bluuebunny/crossref_metadata_2025 huggingface dataset:
Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeats by protein-protein interaction.
Authors: Zhao L J , Giam C Z
Published: May 2006

HTLV-I Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer. The HTLV-I 21-bp repeat contains a TGACGT motif that is homologous to the cAMP-responsive element (CRE) and crucial for tax transactivation. Tax exhibits marginal affinity for DNA but rather interacts with cellular CRE-binding proteins to enhance their affinity for the HTLV-I 21-bp repeats. Using the HTLV-I 21-bp repeat and Jurkat T-lymphocyte nuclear extract in a gel electrophoretic mobility-shift assay, we previously detected three protein-DNA complexes that are specific for the CRE in the 21-bp repeat (complexes I, II, and IV). Complexes I and II but not IV interacted with Tax. We now show that complexes I, II, and IV are composed of CREB (CRE binding protein) homodimer, CREB/ATF-1 (activating transcription factor 1) heterodimer, and ATF-1 homodimer, respectively. Tax stabilizes complexes I and II via a direct interaction with the CREB moiety. In the absence of DNA, CREB and Tax continue to form a complex that can be immunoprecipitated by a Tax-specific antibody. These results suggest that one mechanism by which Tax activates transcription may be mediated through the direct interaction with CREB homodimer and/or CREB/ATF-1 heterodimer to stabilize their assembly on the Tax-responsive CRE motifs in the HTLV-I enhancer.
Diversity among tight junctions in rat kidney: glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1.
Authors: Kurihara H , Anderson J M , Farquhar M G
Published: May 2006

ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.
A family of erythrocyte binding proteins of malaria parasites.
Authors: Adams J H , Sim B K , Dolan S A , Fang X , Kaslow D C , Miller L H
Published: May 2006

Malaria erythrocyte binding proteins use the Duffy blood group antigen (Plasmodium vivax and Plasmodium knowlesi) and sialic acid (Plasmodium falciparum) on the erythrocyte surface as receptors. We had previously cloned the one P. vivax gene, the one P. falciparum gene, and part of one of the three P. knowlesi genes encoding these erythrocyte binding proteins and described the homology between the P. knowlesi and P. vivax genes. We have completed the cloning and sequencing of the three P. knowlesi genes and identified introns in the P. vivax and P. falciparum genes that correct the previously published deduced amino acid sequences. All have similar structures, with one or two exons encoding the signal sequence and the erythrocyte binding domain, an exon encoding the transmembrane domain, and two exons encoding the cytoplasmic domain with the exception of the P. knowlesi beta gene. The regions of amino acid sequence homology among all the genes are the 5' and 3' cysteine-rich regions of the erythrocyte binding domain. On the basis of gene structure and amino acid homology, we propose that the Duffy binding proteins and the sialic acid binding protein are members of a gene family. The level of conservation (approximately 70%) of the deduced amino acid sequences in the 5' cysteine-rich region between the P. vivax protein and the three P. knowlesi proteins is as great as between the three P. knowlesi proteins themselves; the P. knowlesi beta protein just 3' to this cysteine-rich region is homologous to the P. vivax protein but not to the other P. knowlesi proteins. Conservation of amino acid sequences among these organisms, separated in evolution, may indicate the regions where the adhesin function resides.
Synthetic neural modeling applied to a real-world artifact.
Authors: Edelman G M , Reeke G N , Gall W E , Tononi G , Williams D , Sporns O
Published: May 2006

We describe the general design, operating principles, and performance of a neurally organized, multiply adaptive device (NOMAD) under control of a nervous system simulated in a computer. The complete system, Darwin IV, is the latest in a series of models based on the theory of neuronal group selection, which postulates that adaptive behavior is the result of selection in somatic time among synaptic populations. The simulated brain of Darwin IV includes visual and motor areas that are connected with NOMAD by telemetry. Under suitable conditions, Darwin IV can be trained to track a light moving in a random path. After such training, it can approach colored blocks and collect them to a home position. Following a series of contacts with such blocks, value signals received through a "snout" that senses conductivity allow it to sort these blocks on the basis of differences in color associated with differences in their conductivity. Darwin IV represents a new approach to synthetic neural modeling (SNM), a technique in which large-scale computer simulations are employed to analyze the interactions among the nervous system, the phenotype, and the environment of a designed organism as behavior develops. Darwin IV retains the advantages of SNM while avoiding the difficulties and pitfalls of attempting to simulate a rich environment in addition to a brain.
Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.
Authors: Gomez-Cambronero J , Huang C K , Gomez-Cambronero T M , Waterman W H , Becker E L , Sha'afi R I
Published: May 2006

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubule-associated protein kinase (MAPK, p42MAPK) antibody show only one band. The molecular mass of that band exactly matches that of p41. MAPK-reactive protein is present in control and stimulated cells, although the intensity of the band is greater in the latter. GM-CSF-stimulated phosphorylation of p41 is time- and dose-dependent. Anti-MAPK antibody detects a single band of 41 kDa, whose intensity increases with time of incubation and concentration of the agonist. Thus, the anti-MAPK antibody appears to react better to the phosphorylated form of p41 from GM-CSF-stimulated cells than to the dephosphorylated form. The p41 and MAPK proteins are localized in the cytosol. Finally, MAPK immunoprecipitates were probed with anti-PY in Western blots and a band of 41 kDa was found. In summary, these results suggest that this 41-kDa protein in neutrophils that is tyrosine phosphorylated in response to GM-CSF and other stimuli is MAPK. Its phosphorylation may represent an early and crucial signal associated with the GM-CSF neutrophil stimulation cascade.
Degenerating Y chromosome of Drosophila miranda: a trap for retrotransposons.
Authors: Steinemann M , Steinemann S
Published: May 2006

In Drosophila miranda, the larval cuticle protein (Lcp) genes are located on the X2 and Y chromosomes, while in other Drosophila species the Lcp genes are inherited on the autosomes. We chose the D. miranda species as a model system to analyze the molecular bases of Y chromosome degeneration, a phenomenon observed in many species. DNA sequence analysis of the Y chromosomal Lcp gene locus reveals dense clustering of trapped retrotransposons. Once inserted at the Y chromosomal location they cannot easily be eliminated by unequal crossing-over, as recombination is a rare event in Drosophila males. In addition, we have uncovered an example of a completely inactive allele on the degenerating Y chromosome. The existence of such inactive Y-specific alleles was originally predicted in H. J. Muller's model for Y chromosome degeneration. We demonstrate that the Y chromosomal Lcp4 allele is no longer transcribed. From the divergence in DNA sequence organization of former homologous chromosome regions we conclude that changes in chromosome structure and destruction of genetic activity in degenerating Y chromosomes are based on one major mechanism, which operates by means of transposable elements.
In vivo footprint analysis of the HLA-DRA gene promoter: cell-specific interaction at the octamer site and up-regulation of X box binding by interferon gamma.
Authors: Wright K L , Ting J P
Published: May 2006

Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression. We have examined the DRA promoter for protein-DNA interactions in the intact cell, which may mediate transcriptional activation. Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y, X1, and X2 boxes. Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines, while class II-negative T-cell lines exhibited no interactions. In lymphoid cell lines, the octamer site is occupied and required for maximal expression. This is most likely due to the presence of the lymphoid-specific OTF-2 factor. In contrast, the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site. Thus, the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line. Interferon gamma induces class II expression in this glioblastoma cell line and, in parallel, up-regulates X1 and X2 box protein-DNA interactions, while all other interactions remain unchanged. These results suggest that interferon gamma functions on a poised promoter by altering weak, nonproductive interactions at the X boxes to strong interactions. These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway.
The antibiotic azatyrosine suppresses progesterone or [Val12]p21 Ha-ras/insulin-like growth factor I-induced germinal vesicle breakdown and tyrosine phosphorylation of Xenopus mitogen-activated protein kinase in oocytes.
Authors: Campa M J , Glickman J F , Yamamoto K , Chang K J
Published: May 2006

The antibiotic azatyrosine [DL-3-(5-hydroxy-2-pyridyl)alanine] suppressed meiotic maturation in oocytes induced by progesterone or the combination of [Val12]p21Ha-ras microinjection and insulin-like growth factor I. The suppression was dose-dependent in the range of 20-250 microM azatyrosine. In addition, azatyrosine blocked the tyrosine phosphorylation of Xp42, a member of the mitogen-activated protein kinase family, after progesterone or [Val12]p21Ha-ras/insulin-like growth factor I stimulation. Activation of maturation-promoting factor, as shown by a decrease in the tyrosine phosphorylation of the Xenopus homolog of p34cdc2, was also suppressed by azatyrosine. Azatyrosine had no effect in vivo or in vitro on the growth factor-induced autophosphorylation of the oocyte insulin-like growth factor I receptor. Azatyrosine has been shown by others [Shindo-Okada, N., Makabe, O., Nagahara, H. & Nishimura, S. (1989) Mol. Carcinog. 2, 159-167] to inhibit the growth of ras-transformed cells without affecting that of nontransformed cells. In oocytes, the antibiotic exerts an inhibitory action on both a ras-dependent and a ras-independent pathway. Lack of an effect of azatyrosine on germinal vesicle breakdown induced by the microinjection of an extract from mature oocytes, however, suggests that azatryosine is acting upstream of maturation-promoting factor activation.
Magnetite biomineralization in the human brain.
Authors: Kirschvink J L , Kobayashi-Kirschvink A , Woodford B J
Published: May 2006

Although the mineral magnetite (Fe3O4) is precipitated biochemically by bacteria, protists, and a variety of animals, it has not been documented previously in human tissue. Using an ultrasensitive superconducting magnetometer in a clean-lab environment, we have detected the presence of ferromagnetic material in a variety of tissues from the human brain. Magnetic particle extracts from solubilized brain tissues examined with high-resolution transmission electron microscopy, electron diffraction, and elemental analyses identify minerals in the magnetite-maghemite family, with many of the crystal morphologies and structures resembling strongly those precipitated by magnetotactic bacteria and fish. These magnetic and high-resolution transmission electron microscopy measurements imply the presence of a minimum of 5 million single-domain crystals per gram for most tissues in the brain and greater than 100 million crystals per gram for pia and dura. Magnetic property data indicate the crystals are in clumps of between 50 and 100 particles. Biogenic magnetite in the human brain may account for high-field saturation effects observed in the T1 and T2 values of magnetic resonance imaging and, perhaps, for a variety of biological effects of low-frequency magnetic fields.
Skin test to assess virus-specific cytotoxic T-cell activity.
Authors: Kündig T M , Althage A , Hengartner H , Zinkernagel R M
Published: May 2006

A way to assess specific CD8+ T-cell activity in a skin test analogous to the conventional delayed-type hypersensitivity (DTH) reaction for CD4+ T cells is presented. Local injection of viral class I binding peptides caused a specific CD8+ T-cell-mediated DTH in footpads of virally infected mice. The DTH was inducible only during the acute phase of the infection. Apparently because of the short half-life of locally available peptide, only activated CD8+ effector T cells could mediate the reaction. This skin test may prove to be particularly interesting for use in humans to evaluate the activation status of CD8+ T cells during acute viral infections and of memory CD8+ T cells, for example, in chronically active immunopathological disease or infection.
An algorithm for protein engineering: simulations of recursive ensemble mutagenesis.
Authors: Arkin A P , Youvan D C
Published: May 2006

An algorithm for protein engineering, termed recursive ensemble mutagenesis, has been developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Starting from partially randomized "wild-type" DNA sequences, a highly parallel search of sequence space for peptides fitting an experimenter's criteria is performed. Each iteration uses information gained from the previous rounds to search the space more efficiently. Simulations of the technique indicate that, under a variety of conditions, the algorithm can rapidly produce a diverse population of proteins fitting specific criteria. In the experimental analog, genetic selection or screening applied during recursive ensemble mutagenesis should force the evolution of an ensemble of mutants to a targeted cluster of related phenotypes.
Hagfish humoral defense protein exhibits structural and functional homology with mammalian complement components.
Authors: Hanley P J , Hook J W , Raftos D A , Gooley A A , Trent R , Raison R L
Published: May 2006

A genomic clone and cDNA fragment encoding a portion of a humoral recognition molecule from the hagfish were isolated and sequenced. The serum protein has previously been described as having structural features that are immunoglobulin-like. Amino acid sequence obtained from the 77-kDa H1 heavy chain facilitated the isolation of a genomic clone containing at least two coding regions. Through use of primers derived from the genomic sequences, a 231-base-pair cDNA fragment was obtained by PCR from liver RNA. Comparison of the deduced 120-amino acid sequence from the N terminus of H1 with known protein sequences revealed substantial sequence similarity with the beta chain of the murine fourth complement component C4 and with the related third and fifth complement molecules C5 and C3 and the major histocompatibility complex-encoded sex-limited protein. Observation of structural and functional similarities associated with the sequence similarity indicate that these molecules share an evolutionary relationship: the polypeptide chain structure of hagfish complement-like protein (CLP) resembles that of C4; CLP contains a hidden thioester group on the 70-kDa chain; CLP binds to streptococcal cells and enhances the phagocytosis of yeast by hagfish leukocytes. These data suggest that CLP forms part of a non-clonally-derived complement-related humoral defense system in the hagfish.
Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.
Authors: Fearon E R , Finkel T , Gillison M L , Kennedy S P , Casella J F , Tomaselli G F , Morrow J S , Van Dang C
Published: May 2006

We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.
Elevated sister chromatid exchange phenotype of Bloom syndrome cells is complemented by human chromosome 15.
Authors: McDaniel L D , Schultz R A
Published: May 2006

Bloom syndrome (BSx) is a rare autosomal-recessive chromosome-instability disorder manifested by a constellation of clinical features including a significant predisposition to early onset of neoplasia. BSx cells display cytogenetic abnormalities, the pathognomonic feature being an increased rate of spontaneous sister chromatid exchanges (SCEs), 10- to 15-fold more frequent than SCEs seen in control cells. Identification of the primary biochemical defect in BSx and its relationship to SCE frequency and neoplasia have been complicated by reports that BSx cell lines exhibit defects in the structure and/or activity of a number of different enzymes. The rare occurrence of the disorder and lack of informative families have precluded mapping of the primary defect by standard linkage analysis. We have utilized BSx cells as recipients for microcell-mediated chromosome transfer to map a locus that renders complementation of the elevated SCE phenotype. Studies with the BSx cell line GM08505 demonstrated a stable frequency of SCEs 10-fold higher than control values, offering a phenotype suitable for complementation studies. Transfer of different independent human chromosomes from somatic cell hybrids into BSx cells permitted identification of a single chromosome that dramatically reduced the SCE frequency to a level near that seen in control cells. Detailed characterization revealed this complementing element to be human chromosome 15.
Single crystal structure of a mixed-chain triacylglycerol: 1,2-dipalmitoyl-3-acetyl-sn-glycerol.
Authors: Goto M , Kodali D R , Small D M , Honda K , Kozawa K , Uchida T
Published: May 2006

The mixed chain triacylglycerol 1,2-dipalmitoyl-3-acetyl-sn-glycerol was synthesized and its crystal structure was determined to a final reliability factor (R) of 0.11. Two molecules are present in the monoclinic unit cell: space group P2(1); a = 5.375(1), b = 8.286(2), c = 42.96(1) A; beta = 93.30(2) degrees, V = 1910 A3, rho = 1.065 g/cm3, and mu = 5.7 cm-1. The structure is a trilayer: a bilayer of palmitate chains packed in the beta mode (T parallel) and an interdigitated monolayer of acetates. The glycerol backbone and acetate extend roughly linearly from the sn-1 chain. The sn-2 chain bends around the C-2 carbon to lie next to the sn-1 chain. Analysis of the torsion angles indicate that the glycerol conformation of 1,2-dipalmitoyl-3-acetyl-sn-glycerol is markedly different from single acid triacylglycerols and from 1,2-diacyl-sn-glycerols but very similar to 1,2-dimyristoyl-sn-glycero-3-phosphocholine.
Extracellular matrix regulates smooth muscle responses to substance P.
Authors: Bowers C W , Dahm L M
Published: May 2006

Little is known about the extracellular factors that determine a cell's responsiveness to neurotransmitters. This is a particularly important issue for pharmacologically diverse cell types such as neurons and smooth muscle. This report demonstrates that the contractile responses of amniotic smooth muscle to a specific neuropeptide, substance P, is controlled by a molecule(s) intimately associated with the extracellular basement membrane. This molecule(s) normally represses the expression of substance P responsiveness in this tissue. When the amniotic smooth muscle is separated from the basement membrane by dissociation, normally unresponsive cells exhibit a progressive increase in responsiveness to substance P, beginning within the first 24 hr in culture. The induction of substance P responses was completely inhibited when the cells were plated onto isolated amniotic basement membrane rather than onto polyornithine or collagen I. Similar changes in the responsiveness to another agonist, histamine, did not occur. The data demonstrate that extracellular matrix exerts a major instructive influence in determining the responsiveness of avian amniotic smooth muscle to specific ligands. We suggest that similar regulatory mechanisms may operate in other tissues.
Delineation of the dystonia-parkinsonism syndrome locus in Xq13.
Authors: Graeber M B , Kupke K G , Müller U
Published: May 2006

The X chromosome-linked dystonia-parkinsonism syndrome (XDP) is a severe movement disorder, characterized by both dystonia and parkinsonism. XDP is a genetically homogeneous disorder. Known ancestry of all patients has been traced back to Panay, Philippines, where the disease probably originated from a single mutation (founder effect). The gene locus, DYT3, has been previously assigned to the proximal long arm of the X chromosome (Xq12-q21.1). Using four dinucleotide tandem repeat (DNTR) sequences from Xq13-derived yeast artificial chromosomes (YACs), we further delineate DYT3 within Xq13. Observation of a recombination event between DYT3 and DNTR locus 4548-7, derived from a YAC encompassing locus DXS56, establishes 4548-7 as a distal flanking marker. Assignment of DYT3 to a region in Xq13, flanked by loci 4548-7 and DXS159, is further supported by highly significant allelic association between DYT3 and a total of four DNTR loci--PY2-31, PY5-10, 4548-1, and 4548-7--located in a region defined by PGK1 and DXS56. /phi/ and /delta/ values were 0.82/0.35, 0.78/0.42, 0.65/0.34, and 0.88/0.58 for PY2-31, PY5-10, 4548-1, and 4548-7 at P less than 10(-2), P less than 10(-4), P less than 10(-3), and P less than 10(-6).
Tryptophan analogues form adducts by cooperative reaction with aldehydes and alcohols or with aldehydes alone: possible role in ethanol toxicity.
Authors: Austin J E , Fraenkel-Conrat H
Published: May 2006

Previous work showed that the exocyclic amino groups of nucleic acid components react quickly at ambient temperature with acetaldehyde and ethanol to yield mixed acetals [R-NH-CH(CH3)-O-C2H5]. We now find that the same type of reaction occurs readily with the nitrogen of 3-substituted indoles (e.g., indole-3-acetic acid and N-acetyltryptophan), analogues of the amino acid tryptophan. In contrast, unsubstituted indole reacts very rapidly at the carbon in ring position 2 or 3 with acetaldehyde to form bis(indolyl)ethane without ethanol entering into the reaction. Product structures have been confirmed by fast atom bombardment MS and 1H NMR. The former reaction occurs optimally in 30-50% aqueous solution below pH 4. It also proceeds more slowly and with reduced yields in aqueous media at more neutral pH. This reaction may be of biological concern, as it supplies a mechanism for protein modifications with possible toxic effects in human tissues where ethanol is metabolized.
Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons.
Authors: Krsmanović L Z , Stojilković S S , Merelli F , Dufour S M , Virmani M A , Catt K J
Published: May 2006

Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.
De novo generation of neuronal cells from the adult mouse brain.
Authors: Richards L J , Kilpatrick T J , Bartlett P F
Published: May 2006

Cells of neuronal morphology, expressing the 150- and 200-kDa neurofilament proteins, were generated in vitro from populations of neural cells dissociated from adult (greater than 60-day-old) mouse brain. Most of these neurons arose from dividing precursors, as demonstrated by the incorporation of [3H]thymidine during the culture period and autoradiography. Neuronal production was optimal under the conditions in which precursors were initially stimulated with basic fibroblast growth factor and then exposed to medium conditioned by an astrocytic cell line, Ast-1, in serum-free medium. Few, if any, neurons arose in control cultures or in cultures kept in serum and fibroblast growth factor. These results suggest that neuronal precursors exist in the adult mammalian brain, but they require discrete epigenetic signals for their proliferation and differentiation.
Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit.
Authors: Zhang M , Zamore P D , Carmo-Fonseca M , Lamond A I , Green M R
Published: May 2006

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF), an essential mammalian splicing factor, is composed of two subunits: a 65-kDa protein (U2AF65), which binds the pre-mRNA polypyrimidine tract and is required for in vitro splicing, and an associated 35-kDa protein (U2AF35). Here we report the isolation of a cDNA encoding U2AF35. U2AF35 contains sequence motifs found in several mammalian pre-mRNA splicing factors. We show directly that U2AF65 and U2AF35 interact with each other and delineate the regions of both proteins that mediate this interaction. Using anti-peptide antibodies against U2AF35, we show that the protein has the intracellular distribution characteristic of U2AF65. Both U2AF65 and U2AF35 are concentrated in a small number of nuclear foci corresponding to coiled bodies, subnuclear organelles first identified by light microscopy in 1903.
Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs.
Authors: Tsai-Wu J J , Liu H F , Lu A L
Published: May 2006

In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein. DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY.
Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.
Authors: Smeekens S P , Montag A G , Thomas G , Albiges-Rizo C , Carroll R , Benig M , Phillips L A , Martin S , Ohagi S , Gardner P
Published: May 2006

Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.
Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II.
Authors: Huckle W R , Dy R C , Earp H S
Published: May 2006

The cellular effects of numerous hormones and neurotransmitters, including the vasoactive agents angiotensin II (AngII) and [Arg8]vasopressin, are mediated in part by protein-serine threonine kinases activated by increase of cytosolic Ca2+ concentration. In this study, we have tested the ability of Ca(2+)-mobilizing agents to activate cellular tyrosine kinases. Treatment of intact GN4 liver epithelial cells with AngII rapidly (less than or equal to 15 sec) increased tyrosine kinase activity measured either in unfractionated cell lysates or in anti-phosphotyrosine immune complexes from detergent-solubilized cells. Increased phosphorylation of the exogenous substrate poly(Glu80Tyr20) (3- to 4-fold over control) by immunoprecipitated kinases closely paralleled the time- and dose-dependence of the appearance of tyrosine phosphoproteins in intact cells. This effect of AngII was mimicked by thapsigargin, a Ca(2+)-elevating tumor promoter. The ability of AngII, but not epidermal growth factor, to increase tyrosine kinase activity was blocked in cells loaded with the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Dephosphorylation of immunoprecipitated proteins by tyrosine phosphatase treatment was accompanied by a 60-70% loss in in vitro kinase activity, suggesting that the AngII-sensitive kinase(s) are activated by phosphorylation in intact cells. These findings demonstrate a link between two widely occurring signaling pathways, the tyrosine kinases and the Ca2+ second-messenger system, and suggest the possible involvement of Ca(2+)-activated tyrosine kinases in the endocrine actions of AngII and [Arg8]vasopressin.
Emr, an Escherichia coli locus for multidrug resistance.
Authors: Lomovskaya O , Lewis K
Published: May 2006

An Escherichia coli chromosomal DNA fragment cloned on a multicopy plasmid conferred resistance to carbonylcyanide m-chlorophenylhydrazone, nalidixic acid, and a number of other toxic compounds. The sequence of the cloned emr locus located at minute 57.5 of the chromosome revealed two open reading frames, emrA and emrB. emrB encodes a highly hydrophobic 56.2-kDa peptide, with 14 potential alpha-helices to span the inner membrane. The peptide is homologous to QacA, a multidrug-resistant pump from Staphylococcus aureus, and belongs to a gene family that includes tetracycline-resistant pumps of Gram-positive bacteria and the galactose/H+ symporter of E. coli. emrA encodes a putative 42.7-kDa peptide containing a single hydrophobic domain and a large C-terminal hydrophilic domain. An active pho-fusion to the C domain suggested that EmrA is a membrane protein. Disruption of emrB significantly increased sensitivity of cells to uncouplers. The cellular content of uncoupler increased in the order: overexpressed emrB cells greater than wild type greater than emrB-.
Gene transfer of a reserpine-sensitive mechanism of resistance to N-methyl-4-phenylpyridinium.
Authors: Liu Y , Roghani A , Edwards R H
Published: May 2006

The toxin N-methyl-1,2,3,6-tetrahydropyridine produces a model of neural degeneration very similar to idiopathic Parkinson disease. To understand the cellular mechanisms that modulate susceptibility to its active metabolite N-methyl-4-phenylpyridinium (MPP+), we have transfected a cDNA expression library from the relatively MPP(+)-resistant rat pheochromocytoma PC12 cells into MPP(+)-sensitive Chinese hamster ovary (CHO) fibroblasts. Selection of the stable transformants in high concentrations of MPP+ has yielded a clone extremely resistant to the toxin. Reserpine reverses the resistance to MPP+, suggesting that a transport activity protects against this form of toxicity, perhaps by sequestering the toxin within an intracellular compartment. In support of this hypothesis, dopamine loaded into the CHO transformant shows a localized distribution that is distinct from the pattern observed in wild-type cells and is also reversed by reserpine.
Fc epsilon RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells.
Authors: Hutchcroft J E , Geahlen R L , Deanin G G , Oliver J M
Published: May 2006

In RBL-2H3 rat tumor mast cells, cross-linking the high-affinity IgE receptor Fc epsilon RI causes tyrosine phosphorylation of multiple proteins. These phosphoproteins include phospholipase C gamma 1, the beta and gamma subunits of the Fc epsilon RI, the Src family protein-tyrosine kinase Lyn, and a 72-kDa protein that coimmunoprecipitates from lysates of antigen-stimulated cells with antibody to the receptor beta subunit. We now present evidence that the 72-kDa Fc epsilon RI-associated protein is the protein-tyrosine kinase PTK72 that forms part of the antigen receptor complex in B lymphocytes. The identification is based on immunoreactivity with anti-PTK72 antiserum, chromatographic profiles on the affinity resin heparin/agarose, and one-dimensional phosphopeptide mapping studies. Enzymatic activity of the kinase is increased in anti-PTK72 immune complexes prepared from lysates of antigen-activated RBL-2H3 cells. The 72-kDa protein-tyrosine kinase is the principal substrate for in vitro tyrosine phosphorylation in anti-phosphotyrosine immunoprecipitates of RBL-2H3 cells. The discovery that RBL-2H3 mast cells share a receptor-activated protein-tyrosine kinase, PTK72, with B lymphocytes provides additional support for the existence of common signaling pathways initiated by multichain immune recognition receptors.
Different receptor mechanisms mediate the pyrogenic and behavioral effects of interleukin 1.
Authors: Kent S , Bluthe R M , Dantzer R , Hardwick A J , Kelley K W , Rothwell N J , Vannice J L
Published: May 2006

Interleukin 1 (IL-1) is a cytokine released during immune activation that mediates the host's response to infection and inflammation. Peripheral and central injections of IL-1 induce fever and sickness behavior, including decreased food motivation and reduced interest in social activities. To determine the receptor mechanisms responsible for these effects, rats were injected with IL-1 receptor antagonist (IL-1ra), an endogenous cytokine that acts as a pure antagonist of IL-1 receptors. IL-1ra blocked the increased body temperature and oxygen consumption induced by injection of recombinant human IL-1 only when both cytokines were administered i.p. In contrast, i.p. or intracerebroventricular administration of IL-1ra blocked the depressive effect of IL-1 beta on food-motivated behavior and social exploration when this cytokine was administered by the same route as the antagonist. In addition, intracerebroventricular IL-1ra blocked the reduction in social exploration produced by i.p. IL-1 beta but had only partial antagonist effects on the decrease in food-motivated behavior induced by i.p. IL-1 beta. In each case, the dose of IL-1ra was 100- to 1000-fold in excess of the biologically active dose of IL-1. These results suggest that the receptor mechanisms that mediate the behavioral and pyrogenic effects of IL-1 are heterogeneous.
B29 gene products complex with immunoglobulins on B lymphocytes.
Authors: Ishihara K , Wood W J , Damore M , Hermanson G G , Wall R , Kincade P W
Published: May 2006

B29 is a B-lineage-specific gene predicted from sequence information to be a transmembrane member of the immunoglobulin (Ig) superfamily, with a single extracellular Ig-like domain. Its presumptive cytoplasmic region contains a peptide motif present in CD3 and other molecules involved in lymphocyte activation. Affinity-purified goat antibodies were prepared to a TrpE fusion protein of B29 and used to study B29 expression on lymphoid cells. The antiserum precipitated surface-labeled heterodimers from B lymphoma cells. One was 65-88 kDa (unreduced) or 36-47 plus 32-34 kDa (reduced) by SDS/PAGE analysis, regardless of detergent. A smaller heterodimer was detected only with Triton detergent extraction. IgM molecules were coprecipitated by the B29 antiserum when the weak detergent digitonin was used. In addition, cocapping experiments revealed that most B29 molecules codistribute with Ig on the cell surface. Although early B-lineage cells and plasma cells contain B29 mRNA, surface expression was detectable only on B cells that had significant amounts of surface Ig. The surface expression was B-lineage-specific and included cells from mutant xid mice and B-cell lines representing mu, delta, gamma, and alpha heavy-chain isotypes and both kappa and lambda light-chain types. The density of surface B29 protein correlated directly with surface mu heavy-chain density on subclones of a B-cell lymphoma and lipopolysaccharide-stimulated pre-B cells. These findings show that B29 is covalently linked in a heterodimer and are consistent with a recently proposed model of surface Ig complexes.
Differential regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma.
Authors: Lindvall O , Ernfors P , Bengzon J , Kokaia Z , Smith M L , Siesjö B K , Persson H
Published: May 2006

In situ hybridization was used to study expression of mRNAs for members of the nerve growth factor (NGF) family in the rat brain after 2 and 10 min of forebrain ischemia and 1 and 30 min of insulin-induced hypoglycemic coma. Two hours after the ischemic insults, the level of brain-derived neurotrophic factor (BDNF) mRNA was markedly increased in the granule cells of the dentate gyrus, and at 24 h it was still significantly elevated. NGF mRNA showed a pronounced increase 4 h after 2 min of ischemia but had returned to a control level at 24 h. Both 2 and 10 min of ischemia caused a clear reduction of the level of mRNA for neurotrophin 3 (NT-3) in the dentate granule cells and in regions CA2 and medial CA1 of the hippocampus 2 and 4 h after the insults. The increase of BDNF mRNA could be partially blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist NBQX but was not influenced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. Both NBQX and MK-801 attenuated the decrease of NT-3 mRNA after ischemia. One and 30 min of hypoglycemic coma also induced marked increases in BDNF and NGF mRNA in dentate granule cells with maximal levels at 2 h. If the changes of mRNA expression lead to alterations in the relative availability of neurotrophic factors, this could influence functional outcome and neuronal necrosis following ischemic and hypoglycemic insults.
Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.
Authors: Lev S , Givol D , Yarden Y
Published: May 2006

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor.
Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro.
Authors: Luebke J I , Greene R W , Semba K , Kamondi A , McCarley R W , Reiner P B
Published: May 2006

Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons.