Injecting activated Ha-Ras protein also mimicked insulin by increasing dGlc6P; whereas, injecting a Ras protein lacking the COOH-terminal site of fatty acylation required for Ras function was without effect. Introducing the neutralizing Ras antibody Y13-259 into cells attenuated the effect of insulin. These findings implicate Ras in the acute regulation of metabolism by insulin.
Single-strand conformation polymorphism analysis coupled with stratified DNA sequencing reveals reduced sequence variation in the su(s) and su(wa) regions of the Drosophila melanogaster X chromosome.
Authors: Aguadé M , Meyers W , Long A D , Langley C H
Published: May 2006

Single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing of stratified sub-samples was used to survey DNA polymorphism in the su(s) and su(wa) regions in a natural population of Drosophila melanogaster. su(s) and su(wa) are located near the telomere of the X chromosome, where the rate of crossing over per kilobase of DNA monotonically decreases toward the tip. SSCP was assessed in 12 noncoding segments amplified from the su(s) region (3213 bp) and in 8 noncoding segments amplified from the su(wa) region (1955 bp). Sets of segments were multiplexed in a single electrophoretic lane to increase the number of base pairs assayed per lane. Eight segments were monomorphic, and the other 12 segments exhibited two to four SSCP classes. Only four within-SSCP-class DNA sequence differences (a single nucleotide substitution) were observed among 24,360 bp compared within classes. The between-SSCP-class DNA sequence comparisons revealed 27 substitutions and 9 insertion/deletion polymorphisms. The average numbers of substitutional differences per site were 0.0010 and 0.0021 for su(s) and su(wa), respectively. These values are intermediate between those reported for the more distal y-ASC region (0.0004) and the more proximal Pgd locus (0.0024). This observation is consistent with the prediction of the hitchhiking-effect model-i.e., a monotonic increase in polymorphism as a function of crossing over per kilobase.
N- and P-type Ca2+ channels are involved in acetylcholine release at a neuroneuronal synapse: only the N-type channel is the target of neuromodulators.
Authors: Fossier P , Baux G , Tauc L
Published: May 2006

Cholinergic transmission in an identified neuro-neuronal synapse of the Aplysia buccal ganglion was depressed by application of a partially purified extract of the funnel-web-spider venom (FTx) or of its synthetic analog (sFTx). This specific blocker of voltage-dependent P-type Ca2+ channels did not interfere with the effect of the N-type Ca2+ channel blocker omega-conotoxin, which could further decrease synaptic transmission after a previous application of FTx. Similar results were obtained when the reversal order of application of these two Ca2+ channel blockers was used. Both P- and N-type Ca2+ currents trigger acetylcholine release in the presynaptic neuron. The neuromodulatory effects of FMRF-amide, histamine, and buccalin on transmitter release disappeared after the blockade of the N-type Ca2+ channels but remained still effective in the presence of FTx. These results indicate that only N-type Ca2+ channels appear to be sensitive to the neuromodulators we have identified.
Inversion of the Bohr effect upon oxygen binding to 24-meric tarantula hemocyanin.
Authors: Sterner R , Decker H
Published: May 2006

The Bohr effect describes the usually negative coupling between the binding of oxygen and the binding of protons to respiratory proteins. It was first described for hemoglobin and provides for an optimal oxygen supply of the organism under changing physiological conditions. Our measurements of both oxygen and proton binding to the 24-meric tarantula hemocyanin establish the unusual case where a respiratory protein binds protons at low degrees of oxygenation but releases protons at high degrees of oxygenation. In contrast to what is observed with hemoglobin and other respiratory proteins, this phenomenon amounts to the inversion of the Bohr effect in the course of an oxygen-binding curve at a given pH value. Therefore, protons in spider blood can act either as allosteric activators or as allosteric inhibitors of oxygen binding, depending on the degree of oxygenation of hemocyanin. These functional properties of tarantula hemocyanin, which cannot be explained by classical allosteric models, require at least four different conformational states of the subunits. Inspection of the known x-ray structures of closely related hemocyanins suggests that salt bridges between completely conserved histidine and glutamate residues located at particular intersubunit interfaces are responsible for the observed phenomena.
Tumor necrosis factor alpha inhibits signaling from the insulin receptor.
Authors: Hotamisligil G S , Murray D L , Choy L N , Spiegelman B M
Published: May 2006

Insulin resistance is a common problem associated with infections and cancer and, most importantly, is the central component of non-insulin-dependent diabetes mellitus. We have recently shown that tumor necrosis factor (TNF) alpha is a key mediator of insulin resistance in animal models of non-insulin-dependent diabetes mellitus. Here, we investigate how TNF-alpha interferes with insulin action. Chronic exposure of adipocytes to low concentrations of TNF-alpha strongly inhibits insulin-stimulated glucose uptake. Concurrently, TNF-alpha treatment causes a moderate decrease in the insulin-stimulated autophosphorylation of the insulin receptor (IR) and a dramatic decrease in the phosphorylation of IR substrate 1, the major substrate of the IR in vivo. The IR isolated from TNF-alpha-treated cells is also defective in the ability to autophosphorylate and phosphorylate IR substrate 1 in vitro. These results show that TNF-alpha directly interferes with the signaling of insulin through its receptor and consequently blocks biological actions of insulin.
Allele-specific parental imprinting of dzr1, a posttranscriptional regulator of zein accumulation.
Authors: Chaudhuri S , Messing J
Published: May 2006

Parental imprinting describes the phenomenon of unequivalent gene function based on transmission from the female or male parent. We have discovered parental imprinting of an allele of the dzr1 locus that posttranscriptionally regulates the accumulation of 10-kDa zein in the maize endosperm. The imprinted allele of MO17 inbred origin, dzr1 + MO17, conditions low accumulation of the 10-kDa zein and is dominant when transmitted through the female but recessive when transmitted through the male. Analyzing endosperms with equal parental contributions of dzr1 + MO17 ruled out the possibility that the unequivalent phenotype of dzr1 + MO17 was due to parental dosage imbalance in the triploid endosperm. Second-generation studies show that the dominant or recessive phenotype of dzr1 + MO17 is determined at every generation based on immediate parental origin with no grandparental effect.
Stable intrachain and interchain complexes of neurofilament peptides: a putative link between Al3+ and Alzheimer disease.
Authors: Hollósi M , Shen Z M , Perczel A , Fasman G D
Published: May 2006

The etiologic role of Al3+ in Alzheimer disease has been controversial. Circular dichroism (CD) spectroscopic studies on two synthetic fragments of human neurofilament protein mid-sized subunit (NF-M), NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), and their alanine-substituted and/or serine-phosphorylated derivatives were carried out in an attempt to find a molecular mechanism for the effect of Al3+ to induce aggregation of neuronal proteins or their catabolic fragments. Al3+ and Ca2+ ions were found to induce beta-pleated sheet formation in the phosphorylated fragments. The cation sensitivity depended on the length and charge distribution of the sequence and site of phosphorylation. Al3+-induced conformational changes were irreversible to citric acid chelation, whereas Ca(2+)-induced conformational changes were reversible with citric acid. Studies of the alanine derivatives demonstrated which residues affected Al3+ or Ca2+ binding. Peptides containing at least one free (nonphosphorylated) serine residue were shown to form an intramolecular Al3+ complex, rather than an intermolecular one. In the intramolecular (intrachain) complex, the ligand function of the deprotonated serine hydroxyl was delineated [(Al.pepH-1)-type complex]. Ca2+ ions did not show a tendency for intramolecular complexing. The potential role of Al3+ in Alzheimer disease tangle and plaque formation is strongly suggested.
Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.
Authors: Blumer K J , Johnson G L , Lange-Carter C A
Published: May 2006

Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.
Platelet-activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene.
Authors: Bazan N G , Fletcher B S , Herschman H R , Mukherjee P K
Published: May 2006

Platelet-activating factor (PAF), a potent lipid mediator generated in cell injury and in the inflammatory and immune responses, promotes transcriptional activation of several primary response genes. TIS10/PGS-2 is a primary response gene encoding the inducible form of prostaglandin synthase. The inductive effects of PAF and retinoic acid (RA), alone and in combination, were studied with the regulatory region of TIS10/PGS-2 transfected into an exponentially growing glioblastoma-neuroblastoma NG108-15 hybrid in the human SH-SY5Y neuroblastoma or in the NIH 3T3 cell. RA alone exhibited only a small inductive effect. However, in the presence of RA (100 nM), a PAF-dependent (1-50 nM) synergistic activation of luciferase reporter constructs driven by regulatory regions of the TIS10/PGS-2 gene was found. The hetrazepine BN-50730, an antagonist selective for intracellular PAF binding sites, inhibited PAF and RA induction of luciferase from the TIS10/PGS-2 promoter. Thus, the intracellular PAF binding site is involved in TIS10/PGS-2 expression. Induction is rapid, suggesting that the combination of PAF and RA activates a preexisting latent transcription factor(s). Deletion studies restrict the major PAF and RA cis-acting response element of the TIS10/PGS-2 gene to a 70-nucleotide sequence as an intracellular inducer of TIS10/PGS-2 expression. The synergistic effect of RA and PAF represents an unusual convergence of nuclear signaling pathways by which, through the modulation of preexisting transcription factors, specific gene expression can be upregulated. PAF-dependent induction of TIS10/PGS-2 expression may play a role in cell injury, differentiation, inflammation, and immune responses.
p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation.
Authors: Couture C , Baier G , Altman A , Mustelin T
Published: May 2006

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.
Astrocytosis and axonal proliferation in the hippocampus of S100b transgenic mice.
Authors: Reeves R H , Yao J , Crowley M R , Buck S , Zhang X , Yarowsky P , Gearhart J D , Hilt D C
Published: May 2006

S100 beta is a calcium-binding protein that is expressed at high levels in brain primarily by astrocytes. Addition of the disulfide-bonded dimeric form of S100 beta to primary neuronal and glial cultures and established cell lines induces axonal extension and alterations in astrocyte proliferation and phenotype, but evidence that S100 beta exerts the same effects in vivo has not been presented. An 8.9-kb murine S100b genomic clone was used to produce two lines of transgenic mice in which S100 beta RNA is increased in a dose-related manner to 2-fold and 7-fold above normal. These lines show concomitant increased S100 beta protein throughout the brain. Expression in both lines is cell type- and tissue-appropriate, and expression levels are correlated with the transgene copy number, demonstrating that sequences necessary for normal regulation of the gene are included within the cloned segment. In the hippocampus of adult transgenic mice, Western blotting detects elevated levels of glial fibrillary acidic protein and several markers of axonal sprouting, including neurofilament L, phosphorylated epitopes of neurofilament H and M, and beta-tubulin. Immunocytochemistry demonstrates alterations in astrocyte morphology and axonal sprouting, especially in the dentate gyrus. Thus, both astrocytosis and neurite proliferation occur in transgenic mice expressing elevated levels of S100 beta. These transgenic mice provide a useful model for studies of the role of S100 beta in glial-neuronal interactions in normal development and function of the brain and for analyzing the significance of elevated levels of S100 beta in Down syndrome and Alzheimer disease.
The cytoplasmic domain of the Na+/H+ exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHE1/trout beta NHE antiporter.
Authors: Borgese F , Malapert M , Fievet B , Pouyssegur J , Motais R
Published: May 2006

Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) are difficult to interpret as variable results have been reported for the different isoforms when expressed in various cell types. We took advantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the molecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate the critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced by the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, is essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decreased by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP-mediated activation by 72%. The residual activation required a cytoplasmic fragment (aa 559-661) that contains four sequences considered likely as putative PKA consensus sites. The results obtained with the chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exchange so induced can be modulated by the nature of the interaction between the N- and C-terminal domains.
Cell fates in leech embryos with duplicated lineages.
Authors: Lans D , Ho R K , Weisblat D A
Published: May 2006

We have examined the fates of the progeny of supernumerary embryonic stem cells (O/P teloblasts) generated by microinjecting polyadenylic acid into newborn O/P teloblasts in embryos of the leech, Helobdella triserialis. In normal development, each O/P teloblast generates a rostrocaudal column of daughter cells (primary blast cells) that contribute distinct segmentally iterated O or P sets of epidermal and neural progeny to the mature leech. Previous results suggest that primary blast cells derived from ipsilateral pairs of O/P teloblasts are equipotent and equivalent at birth; that they and their progeny assume distinct O or P fates according to hierarchical and position-dependent interactions; and that the P fate is the primary, or default, fate and the O fate is the secondary fate. In the work presented here, one O/P teloblast was experimentally induced to undergo a supernumerary equal division, and the developmental fates of the progeny of the three (two "duplicate" and one "nonduplicate") ipsilateral O/P teloblasts were determined at stages 8 and 10. We find that some supernumerary O/P teloblasts produce supernumerary P progeny, whereas others generate supernumerary O progeny. When three O/P-derived bandlets are present, bandlets derived from the duplicate O/P teloblasts give rise to progeny of the same (O or P) fate. When the nonduplicate bandlet is absent, the duplicate bandlets assume distinct O and P fates. These results suggest that ipsilateral sister O/P teloblasts, while equipotent, might not be equivalent.
Three tumor-suppressor regions on chromosome 11p identified by high-resolution deletion mapping in human non-small-cell lung cancer.
Authors: Bepler G , Garcia-Blanco M A
Published: May 2006

Non-small-cell lung cancer is the leading cause of cancer death for men and women in the industrialized nations. Identification of regions for genes involved in its pathogenesis has been difficult. Data presented here show three distinct regions identified on chromosome 11p. Two regions on 11p13 distal to the Wilms tumor gene WT1 and on 11p15.5 between the markers HBB and D11S860 are described. The third region on the telomere of 11p15.5 has been previously described and is further delineated in this communication. By high-resolution mapping the size of each of these regions was estimated to be 2-3 megabases. The frequency of somatic loss of genetic information in these regions (57%, 71%, and 45%, respectively) was comparable to that seen in heritable tumors such as Wilms tumor (55%) and retinoblastoma (70%) and suggests their involvement in pathogenesis of non-small-cell lung cancer. Gene dosage analyses revealed duplication of the remaining allele in the majority of cases in the 11p13 and the proximal 11p15.5 region but rarely in the distal 11p15.5 region. In tumors with loss of heterozygosity in all three regions any combination of duplication or simple deletion was observed, suggesting that loss of heterozygosity occurs independently and perhaps at different points in time. These results provide a basis for studies directed at cloning potential tumor-suppressor genes in these regions and for assessing their biological and clinical significance in non-small-cell lung cancer.
Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.
Authors: Quon M J , Guerre-Millo M , Zarnowski M J , Butte A J , Em M , Cushman S W , Taylor S I
Published: May 2006

Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.
Dynamic instability of microtubules as an efficient way to search in space.
Authors: Holy T E , Leibler S
Published: May 2006

During the formation of the mitotic spindle, dynamic microtubules search for chromosomes in the surrounding cytosol. The average time required to reach such "targets" is calculated within a simple model of dynamic instability. The values of the dynamic parameters that minimize this search time are also obtained. The results approximate well observations of the capture of chromosomes in prometaphase cells. Compared to usual reversible polymerization, dynamic instability reduces the time required to find a target by several orders of magnitude.
Different event-related patterns of gamma-band power in brain waves of fast- and slow-reacting subjects.
Authors: Jokeit H , Makeig S
Published: May 2006

Fast- and slow-reacting subjects exhibit different patterns of gamma-band electroencephalogram (EEG) activity when responding as quickly as possible to auditory stimuli. This result appears to confirm long-standing speculations of Wundt that fast- and slow-reacting subjects produce speeded reactions in different ways and demonstrates that analysis of event-related changes in the amplitude of EEG activity recorded from the human scalp can reveal information about event-related brain processes unavailable using event-related potential measures. Time-varying spectral power in a selected (35- to 43-Hz) gamma frequency band was averaged across trials in two experimental conditions: passive listening and speeded reacting to binaural clicks, forming 40-Hz event-related spectral responses. Factor analysis of between-subject event-related spectral response differences split subjects into two near-equal groups composed of faster- and slower-reacting subjects. In faster-reacting subjects, 40-Hz power peaked near 200 ms and 400 ms poststimulus in the react condition, whereas in slower-reacting subjects, 40-Hz power just before stimulus delivery was larger in the react condition. These group differences were preserved in separate averages of relatively long and short reaction-time epochs for each group. gamma-band (20-60 Hz)-filtered event-related potential response averages did not differ between the two groups or conditions. Because of this and because gamma-band power in the auditory event-related potential is small compared with the EEG, the observed event-related spectral response features must represent gamma-band EEG activity reliably induced by, but not phase-locked to, experimental stimuli or events.
Fatty acid synthesis: a potential selective target for antineoplastic therapy.
Authors: Kuhajda F P , Jenner K , Wood F D , Hennigar R A , Jacobs L B , Dick J D , Pasternack G R
Published: May 2006

OA-519 is a prognostic molecule found in tumor cells from breast cancer patients with markedly worsened prognosis. We purified OA-519 from human breast carcinoma cells, obtained its peptide sequence, and unambiguously identified it as fatty acid synthase through sequence homology and enzymology. Tumor fatty acid synthase is an approximately 270-kDa polypeptide which specifically abolished immunostaining of human breast cancers by anti-OA-519 antibodies. Tumor fatty acid synthase oxidized NADPH in a malonyl-CoA-dependent fashion and synthesized fatty acids composed of 80% palmitate, 10% myristate, and 10% stearate from acetyl-CoA, malonyl-CoA, and NADPH with a specific activity of 624 nmol of NADPH oxidized per min per mg. Tumor cell lines with elevated fatty acid synthase showed commensurate increases in incorporation of [U-14C]acetate into acylglycerols demonstrating that fatty acid synthase increases occur in the context of overall increases in endogenous fatty acid synthesis. Cerulenin inhibited acylglycerol synthesis in tumor cells and fibroblast controls in a dose-dependent fashion and also caused a growth inhibition which generally paralleled the level of endogenous fatty acid synthesis. Supraphysiologic levels of palmitate, 14 microM in dimethyl sulfoxide, significantly reversed the growth inhibition caused by cerulenin at concentrations of up to 5 micrograms/ml, indicating that cerulenin-mediated growth inhibition was due to fatty acid synthase inhibition.
Sak, a murine protein-serine/threonine kinase that is related to the Drosophila polo kinase and involved in cell proliferation.
Authors: Fode C , Motro B , Yousefi S , Heffernan M , Dennis J W
Published: May 2006

We have isolated murine cDNAs encoding two isoforms of a putative protein-serine/threonine kinase, designated Sak-a and Sak-b, which differ in their noncatalytic C-terminal ends. The kinase domain of Sak is related to the catalytic domains of the Drosophila polo, Saccharomyces cerevisiae CDC5, and murine Snk and Plk kinases, a family of proteins for which a role in controlling cell proliferation has been established (polo, CDC5) or implicated (Snk, Plk). Northern and in situ RNA analyses of Sak gene expression in mouse embryos and adult tissues revealed that expression was associated with mitotic and meiotic cell division. In addition, during embryogenesis, Sak expression was prominent in the respiratory and olfactory mucosa. The pattern of Sak expression and its sequence homology with the polo gene family suggest that the Sak kinase may play a role in cell proliferation. In support of this, cell growth was suppressed by expression of a Sak-a-antisense fragment in CHO cells.
Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I.
Authors: McPherson P S , Czernik A J , Chilcote T J , Onofri F , Benfenati F , Greengard P , Schlessinger J , De Camilli P
Published: May 2006

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (through its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions. These proteins interact with wild-type Grb2 but not with Grb2 containing point mutations in each of its two SH3 domains corresponding to the loss of function mutants in the Caenorhabditis elegans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were previously shown to bind Grb2. The third protein of 145 kDa is brain specific and to our knowledge has not been previously described. The fourth protein is synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I is thought to mediate the interaction of synaptic vesicles with the presynaptic cytomatrix. These data suggest that Grb2, or other proteins containing SH3 domains, may play a role in the regulation of the exo/endocytotic cycle of synaptic vesicles and therefore of neurotransmitter release.
Peptide segment ligation strategy without use of protecting groups.
Authors: Liu C F , Tam J P
Published: May 2006

We describe the concept and the verification of a chemical ligation approach to the synthesis of proteins using peptide segments with no protecting groups and no activation of the C-terminal alpha-carboxyl group. This approach consists of three steps: (i) aldehyde introduction, in which a masked glycolaldehyde ester is linked to the carboxyl terminus of an unprotected peptide by reverse proteolysis; (ii) ring formation, in which the unmasked aldehyde reacts with the N-terminal alpha-amino group of the second unprotected peptide containing either a cysteine or a threonine residue to form a thiazolidine or oxazolidine ring at an acidic pH; and (iii) rearrangement in which O-acyl ester linkage is transferred to N-acyl amide linkage to form a peptide bond with a pseudoproline structure at higher pH. The feasibility of this scheme was verified by a model study on small compounds and its potential was demonstrated by the synthesis of a 50-residue epidermal growth factor-like peptide containing a preformed disulfide bond.
Bloom syndrome: an analysis of consanguineous families assigns the locus mutated to chromosome band 15q26.1.
Authors: German J , Roe A M , Leppert M F , Ellis N A
Published: May 2006

By the principle of identity by descent, parental consanguinity in individuals with rare recessively transmitted disorders dictates homozygosity not just at the mutated disease-associated locus but also at sequences that flank that locus closely. In 25 of 26 individuals with Bloom syndrome examined whose parents were related, a polymorphic tetranucleotide repeat in an intron of the protooncogene FES was homozygous, far more often than expected (P < 0.0001 by chi 2). Therefore, BLM, the gene that when mutated gives rise to Bloom syndrome, is tightly linked to FES, a gene whose chromosome position is known to be 15q26.1. This successful approach to the assignment of the Bloom syndrome locus to one short segment of the human genome simultaneously (i) demonstrates the power of homozygosity mapping and (ii) becomes the first step in a "reverse" genetics definition of the primary defect in Bloom syndrome.
The absence of IgE antibody-mediated augmentation of immune responses in CD23-deficient mice.
Authors: Fujiwara H , Kikutani H , Suematsu S , Naka T , Yoshida K , Yoshida K , Tanaka T , Suemura M , Matsumoto N , Kojima S
Published: May 2006

The CD23 antigen, a low-affinity receptor for IgE (Fc epsilon RII), is a type II membrane-bound glycoprotein expressed on various cells, particularly mature B cells. A number of functions have been ascribed to CD23, including specific regulation of IgE production, IgE-mediated cytotoxicity and release of mediators, IgE-dependent antigen focusing, promotion of B-cell growth, prevention of germinal center B cells from apoptosis, proliferation of myeloid precursors, and maturation of early thymocytes. It is not clear whether these activities represent in vivo functions. To explore in vivo functions of CD23, we have produced CD23-deficient mice. These mice displayed normal lymphocyte differentiation and could mount normal antibody responses, including IgE responses upon immunization with T-dependent antigens and infection with Nippostrongyrus brasiliensis. Germinal center formation after immunization and in vitro proliferative response of B cells were not affected in mutant mice. However, antigen-specific IgE-mediated enhancement of antibody responses was severely impaired.
Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ thymocytes into mature T cells.
Authors: Benveniste P , Takahama Y , Wiest D L , Nakayama T , Sharrow S O , Singer A
Published: May 2006

Immature precursor cells are induced in the thymus to express clonotypic T-cell antigen receptors (TCRs) and to differentiate into mature T cells. Perhaps the least understood event which occurs during intrathymic development is the positive selection of immature CD4+CD8+ thymocytes for differentiation into mature CD4+ and CD8+ T cells based on the TCR specificity individual thymocytes express. TCR expression by CD4+CD8+ thymocytes is quantitatively regulated by CD4-mediated activation of p56lck protein-tyrosine kinase whose activity can in turn be regulated by the membrane-bound protein-tyrosine-phosphatase CD45. Here we show that antibody engagement of CD45 external domains enhances Lck tyrosine kinase activity in CD4+CD8+ thymocytes, inhibits TCR expression, and inhibits differentiation of immature CD4+CD8+ thymocytes into mature T cells. Thus, engagement of the external domains of CD45 tyrosine phosphatase can regulate the ability of immature CD4+CD8+ thymocytes to undergo positive selection, suggesting an important regulatory role for intrathymic ligands that are capable of engaging CD45 within the thymus.
Relief of opsin desensitization and prolonged excitation of rod photoreceptors by 9-desmethylretinal.
Authors: Corson D W , Cornwall M C , MacNichol E F , Tsang S , Derguini F , Crouch R K , Nakanishi K
Published: May 2006

The 9-methyl group of 11-cis-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretinal, combines with opsin to form a pigment that produces abnormal photoproducts and diminished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by approximately 7.5-fold to 0.15 +/- 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 +/- 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is approximately 30-fold smaller and decays approximately 5 times more slowly than that of native pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.
Tumor necrosis factor alpha is an autocrine growth factor for normal human B cells.
Authors: Boussiotis V A , Nadler L M , Strominger J L , Goldfeld A E
Published: May 2006

Transcription of the human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest events that occurs after stimulation of B or T cells via their antigen receptors. Antibody directed at surface immunoglobulin (anti-Ig) on B cells has previously been shown to induce a rapid burst of TNF-alpha gene transcription, which can be blocked by the immunosuppressants cyclosporin A (CsA) and FK506. Here, TNF-alpha gene transcription is shown also to be highly and rapidly induced in human B cells after stimulation via the CD40 and interleukin 4 pathways, which similarly is inhibited by CsA and a panel of CsA or FK506 analogues that block calcineurin phosphatase activity. Endogenous TNF-alpha produced after stimulation was involved in B-cell proliferation since anti-TNF-alpha monoclonal antibody inhibited both anti-Ig- and anti-CD40-induced B-cell proliferative responses. Moreover, addition of TNF-alpha during stimulation resulted in augmentation of B-cell proliferation, which was also inhibited by anti-TNF-alpha monoclonal antibody. Although lymphotoxin alpha (LT-alpha) mRNA is induced by both pathways, it is not blocked by CsA, whereas LT-beta mRNA is constitutively expressed in B cells. Thus, TNF-alpha is a necessary autocrine growth factor for human B cells stimulated via two independent CsA-sensitive pathways and plays a role similar to that of interleukin 2 in T-cell proliferation. The autocrine nature of TNF-alpha in activated B cells implies a potential role for this cytokine in infection-related polyclonal B-cell expansion and in B-cell malignancies.
Nuclear localization signals also mediate the outward movement of proteins from the nucleus.
Authors: Guiochon-Mantel A , Delabre K , Lescop P , Milgrom E
Published: May 2006

Several nuclear proteins, including steroid hormone receptors, have been shown to shuttle continuously between the nucleus and the cytoplasm. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted the nuclear localization signals of the progesterone receptor or the simian virus 40 large tumor antigen onto beta-galactosidase. These additions were shown to impart to the protein the ability to shuttle between the nucleus and the cytoplasm. Microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The same nuclear localization signals are thus involved in both the inward and the outward movement of proteins through the nuclear membrane. We also show that although the nuclear import requires energy, the nuclear export does not. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally.
CD8+ T lymphocytes of African green monkeys secrete an immunodeficiency virus-suppressing lymphokine.
Authors: Ennen J , Findeklee H , Dittmar M T , Norley S , Ernst M , Kurth R
Published: May 2006

Following natural and experimental infection by simian immunodeficiency virus SIVagm of African green monkeys (AGMs), the natural host, there is no evidence for the development of an immunodeficiency. Within the framework of our studies on human immunodeficiency virus (HIV)/SIV pathogenesis, we investigated the influence of CD8 T lymphocytes on SIVagm replication in AGM CD4 T lymphocytes in vitro. The following observations were made: (i) Peripheral blood mononuclear cells from both seronegative and seropositive AGMs contained only a low proportion (i.e., 10%) of CD4+ lymphocytes, whereas a high proportion (80%) of CD8+ cells was detected. Even after persistent SIVagm infection, CD4 T lymphocytes do not decrease in number. (ii) The target of in vitro infection of peripheral blood cells is the CD4+ mononuclear cell (T lymphocytes, monocytes) and SIVagm infects by binding to the CD4 molecule. (iii) In both naturally and experimentally SIVagm-infected AGMs the CD4+ T cells and monocytes, but not the CD8+ T cells, harbor DNA provirus. (iv) Virus reisolation and virus replication of SIVagm in CD4 T lymphocytes from seropositive AGMs is suppressed in the presence of autologous CD8 T lymphocytes or a soluble factor produced by these cells. Taken together, one possible reason for the apathogenicity of the SIVagm infection in AGMs may be the suppression of virus replication by a soluble, yet unidentified factor secreted by CD8 lymphocytes quantitatively dominating among peripheral blood cell populations. We have tentatively termed this factor "immunodeficiency virus-suppressing lymphokine." In addition, we show that immunodeficiency virus-suppressing lymphokine from AGMs is able to suppress HIV-1 replication in human CD4+ T cells.
Genetic variability for stomatal conductance in Pima cotton and its relation to improvements of heat adaptation.
Authors: Radin J W , Lu Z , Percy R G , Zeiger E
Published: May 2006

Responses of stomata to environment have been intensively studied, but little is known of genetic effects on stomatal conductance or their consequences. In Pima cotton (Gossypium barbadense L.), a crop that is bred for irrigated production in very hot environments, stomatal conductance varies genetically over a wide range and has increased with each release of new higher-yielding cultivars. A cross between heat-adapted (high-yielding) and unadapted genotypes produced F2 progeny cosegregating for stomatal conductance and leaf temperature. Within segregating populations in the field, conductance was negatively correlated with foliar temperature because of evaporative cooling. Plants were selected from the F2 generation specifically and solely for differing stomatal conductance. Among F3 and F4 populations derived from these selections, conductance and leaf cooling were significantly correlated with fruiting prolificacy during the hottest period of the year and with yield. Conductance was not associated with other factors that might have affected yield potential (single-leaf photosynthetic rate, leaf water potential). As breeders have increased the yield of this crop, genetic variability for conductance has allowed inadvertent selection for "heat avoidance" (evaporative cooling) in a hot environment.
The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.
Authors: Black S M , Harikrishna J A , Szklarz G D , Miller W L
Published: May 2006

Steroidogenesis is initiated by the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc [cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving); EC 1.14.15.6]. Several subsequent steroidal conversions occur in the endoplasmic reticulum (ER), but the last step in the production of glucocorticoids and mineralocorticoids again occurs in the mitochondria. Although cellular compartmentalization of steroidogenic enzymes appears to be a feature of all steroidogenic pathways, some reports indicate that cholesterol can be converted to pregnenolone outside the mitochondria. To investigate whether P450scc can function outside the mitochondria, we constructed vectors producing P450scc and various fusion enzymes of P450scc with electron-transport proteins and directed their expression to either the ER or the mitochondria. Whether targeted to mitochondria or to the ER, plasmid vectors encoding P450scc and fusion proteins of P450scc with either mitochondrial or microsomal electron-transport proteins produced immunodetectable protein. When expressed in mitochondria, all of these constructions converted 22-hydroxycholesterol to pregnenolone, but when expressed in the ER none of them produced pregnenolone. These results show that P450scc can function only in the mitochondria. Furthermore, it appears to be the mitochondrial environment that is required, rather than the specific mitochondrial electron-transport intermediates.
Hippocampal growth and attrition in birds affected by experience.
Authors: Clayton N S , Krebs J R
Published: May 2006

Hand-raised marsh tits (Parus palustris) were exposed to experience of storing and retrieving food at three different ages (35-59, 60-83, 115-138 days posthatch). At equivalent ages, control birds were given identical experience except for storing and retrieving food. Volumetric analysis was carried out to measure the hippocampal region, ectostriatum, and telencephalon of experienced and control birds. Individuals with experience of storing and retrieving food had a larger hippocampal region relative to the rest of the telencephalon than did controls, independent of age. The hippocampal region of experienced birds also contained more neurons and fewer apoptotic cells than that of controls. No volumetric differences were observed in ectostriatum, which served as a control brain region. The results suggest that some aspect of food-storing and retrieval directly influences growth and attrition of the hippocampal region in food-storing birds.