Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules.

While the strong biotin-avidin interaction has been widely used for the detection of biomolecules, its irreversibility complicates their isolation. We report the synthesis of a photocleavable biotin derivative (PCB) which eliminates many limitations of existing methods. This reagent contains a biotin moiety linked through a spacer arm to a photocleavable moiety, which reacts selectively with primary amino groups on any substrate. In experiments using [leucine]-enkephalin as a model substrate, we show that PCB retains its high affinity toward avidin/streptavidin and allows rapid (< 5 min) and efficient (> 99%) photorelease of the substrate in a completely unaltered form. Photocleavable biotins should be useful in numerous applications involving the isolation of proteins, nucleic acids, lipids, and cells.

Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.

Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.

A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.

The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.

Ganglioside GM1 binds to the Trk protein and regulates receptor function.

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins.

Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores called exochelins. Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium. In this paper, we describe the purification of exochelins of M. tuberculosis and their characterization by mass spectrometry. Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state. The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain. These series further subdivide into serine- or threonine-containing species. The virulent M. tuberculosis Erdman strain and the avirulent M. tuberculosis H37Ra strain produce a similar set of exochelins. Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins. However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester. These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.