Retinoid X receptor-selective ligands produce malformations in Xenopus embryos.

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Pigments destroyed during photoinhibition of water-splitting photosystem II core complexes from the green alga Chlamydomonas reinhardtii were studied. Under conditions of a transiently inactivated donor side, illumination leads to an irreversible inhibition of the electron transfer at the donor side that is paralleled by the destruction of chlorophylls a absorbing maximally around 674 and 682 nm. The observed stochiometry of 1 +/- 0.1 destroyed chlorophyll per inhibited photosystem II suggests that chlorophyll destruction could be the primary photodamage causing the inhibition of photosystem II under these conditions.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands.

Two dodecapeptides belonging to distinct classes of Src homology 3 (SH3) ligands and selected from biased phage display libraries were used to investigate interactions between a specificity pocket in the Src SH3 domain and ligant residues flanking the proline-rich core. The solution structures of c-Src SH3 complexed with these peptides were solved by NMR. In addition to proline-rich, polyproline type II helix-forming core, the class I and II ligands each possesses a flanking sequence that occupies a large pocket between the RT and n-Src loops of the SH3 domain. Structural and mutational analyses illustrate how the two classes of SH3 ligands exploit a specificity pocket on the receptor differently to increase binding affinity and specificity.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal glycine of an invariably conserved GXXGXXK motif present in all steroid and phenol sulfotransferases for which primary structures are known. This consistency strongly suggests that all steroid and phenol sulfotransferase genes will be similarly spliced. The GXXGXXK motif forms the active binding site for the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate. Amino acid sequence alignment of 19 cloned steroid and phenol sulfotransferases starting with the GXXGXXK motif indicates that the 3'-terminal exon for each steroid and phenol sulfotransferase gene encodes a similarly sized C-terminal fragment of the protein. Interestingly, on further analysis of the alignment, three distinct amino acid sequence patterns emerge. The presence of the conserved functional GXXGXXK motif suggests that the protein domains encoded by steroid and phenol sulfotransferase 3'-terminal exons have evolved from a common ancestor. Furthermore, it is hypothesized that during the course of evolution, the 3'-terminal exon further diverged into at least three sulfotransferase subdivisions: a phenol or aryl group, an estrogen or phenolic steroid group, and a neutral steroid group.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.

A multimer model for P680, the primary electron donor of photosystem II.

We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins. Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair. We find that this model is consistent with many experimental studies of PS II. The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous. This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor. We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.