bluuebunny/crossref_metadata_2025 huggingface dataset:
Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.
Authors: Dvir A , Peterson S R , Knuth M W , Lu H , Dynan W S
Published: May 2006

The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.
Gene-culture coevolution: toward a general theory of vertical transmission.
Authors: Feldman M W , Zhivotovsky L A
Published: May 2006

A general formulation of cultural and genetic transmission is developed. The cultural transmission is vertical and the genetics may involve multiple loci. Each individual is represented by a phenogenotype, and conditions are given under which the evolutionary dynamics of phenogenotype frequencies are reducible to phenogametic or phenoallelic frequencies. The interaction between genes and culture is specified by an association measure, and results on the order of magnitude of this association at equilibrium are presented.
Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.
Authors: Venkitaraman A R , Cowling R J
Published: May 2006

Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-dependent stimulation of the enzyme phosphatidylinositol 3-kinase, a tyrosine kinase substrate, provides further evidence suggestive of p59fyn activation. We demonstrate that p59fyn forms part of a protein complex with the IL-7R. A chimeric receptor comprising the CD8 extracellular domain and the IL-7R cytoplasmic tail (CD8/IL-7R) recruits tyrosine kinase activity in transfected myeloma cells, and p59fyn can be detected in association with it by immunoprecipitation and immunoblotting. Conversely, p59fyn immunoprecipitates contain the phosphorylated CD8/IL-7R. We have identified a segment of the IL-7R cytoplasmic tail which mediates p59fyn recruitment: a truncated CD8/IL-7R containing only this segment recruits tyrosine kinase activity, associates with p59fyn, and activates phosphatidylinositol 3-kinase. Interestingly, this segment contains no tyrosine residues, although it is the phosphotyrosine-binding src homology domains of p59fyn and phosphatidylinositol 3-kinase which mediate their association with many growth factor receptors. Thus our results suggest that an unusual interaction links IL-7R to these two important signaling pathways.
Dopamine enhances both electrotonic coupling and chemical excitatory postsynaptic potentials at mixed synapses.
Authors: Pereda A , Triller A , Korn H , Faber D S
Published: May 2006

The transmitter dopamine reduces electrotonic coupling between retinal horizontal cells and increases their sensitivity to glutamate. Since in other systems single afferents establish mixed electrotonic and chemical excitatory synapses with their targets, dopamine might be expected there to depress one component of excitation while enhancing the other. This hypothesis was tested by applying dopamine locally in the vicinity of the lateral dendrite of the goldfish Mauthner cell (M cell) and monitoring the composite electrotonic and chemical excitatory postsynaptic potentials and currents evoked by ipsilateral eighth nerve stimulation. Dopamine produces persistent enhancements of both components of the postsynaptic response while it also increases input conductance. All these dopamine actions are prevented by superfusing the brain with saline containing the dopamine D1 receptor antagonist SCH-23390. Postsynaptic injections of the cAMP-dependent protein kinase inhibitor (Walsh inhibitor, or PKI5-24) block the dopamine-induced changes in synaptic transmission, implicating a cAMP-dependent mechanism. Furthermore, there is a dopaminergic innervation of the M cell, as demonstrated immunohistochemically with antibodies against dopamine and the rate-limiting enzyme in its synthetic pathway, tyrosine hydroxylase. Varicose immunoreactive fibers lie in the vicinity of the distal part of the lateral dendrite between the large myelinated club endings that establish the mixed synapses. As determined with electron microscopy, the dopaminergic fibers contain small vesicles, and they do not have synaptic contacts with either the afferents or the M cell, remaining instead in the synaptic bed. Taken together, these results suggest that dopamine released at a distance from these terminals increases the gain of this primary sensory input to the M cell, most likely through a phosphorylation mechanism.
Empty and peptide-containing conformers of class I major histocompatibility complex molecules expressed in Drosophila melanogaster cells.
Authors: Jackson M R , Song E S , Yang Y , Peterson P A
Published: May 2006

Transfected Drosophila melanogaster cells can express large quantities of class I major histocompatibility complex molecules. Such molecules lack endogenous peptides because the Drosophila cells are devoid of proteins necessary for intracellular peptide loading. The empty molecules are efficiently expressed on the cell surface and can acquire extracellular peptides. The conformation and stability of empty murine class I molecules are determined by the source of beta 2-microglobulin. All beta 2-microglobulin-induced conformers of empty heavy chains seem to be unified in a common rigid conformation on peptide binding.
Overexpression of an Aplysia shaker K+ channel gene modifies the electrical properties and synaptic efficacy of identified Aplysia neurons.
Authors: Kaang B K , Pfaffinger P J , Grant S G , Kandel E R , Furukawa Y
Published: May 2006

Although potassium channels play a variety of roles in shaping the electrical properties of neurons, little is known about how these channels are constituted in neurons. To examine the assembly and physiological function of A-type K+ channels in mature differentiated neurons, we have developed a highly efficient gene transfer method for Aplysia neurons that has allowed us to express about 10(7) copies of the cloned Aplysia Shaker (Sh) K+ channel (AK01a) in single identified cells. We find that expression of AK01a phenocopies one of the native transient K+ currents (IAdepol), suggesting that the native channel carrying IAdepol is assembled as a homooligomer of AK01a. Overexpression of AK01a has substantial effect on the action potential, shortening its duration, enhancing its hyperpolarizing afterpotential, and depressing by more than half the amount of transmitter release by the action potential from the terminals. Thus, the AK01a channel not only contributes to the firing properties within a given neuron but also can regulate the signaling between interconnected cells.
Industrial ecology: concepts and approaches.
Authors: Jelinski L W , Graedel T E , Laudise R A , McCall D W , Patel C K
Published: May 2006

Industrial ecology is a new approach to the industrial design of products and processes and the implementation of sustainable manufacturing strategies. It is a concept in which an industrial system is viewed not in isolation from its surrounding systems but in concert with them. Industrial ecology seeks to optimize the total materials cycle from virgin material to finished material, to component, to product, to waste product, and to ultimate disposal. To better characterize the topic, the National Academy of Sciences convened a colloquium from which were derived a number of salient contributions. This paper sets the stage for the contributions that follow and discusses how each fits into the framework of industrial ecology.
Efficient energy use in manufacturing.
Authors: Ross M
Published: May 2006

The potential for improved industrial efficiency is great and a substantial part of that potential is being realized in the course of events. With new technology and increasing affluence, the composition of production is changing such that energy and materials consumption is growing more slowly than the economy. Through new technologies and appropriate public policies, the energy intensities of all production processes should also continue to decline. Potential difficulties facing this relatively rosy scenario are also discussed.
Materials derived from biomass/biodegradable materials.
Authors: Luzier W D
Published: May 2006

Interest in biodegradable plastics made from renewable resources has increased significantly in recent years. PHBV (polyhydroxybutyrate-polyhydroxyvalerate) copolymers are good examples of this type of materials. This paper provides an overview of the manufacturing process, properties, biodegradability, and application/commercial issues associated with PHBV copolymers. They are naturally produced by bacteria from agricultural raw materials, and they can be processed to make a variety of useful products, where their biodegradability and naturalness are quite beneficial. PHBV copolymers are still in the first stage of commercialization. But they are presented in this paper as an example of how new technology can help meet society's needs for plastics and a clean environment.
Industrial input-output analysis: implications for industrial ecology.
Authors: Duchin F
Published: May 2006

Industrial ecology will need to develop fundamentally new approaches to reducing, reusing, and recycling wastes. Industrial ecology will also require an analytic framework for examining the implications for the economic system as a whole of each potential web of industrial changes. A suitable framework is furnished by structural economics, which situates the economy within the physical world. This approach is based on dynamic analysis rather than static concepts of equilibrium, and optimization assumptions are used selectively rather than as the general solution mechanism. Input-output economics, an important formal model within structural economics, can trace the stocks and flows of energy and other materials from extraction through production and consumption to recycling or disposal. An input-output computation, including wastes, is presented; it illustrates the separate but integrated analysis of physical stocks and flows and of prices and costs. This paper also describes the major advances that have been made in the last decade in the extension of input-output economics to address increasingly complex questions, notably the fully dynamic physical/price/income model and the engineering/input-output data base. Economists need to be able to assess the costs of cleaning up and to develop incentive schemes to increase the likelihood this will happen. To do this, economists need to take on the difficult "how" questions that concern industrial ecologists since the cost, and indeed the wider implications, of cleaning up depends upon how it is done. Structural economics, and modern input-output models and data bases, in particular, can help meet this challenge.
Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction.
Authors: Sandy M S , Chiocca S M , Cerutti P A
Published: May 2006

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.
Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells.
Authors: Leppä S , Mali M , Miettinen H M , Jalkanen M
Published: May 2006

S115 mouse mammary epithelial cells lose their epithelial morphology and become tumorigenic when exposed to steroids. We have recently reported that testosterone exposure results in the suppression of syndecan expression, suggesting that this cell surface proteoglycan may influence S115 cell phenotype. We now report that a similar suppression and morphological response of S115 cells can be achieved by glucocorticoid exposure. We introduced into S115 cells an exogenous gene construct containing the full-length human syndecan cDNA under the control of a glucocorticoid-inducible retroviral promoter, in order to study the effect of syndecan expression on S115 cell behavior. Glucocorticoid-induced re-expression of syndecan in S115 cells restored an epithelial phenotype, while control transfectants and parental S115 cells exhibited an altered, nonepithelial phenotype. Moreover, the S115 cells expressing exogenous syndecan revealed a reduced ability to form colonies in soft agar. Therefore, the maintenance of epithelial morphology and normal growth of S115 cells are dependent on syndecan expression.
Guanylin: an endogenous activator of intestinal guanylate cyclase.
Authors: Currie M G , Fok K F , Kato J , Moore R J , Hamra F K , Duffin K L , Smith C E
Published: May 2006

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.
Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A.
Authors: Burstein E S , Macara I G
Published: May 2006

The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.
Testosterone facilitates some conspecific song discriminations in castrated zebra finches (Taeniopygia guttata).
Authors: Cynx J , Nottebohm F
Published: May 2006

An experiment was designed to test for the influence of testosterone on song discriminations. We found that testosterone did have an effect, which interacted with practice and the nature of the stimuli. Fourteen adult castrated zebra finches (Taeniopygia guttata) were grouped into seven pairs. In each pair, one bird was implanted with a testosterone-filled silastic tube and the other was implanted with an empty silastic tube. They were then trained on a go/no-go operant task to discriminate between bird songs in six consecutive experiments. The songs to be discriminated were as follows: experiment 1, two canary song segments; experiment 2, the bird's own song and that of the other member of the pair; experiment 3, the same two songs as in experiment 2 but with reversed stimulus-response contingencies; experiment 4, two other zebra finch songs; experiment 5, another two zebra finch songs; and experiment 6, another two canary song segments. There were no reliable learning differences between birds treated with testosterone or with an empty silastic in experiments 1 and 3-6. However, in experiment 2, testosterone-treated birds mastered the discrimination between their own song and the song of the other member of the pair in fewer trials than birds treated with empty silastics. We suggest that a song's ability to control the behavior of male zebra finches is influenced by the nature of the song, prior experience with the training paradigm, and hormone levels.
Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells.
Authors: Gillies S D , Reilly E B , Lo K M , Reisfeld R A
Published: May 2006

A genetically engineered fusion protein consisting of a chimeric anti-ganglioside GD2 antibody (ch14.18) and interleukin 2 (IL2) was tested for its ability to enhance the killing of autologous GD2-expressing melanoma target cells by a tumor-infiltrating lymphocyte line (660 TIL). The fusion of IL2 to the carboxyl terminus of the immunoglobulin heavy chain did not reduce IL2 activity as measured in a standard proliferation assay using either mouse or human T-cell lines. Antigen-binding activity was greater than that of the native chimeric antibody. The ability of resting 660 TIL cells to kill their autologous GD2-positive target cells was enhanced if the target cells were first coated with the fusion protein. This stimulation of killing was greater than that of uncoated cells in the presence of equivalent or higher concentrations of free IL2. Such antibody-cytokine fusion proteins may prove useful in targeting the biological effect of IL2 and other cytokines to tumor cells and in this way stimulate their immune destruction.
Receptors for IgG complexes activate synthesis of monocyte chemoattractant peptide 1 and colony-stimulating factor 1.
Authors: Hora K , Satriano J A , Santiago A , Mori T , Stanley E R , Shan Z , Schlondorff D
Published: May 2006

The chemotactic factors responsible for complement-independent macrophage accumulation in immune-complex diseases such as glomerulonephritis remain unknown. Fc receptors for IgG complexes are found on mesangial cells of the kidney, which produce the macrophage growth factor colony-stimulating factor 1 (CSF-1). We therefore investigated the possible stimulation of mesangial-cell expression of CSF-1 and the recently identified monocyte-specific chemoattractant protein 1 (MCP-1) by IgG complexes. IgG complexes, but not monomeric IgG or F(ab')2 fragments of IgG, rapidly (2-8 h) increased mRNA for both CSF-1 (10-fold) and MCP-1 (20-fold) in cultured mouse mesangial cells. The increase of mRNA for CSF-1 and MCP-1 was not reduced by either cytochalasin B or D, indicating that Fc receptor occupancy is sufficient for signaling and that phagocytosis is not required to elicit this response. IgG complexes also caused a 10-fold increase in the secretion of CSF-1 and a 3- to 5-fold increase in secretion of MCP-1 into the cell culture medium. The synthesis and release of CSF-1 and MCP-1 by mesangial cells as a consequence of Fc receptor occupancy may be responsible for macrophage recruitment and activation at sites of immune-complex deposition.
The molecular mechanism of "ecstasy" [3,4-methylenedioxy-methamphetamine (MDMA)]: serotonin transporters are targets for MDMA-induced serotonin release.
Authors: Rudnick G , Wall S C
Published: May 2006

MDMA ("ecstasy") has been widely reported as a drug of abuse and as a neurotoxin. This report describes the mechanism of MDMA action at serotonin transporters from plasma membranes and secretory vesicles. MDMA stimulates serotonin efflux from both types of membrane vesicle. In plasma membrane vesicles isolated from human platelets, MDMA inhibits serotonin transport and [3H]imipramine binding by direct interaction with the Na(+)-dependent serotonin transporter. MDMA stimulates radiolabel efflux from plasma membrane vesicles preloaded with [3H]serotonin in a stereo-specific, Na(+)-dependent, and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, which contain the vesicular biogenic amine transporter, MDMA inhibits ATP-dependent [3H]serotonin accumulation and stimulates efflux of previously accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due to dissipation of the transmembrane pH difference generated by ATP hydrolysis and to direct interaction with the vesicular amine transporter.
Hormonal regulation of major histocompatibility complex class I genes in rat thyroid FRTL-5 cells: thyroid-stimulating hormone induces a cAMP-mediated decrease in class I expression.
Authors: Saji M , Moriarty J , Ban T , Kohn L D , Singer D S
Published: May 2006

Thyrocytes normally express major histocompatibility complex (MHC) class I, but not class II, cell surface antigens. A rat thyrocyte cell line, FRTL-5, also expresses MHC class I antigens, in addition to a variety of thyroid-specific genes. Treatment of FRTL-5 thyrocytes with physiological concentrations of thyroid-stimulating hormone (TSH) has been shown to induce increased expressed of thyroglobulin and thyroid peroxidase but to simultaneously decrease expression of the TSH receptor. The reduction in TSH receptor expression by TSH is cAMP mediated. In the present study, it is demonstrated that, in thyrocytes treated with TSH, MHC class I expression decreases concomitant with the decrease in TSH receptor expression. This decreased expression is evidenced by reduced cell surface levels of MHC class I antigens, by reduced steady-state RNA levels, and by reduced transcription of the class I genes. TSH-mediated reduction of MHC class I gene transcription in FRTL-5 cells was mapped to a region within 135 base pairs of the promoter.
Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin.
Authors: Hanson M S , Pelzel S E , Latimer J , Muller-Eberhard U , Hansen E J
Published: May 2006

The mechanism(s) used by Haemophilus influenzae to acquire the essential nutrient heme from its human host has not been elucidated. The heme carried by the high-affinity serum protein hemopexin is one potential source of this micronutrient in vivo. A colony-blot assay revealed that heme-human hemopexin-binding activity was shared among most capsular serotype b strains of H. influenzae but was uncommon among other strains. We have identified a recombinant clone binding heme-human hemopexin from a H. influenzae type b (Hib) genomic library expressed in Escherichia coli. Both the Hib strain and the heme-hemopexin-binding clone expressed a polypeptide of approximately 100 kDa that bound radiolabeled heme-hemopexin. Oligonucleotide linker insertion mutagenesis of the plasmid DNA from this recombinant clone was used to confirm that expression of the 100-kDa protein correlated with the heme-hemopexin-binding activity. Exchange of one of these mutant alleles into the Hib chromosome eliminated expression of both the 100-kDa protein and the heme-hemopexin-binding activity. Furthermore, this Hib mutant was unable to utilize heme-human hemopexin as a heme source.
Opioid enhancement of evoked [Met5]enkephalin release requires activation of cholinergic receptors: possible involvement of intracellular calcium.
Authors: Xu H , Gintzler A R
Published: May 2006

Previous work from this laboratory has shown that the electrically evoked release of enkephalin from the guinea pig myenteric plexus is regulated by an opiate receptor-mediated, concentration-dependent mechanism. Low concentrations (nanomolar) of opioids enhance release, whereas higher concentrations (10-100 nM) inhibit release. Each opioid effect is mediated by a different guanine nucleotide-binding protein. We now demonstrate that activation of cholinergic receptors in the myenteric plexus is a prerequisite for opioid excitatory effects, but not inhibitory effects, on enkephalin release. Pretreatment with the muscarinic cholinergic receptor antagonist atropine abolishes the opioid facilitation of stimulated enkephalin release but does not alter the inhibition of release that is observed with higher concentrations of opioid agonist. Exposure to the calcium ionophore A23187 overcomes the abolishment of opioid enhancement of enkephalin release produced by cholinergic receptor blockade. In tissue treated with both atropine and A23187, the magnitude of the opioid enhancement of release is indistinguishable from that observed in untreated preparations. This suggests that the lack of stimulation-induced generation of elevated cytosolic calcium is responsible for the abolishment of facilitory opioid effects when cholinergic receptors are blocked. The known coupling of muscarinic receptors to phospholipase C activation and the generation of inositol trisphosphate (which elevates cytosolic calcium) could suggest that this second messenger is critical for the manifestation of opioid facilitation of enkephalin release.
Mutations in the bZIP domain of yeast GCN4 that alter DNA-binding specificity.
Authors: Tzamarias D , Pu W T , Struhl K
Published: May 2006

The bZIP class of eukaryotic transcriptional regulators utilize a distinct structural motif that consists of a leucine zipper that mediates dimerization and an adjacent basic region that directly contacts DNA. Although models of the protein-DNA complex have been proposed, the basis of DNA-binding specificity is essentially unknown. By genetically selecting for derivatives of yeast GCN4 that activate transcription from promoters containing mutant binding sites, we isolate an altered-specificity mutant in which the invariant asparagine in the basic region of bZIP proteins (Asn-235) has been changed to tryptophan. Wild-type GCN4 binds the optimal site (ATGACTCAT) with much higher affinity than the mutant site (TTGACTCAA), whereas the Trp-235 protein binds these sites with similar affinity. Moreover, the Trp-235, Ala-235, and Gln-235 derivatives differ from GCN4 in their strong discrimination against GTGACTCAC. These results suggest a direct interaction between Asn-235 and the +/- 4 position of the DNA target site and are discussed in terms of the scissors-grip and induced-fork models of bZIP proteins.
Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway.
Authors: Spanagel R , Herz A , Shippenberg T S
Published: May 2006

The mesolimbic dopaminergic system has been implicated in mediating the motivational effects of opioids and other drugs of abuse. The site of action of opioids within this system and the role of endogenous opioid peptides in modulating dopamine activity therein remain unknown. Employing the technique of in vivo microdialysis and the administration of highly selective opioid ligands, the present study demonstrates the existence of tonically active and functionally opposing mu and kappa opioid systems that regulate dopamine release in the nucleus accumbens, the major terminal area of A10 dopaminergic neurons. Thus, stimulation of mu-type receptors in the ventral tegmental area, the site of origin of A10 dopaminergic neurons, increases dopamine release whereas the selective blockade of this opioid receptor type results in a significant decrease in basal dopamine release. In contrast, stimulation of kappa-type receptors within the nucleus accumbens decreases dopamine release whereas their selective blockade markedly increases basal dopamine release. These data show that tonic activation of mu and kappa receptors is required for the maintenance of basal dopamine release in the nucleus accumbens. In view of the postulated role of the mesolimbic system in the mediation of drug-induced alterations in mood and affect, such findings may have implications for the treatment of opiate dependence and affective disorders.
A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.
Authors: Pierce J C , Sauer B , Sternberg N
Published: May 2006

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.
Paradoxical transcriptional activation of rat liver cytochrome P-450 3A1 by dexamethasone and the antiglucocorticoid pregnenolone 16 alpha-carbonitrile: analysis by transient transfection into primary monolayer cultures of adult rat hepatocytes.
Authors: Burger H J , Schuetz J D , Schuetz E G , Guzelian P S
Published: May 2006

The family 3A cytochromes P-450, among the most abundant members of this supergene family of microsomal hemoproteins expressed in animal and human liver, are inducible by glucocorticoids but also by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). To investigate the mechanism for this nonclassical glucocorticoid effect, we analyzed the ability of 1.5 kilobases of DNA or of its successive subsegments isolated from the 5' flanking region of the rat CYP3A1 structural gene to modulate transcription of a reporter gene consisting of a viral promoter coupled to the chloramphenicol acetyltransferase (CAT) structural gene (expression vector pBLCAT2) and transiently expressed in a homologous cell system consisting of primary monolayer cultures of adult rat hepatocytes in which CYP3A1 mRNA and protein are inducible. The CAT activity measured after chimeric gene constructions were transferred into the cultured rat hepatocytes by lipofection increased as much as 7.2-fold if the cells were treated with dexamethasone (DEX). One CYP3A1 fragment (positions -220 to -56; 164 base pairs), which does not contain a traditional glucocorticoid responsive element, conferred dose-dependent DEX responsiveness independent of its orientation but not its position in pBLCAT2. This construction was activated by addition of PCN to the cultures and was synergistically induced by PCN plus DEX. In contrast, induction of CAT activity in cultures containing MMTVCAT, a plasmid containing the CAT gene controlled by the mouse mammary tumor virus long terminal repeat, was unaffected by PCN treatment, required lower concentrations of DEX for a maximal response, and was inhibited by treatment with DEX plus PCN. We conclude that a primary mechanism for induction of CYP3A1 is stimulated transcription through a pathway activated by steroid hormones.
Preproenkephalin promoter "cassette" confers brain expression and synaptic regulation in transgenic mice.
Authors: Donovan D M , Takemura M , O'Hara B F , Brannock M T , Uhl G R
Published: May 2006

The preproenkephalin A gene is a neurotransmitter gene whose expression can be modulated "trans-synaptically" by changes in neuronal activity. DNA sequences lying within 200 base pairs of this gene's transcription start site resemble consensus binding sites for several transcription factor families. In nonneuronal cell cultures, this promoter region is sufficient to mediate gene responses to depolarization, phorbol esters, adenylate cyclase, and calcium fluxes. To assess the role that these cis-acting elements could play in preproenkephalin expression and regulation in vivo, the expression of a construct containing this 200-base-pair region fused to the chloramphenicol acetyltransferase gene was examined in transgenic mice. This promoter confers modest expression in brain, adrenal, and small intestine, with substantially higher levels in testis. These elements confer trans-synaptic regulation in two well-studied models of trans-synaptic preproenkephalin upregulation but not in a third system, underscoring the specificity of the regulatory sequence elements implicated in the synaptic regulation of neuronal genes.
Analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) as potential anti-platelet-aggregation agents.
Authors: Zamecnik P C , Kim B , Gao M J , Taylor G , Blackburn G M
Published: May 2006

Dense granules of platelets contain a high content of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A). We have previously demonstrated an antithrombotic effect of this compound in a live rabbit model. In the present study we find that certain synthetic Ap4A analogues are superior to Ap4A in inhibiting ADP-induced aggregation of human platelets. Analogues having a P--C--P bridge located in the P2,P3 position of Ap4A are the most potent inhibitors. These analogues are also resistant to hydrolytic enzymes. Analogues having the above characteristics exhibit competitive inhibition with ADP in the ADP-induced platelet aggregation reaction. These compounds, such as AppCHFppA, may be useful as antithrombotic agents. The analogues ApSppSpA and ApSpCHFpSpA also showed good inhibitory effects on ADP-induced platelet aggregation. In addition, this action of Ap4A and its analogues provides an example of a dinucleotide inducing an antagonistic effect by occupying an extracellular mononucleotide binding site on platelets. It calls attention to the possibility that Ap4A and its analogues may act in a similar way in whole organisms, triggering effector or inhibitory responses in any one of a variety of cells.
A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure.
Authors: Zhang Y , Talalay P , Cho C G , Posner G H
Published: May 2006

Consumption of vegetables, especially crucifers, reduces the risk of developing cancer. Although the mechanisms of this protection are unclear, feeding of vegetables induces enzymes of xenobiotic metabolism and thereby accelerates the metabolic disposal of xenobiotics. Induction of phase II detoxication enzymes, such as quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases (EC 2.5.1.18) in rodent tissues affords protection against carcinogens and other toxic electrophiles. To determine whether enzyme induction is responsible for the protective properties of vegetables in humans requires isolation of enzyme inducers from these sources. By monitoring quinone reductase induction in cultured murine hepatoma cells as the biological assay, we have isolated and identified (-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane [CH3-SO-(CH2)4-NCS, sulforaphane] as a major and very potent phase II enzyme inducer in SAGA broccoli (Brassica oleracea italica). Sulforaphane is a monofunctional inducer, like other anticarcinogenic isothiocyanates, and induces phase II enzymes selectively without the induction of aryl hydrocarbon receptor-dependent cytochromes P-450 (phase I enzymes). To elucidate the structural features responsible for the high inducer potency of sulforaphane, we synthesized racemic sulforaphane and analogues differing in the oxidation state of sulfur and the number of methylene groups: CH3-SOm-(CH2)n-NCS, where m = 0, 1, or 2 and n = 3, 4, or 5, and measured their inducer potencies in murine hepatoma cells. Sulforaphane is the most potent inducer, and the presence of oxygen on sulfur enhances potency. Sulforaphane and its sulfide and sulfone analogues induced both quinone reductase and glutathione transferase activities in several mouse tissues. The induction of detoxication enzymes by sulforaphane may be a significant component of the anticarcinogenic action of broccoli.
Structure of the histidine-containing phosphocarrier protein HPr from Bacillus subtilis at 2.0-A resolution.
Authors: Herzberg O , Reddy P , Sutrina S , Saier M H , Reizer J , Kapadia G
Published: May 2006

The crystal structure of the histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.0-A resolution and refined to a crystallographic residual error R-factor of 0.150. The secondary-structure folding topology of the molecule is that of an open-face beta-sandwich formed by four antiparallel beta-strands packed against three alpha-helices. The active-site histidine, His-15, caps the N terminus of the first helix, suggesting that the helix dipole plays a role in stabilizing the phosphorylated state of the histidine. A sulfate anion located between His-15 and the neighboring Arg-17 has been identified in the electron-density map. Association of this negatively charged species with the two key catalytic residues implies that the crystal structure resembles the phosphorylated state of the protein. A model of the phosphorylated form of the molecule is proposed, in which the negatively charged phosphoryl group interacts with two main-chain nitrogen atoms of the following helix and with the guanidinium group of Arg-17. It is also proposed that the phosphoryl transfer from HPr to the IIA domain of the glucose permease involves Arg-17 switching between two salt bridges: one with the phosphorylated histidyl of HPr and the other with two aspartyl residues associated with the active site of the IIA domain of glucose permease, which are accessible upon complex formation.
A lattice model for protein structure prediction at low resolution.
Authors: Hinds D A , Levitt M
Published: May 2006

The prediction of the folded structure of a protein from its sequence has proven to be a very difficult computational problem. We have developed an exceptionally simple representation of a polypeptide chain, with which we can enumerate all possible backbone conformations of small proteins. A protein is represented by a self-avoiding path of connected vertices on a tetrahedral lattice, with several amino acid residues assigned to each lattice vertex. For five small structurally dissimilar proteins, we find that we can separate native-like structures from the vast majority of non-native folds by using only simple structural and energetic criteria. This method demonstrates significant generality and predictive power without requiring foreknowledge of any native structural details.
Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury.
Authors: Laywell E D , Dörries U , Bartsch U , Faissner A , Schachner M , Steindler D A
Published: May 2006

Tenascin is an extracellular matrix molecule synthesized and released by young astrocytes during embryonic and early postnatal development of the nervous system, and it is concentrated in boundaries around emerging functional neuronal units. In the adult nervous system, tenascin can be detected only in very low levels. Distinct spatial and temporal distributions of tenascin during developmental events suggest a role in the guidance and/or segregation of neurons and their processes within incipient functional patterns. We show here, using in situ hybridization and immunocytochemistry, that stab wounds of the adult mouse cerebellar and cerebral cortices result in an enhanced expression of tenascin in a discrete region around the lesion site that is associated with a subset of glial fibrillary acidic protein-positive astrocytes. Tenascin up-regulation in the lesioned adult brain may be directly involved in failed regeneration or indirectly involved through its interactions with other glycoconjugates that either inhibit or facilitate neurite growth.
Expression of a cloned rat histamine H2 receptor mediating inhibition of arachidonate release and activation of cAMP accumulation.
Authors: Traiffort E , Ruat M , Arrang J M , Leurs R , Piomelli D , Schwartz J C
Published: May 2006

A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schäffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc. Natl. Acad. Sci. USA 88, 429-433], was used to prepare a probe for Northern blot analysis and to transfect Chinese hamster ovary (CHO) cells. Distribution of the gene transcripts in guinea pig tissues was consistent with that of H2 receptors. Transfected CHO cells expressed a high density of sites binding [125I]iodoaminopotentidine, a selective H2-receptor ligand. These sites were characterized as typical H2 receptors by using a series of competing agents that displayed apparent dissociation constants closely similar to corresponding values at a reference biological system. In transfected cells, histamine stimulated, with high potency and large receptor reserve, the accumulation of cAMP. In addition, in the same cells, histamine potently inhibited the release of arachidonic acid induced either by stimulation of constitutive purinergic receptors or by application of a Ca2+ ionophore. This inhibition was independent of either cAMP or Ca2+ levels. The results suggest that a single H2 receptor may be linked not only to adenylyl cyclase activation but also to reduction of phospholipase A2 activity. Because H1 receptors have been reported to stimulate arachidonic acid release, inhibition of this release, an unexpected signaling pathway for H2 receptors, may account for the opposing physiological responses elicited in many tissues by stimulation of these two receptors subtypes.
AlgR2 is an ATP/GTP-dependent protein kinase involved in alginate synthesis by Pseudomonas aeruginosa.
Authors: Roychoudhury S , Sakai K , Chakrabarty A M
Published: May 2006

The exopolysaccharide alginate is a major virulence factor in the pathogenicity of Pseudomonas aeruginosa infecting the lungs of cystic fibrosis (CF) patients. Alginate synthesis by P. aeruginosa is believed to occur in response to environmental signals present in the CF lung. Transcription of a critical alginate biosynthetic gene, algD, is triggered by environmental signals and is known to be controlled by regulatory proteins AlgR1, AlgR2, and AlgR3. AlgR1 is a member of the family of response regulators of the phosphorylation-dependent two-component bacterial signal transduction systems. In this report, we describe the characterization of AlgR2 as the kinase involved in phosphorylation of AlgR1. AlgR2, an 18-kDa soluble protein undergoes rapid autophosphorylation in the presence of either ATP or GTP and transfers the phosphate to AlgR1. AlgR2 retains high affinity for both ATP and GTP with an apparent Km of 137 and 249 nM, respectively, for phosphorylation by these two substrates. ADP and GDP exhibit competitive inhibition with an apparent Ki of 94 and 314 nM, respectively, during phosphorylation by ATP and 481 and 273 nM during phosphorylation by GTP. AlgR1 and AlgR2 can be isolated in the form of an 80-kDa complex that is capable of undergoing phosphorylation and intracomplex phosphotransfer in vitro. A 16-kDa AlgR2 analog, capable of autophosphorylation in the presence of ATP or GTP and transferring the phosphate moiety to AlgR1, has been characterized in Escherichia coli.