Regional distribution and alterations of lectin binding to colorectal mucin in mucosal biopsies from controls and subjects with inflammatory bowel diseases.

Glycoconjugate composition of colorectal goblet cell mucin was characterized according to the anatomical distribution of lectin-binding sites in mucosal biopsies from 35 control subjects and 55 patients with inflammatory bowel disease. 24 of the controls had mucosal inflammation on biopsy, without clinical evidence of inflammatory bowel disease. These inflamed controls showed a similar rate of presence of lectin-binding sites as the normal noninflamed group. In the controls, the frequency of binding of Ricinus communis agglutinin I to galactosyl residues was consistently higher than that found with either Ulex europaeus agglutinin I to fucosyl or Dolichus biflorus agglutinin to N-acetyl galactosyl groups. A significant proximal to distal gradient for Ulex europaeus agglutinin I binding sites was identified in the controls group. These binding sites were present four times more often in the proximal colon than in the distal colon (P less than 0.025). In the ulcerative and Crohn's colitis groups, this gradient effect was lost, predominantly as a result of decreased availability of fucosyl residues in the proximal colon. In the descending colon of Crohn's colitis tissues, there was a complete absence of Dolichus biflorus agglutinin binding sites compared with the 62.5% incidence in the control group (P less than 0.05). These results demonstrate that the expression of lectin-binding sites in human large intestinal goblet mucin is specifically altered in inflammatory bowel disease, indicating that there are changes in glycosylation of colorectal mucin consistent with alterations in goblet cell differentiation.

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge.

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.

Hemodynamic effects of morphine and nalbuphine in acute myocardial infarction.

Hemodynamic effects of morphine and the new narcotic analgesic, nalbuphine, were compared in a randomized, double-blind study in 15 patients with acute myocardial infarction (11 men and four women, average age 56.2 yr) and normal group mean hemodynamic function. During a 1-hr evaluation the hemodynamic effects were small but there were changes in several parameters. Morphine reduced heart rate (78 to 72 bpm, p less than 0.01) and diastolic and mean arterial pressures (69 to 64 mm Hg, p less than 0.05; and 91 to 84 mm Hg, p less than 0.05); nalbuphine was associated with a decrease in heart rate (82 to 72 bpm, p less than 0.01), decrease in cardiac index, which remained within the normal range (3.16 to 2.75 l/min/m(2), p less than 0.01), and an increase in systemic vascular resistance (1,204 to 1,461 dynes . sec . cm(-5), p less than 0.05). Neither drug altered systolic arterial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, stroke index, stroke work index, or pulmonary vascular resistance. Echocardiographic assessment revealed diminution of left ventricular mean velocity of circumferential fiber shortening after nalbuphine (1.26 to 1.08 circ/sec, p less than 0.05). Both drugs induced small reductions in respiratory rate and arterial pH and increases in PAO2. There were no changes in PaO2. Because of the absence of clinically important deleterious effects on cardiac pump function, nalbuphine merits further investigation as an analgesic in acute myocardial infarction.

Chemical nucleases: their use in studying RNA structure and RNA-protein interactions.

Metal complexes that cleave nucleic acids provide a new means to study RNA structure and RNA-protein interactions. Methods that use these chemical nucleases help compensate for the limitations of other techniques used to determine structure. Because the ligands that coordinate the metal generally control the cleavage selectivity of these complexes, it has become possible to design nucleolytic reagents that target specific higher-order structures. In combination with site-directed mutagenesis these conformation-specific probes can be used to delineate long-range interactions. Alternatively, complexes that cut irrespective of sequence and secondary structure have been used in protection (foot-printing) experiments to locate protein binding sites. Because each position of the nucleic acid is susceptible to cleavage, the protection pattern yields a highly resolved definition of the contact site between the protein and RNA. In other applications, metal complexes have been conjugated to functional moieties such as oligonucleotides, peptides, or substrate analogs to direct their binding to a distinct site on a specific RNA molecule. This latter strategy holds significant therapeutic promise for the destruction of pathogenic RNAs.

Little risks and big fears.

Various aspects of regulatory decisions involving the concepts of "unacceptably hazardous" and "acceptably safe" as related to the growing emphasis on the legal terminology of de minimis are outlined. It is concluded that with rare exceptions a proper public policy for risk evaluations is far from implementation.

Kinetic studies of the reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate.

The reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate (NHxDPH) has been examined by stopped-flow kinetic methods. Like reduced glutathione or NADPH, this pyridine nucleotide generates the catalytically active two-electron reduced form of the enzyme. This reductive half-reaction with NHxDPH has only one detectable kinetic step which shows saturation kinetics (Kd = 76 microM), and has a limiting rate constant of 56 s-1. Comparison of stopped-flow and steady-state turnover data indicates that the reductive half-reaction is rate-limiting in the overall catalytic reaction. No evidence was found for a preequilibrium charge-transfer complex between NHxDPH and the active site FAD, like that seen when NADPH is the electron donor.

Identification of the binding site on 5S rRNA for the transcription factor IIIA: proposed structure of a common binding site on 5S rRNA and on the gene.

Transcription factor IIIA interacts specifically with an internal control region of Xenopus 5S ribosomal RNA genes and is also a component, along with 5S rRNA, of a 7S ribonucleoprotein particle present in previtellogenic oocytes. We have determined the region of the 5S rRNA in the 7S ribonucleoprotein complex that is protected by the transcription factor from digestion with the ribonuclease alpha-sarcin. The binding site for factor IIIA extends from nucleotide 64 through nucleotide 116; the protected region includes two CCUGG helices separated by 11 nucleotides. The same helices occur in the factor IIIA binding site in the 5S rRNA gene and may constitute a common structural feature recognized by the protein in the gene and in the gene product.

Use of the cytotoxic nuclease alpha-sarcin to identify the binding site on eukaryotic 5 S ribosomal ribonucleic acid for the ribosomal protein L5.

Treatment of the large subunit of eukaryotic ribosomes with EDTA releases a 7 S ribonucleoprotein complex that contains protein L5 and 5 S rRNA. The region of rat 5 S rRNA that is protected by protein L5 from digestion with the ribonuclease alpha-sarcin includes nucleotides 1-10, 12-22, and 64-116. This domain approximates the combined binding sites for the three proteins (L5, L18, and L25) that associate with Escherichia coli 5 S rRNA. In addition, the region of 5 S rRNA protected by rat ribosomal protein L5 corresponds closely to that occupied by Xenopus transcription factor IIIA.

A role for aromatic amino acids in the binding of Xenopus ribosomal protein L5 to 5S rRNA.

The formation of the Xenopus L5-5S rRNA complex depends on nonelectrostatic interactions. Fluorescence assays with 1-anilino-8-naphthalenesulfonate demonstrate that a hydrophobic region on L5 becomes exposed upon removal of bound 5S rRNA by treatment with ribonucleases. Several conserved aromatic amino acids, mostly tyrosines, were identified by comparative sequence analysis and changed individually to alanine. Substitution with alanine at any of three positions, Y86, Y99, or Y226, essentially abolishes RNA-binding activity, whereas those made at Y95 and Y207 have more modest effects. Replacement with phenylalanine at Y86 and Y226 does not change binding affinity, indicating that the aromatic ring of the side chain, not the hydroxyl group, is the critical functionality for binding. Alternatively, the phenolic hydroxyls at Y99 and Y207 do contribute to binding. The structural integrity of the mutant proteins was assessed using thermal denaturation and limited digestion with proteases. The T(m) of Y99A is 10 degrees C lower than that of the wild-type protein, and there are some differences in the protease digestion patterns that together indicate the structure of this mutant has been significantly perturbed. The structures of the other variants are not detectably different from the wild-type protein. These results provide evidence that intermolecular stacking interactions involving at least two tyrosine residues, Y86 and Y226, are necessary for formation of the L5-5S rRNA complex and can account, at least in part, for the contribution nonelectrostatic interactions make to the free energy of binding.

The use of chemical nucleases to analyze RNA-protein interactions. The TFIIIA-5 S rRNA complex.

The complex of Xenopus transcription factor IIIA (TFIIIA) with 5 S rRNA was analyzed in nuclease protection experiments using hydroxyl radical. The protection pattern reveals that TFIIIA interacts with a substantial amount of the RNA molecule, with close association between the factor and the arm of the RNA composed of helix IV-loop E-helix V. Additional sites of protection punctuate the other arm of the molecule. Important contact sites within the complex were identified in "missing nucleoside" experiments. Random single-nucleoside gaps were introduced into 5 S rRNA using either Fe[EDTA]2-/H2O2 or bis(1,10-phenanthroline)copper(I). This modified RNA was allowed to bind to TFIIIA in an exchange reaction, and, afterward, bound and unbound fractions were separated on nondenaturing polyacrylamide gels. Missing nucleoside positions specifically enriched in the unbound fraction of RNA are located in the two strands that comprise loop E. These are not necessarily sites of sequence-specific contacts, but rather may constitute a region of secondary structure essential to recognition and binding of TFIIIA to 5 S rRNA.

Analysis of the binding of Xenopus ribosomal protein L5 to oocyte 5 S rRNA. The major determinants of recognition are located in helix III-loop C.

Xenopus ribosomal protein L5 was expressed in Escherichia coli and exhibits high affinity (Kd = 2 nM) and specificity for oocyte 5 S rRNA. The pH dependence of the association constant for the complex reveals an ionization with a pK alpha value of 10.1, indicating that tyrosine and/or lysine residues are important for specific binding of L5 to the RNA. Formation of the L5.5 S rRNA complex is remarkably insensitive to ionic strength, providing evidence that nonelectrostatic interactions make significant contributions to binding. Together, these results suggest that one or more tyrosine residues may form critical contacts through stacking interactions with bases in the RNA. In order to locate recognition elements within 5 S rRNA, we measured binding of L5 to a collection of site-specific mutants. Mutations in the RNA that affected the interaction are confined to the hairpin structure comprised of helix III and loop C. Earlier experiments with a rhodium structural probe had shown that the two-nucleotide bulge in helix III and the intrinsic structure of loop C create sites in the major groove that are opened and accessible to stacking interactions with the metal complex. In the present studies, we detect a correlation between the intercalative binding of the rhodium complex to mutants in the hairpin and binding of L5, supporting the proposal that binding of the protein is mediated, in some part, by stacking interactions. Furthermore, the results from mutagenesis establish that, despite overlapping binding sites on 5 S rRNA, L5 and transcription factor IIIA utilize distinct structural elements for recognition.

Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

A rapid and convenient method has been devised to determine the binding sites for proteins on RNA. The procedure is an adaptation of one used to map DNA-protein complexes by protection against nuclease digestion. The method uses the cytotoxic ribonuclease alpha-sarcin, which hydrolyzes purines in both single- and double-stranded regions of RNA. It has been authenticated by confirming the binding sites for the Escherichia coli ribosomal proteins L18 and L25 on 5S rRNA and its value has been established by identifying the attachment site for protein L5. The procedure should be useful for the analysis of other ribonucleoprotein complexes.

Conformational studies of the nucleic acid binding sites for Xenopus transcription factor IIIA.

The CD spectrum of a restriction fragment that contains a single copy of a Xenopus borealis somatic 5 S rRNA gene, like those of two smaller fragments from the binding site for transcription factor IIIA (TFIIIA), is that of B-form DNA. Under dehydrating conditions (76% ethanol) the 5 S rDNA undergoes a transition into an A conformation. The spectra of the three fragments, however, do exhibit some perturbation in that the longwave positive peaks are shifted to shorter wavelengths and have enhanced rotational strength compared with reference B-form spectra. The helical repeats measured for the smaller fragments indicate that the helix winding angle can account for these features in the CD spectra. We suggest that G:C-rich boxes that punctuate not only the TFIIIA binding site but the whole 5 S gene are responsible for the conformational perturbation manifest in the CD spectra and may play a role in the recognition of the DNA by the factor. The spectrum of the gene is unchanged in the presence of TFIIIA, indicating that the structural heterogeneity of the DNA persists upon complex formation. The CD spectra of native TFIIIA.5 S rRNA particles isolated from oocytes and of particles reconstituted in vitro are identical and only moderately different from the spectrum of free 5 S rRNA, suggesting that the protein effects only limited changes in the secondary and/or tertiary structure of the RNA. The helical structure of the two binding sites is discussed with respect to a common mode of interaction of TFIIIA with DNA and RNA.

Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity.

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.

Analysis of the binding of Xenopus transcription factor IIIA to oocyte 5 S rRNA and to the 5 S rRNA gene.

Binding of transcription factor IIIA (TFIIIA) to site-specific mutants of Xenopus oocyte 5 S rRNA has been used to identify important recognition elements in the molecule. The putative base triple G75:U76:A100 appears to determine the conformation of the loop E region whose integrity is especially important for binding of the factor. Proximal substitutions in helices IV and V indicate that the proper folding of loop E is also dependent on these structures. Mutations in helix V affect binding of TFIIIA to 5 S rRNA and to the gene similarly and provide evidence that zinc finger 5 makes sequence-specific contact through the major groove of both nucleic acids. Although fingers 1-3 are positioned along helix IV and loop D, mutations in this region, including those that disrupt the tetraloop or close the opening in the major groove of the helix created by the U80:U96 mismatch, have no impact on binding. Substitutions made at stem-loop junctions in the arm of the RNA comprised of helix II-loop B-helix III display minor decreases in affinity for TFIIIA. Despite the alignment of the factor along nearly the entire length of 5 S rRNA, the essential elements for high affinity binding are limited to the central region of the molecule. Analysis of the corresponding mutations in the gene confirm that box C and the intermediate element provide the high affinity sites for binding of the factor to the DNA. Despite the small thermodynamic contribution made by contacts to box A, mutations made in this element can cause substantial changes in the orientation of the carboxyl-terminal fingers along the 5'-end of the internal control region.

The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.

The ribonuclease activity of the cytotoxic protein alpha-sarcin has been characterized. When rat liver ribosomes or 60 S ribosomal subunits were the substrates, alpha-sarcin cleaved a single oligonucleotide of about 488 residues, the alpha-fragment, from the 3' end of 28 S rRNA. In contrast, 40 S ribosomal subunits were not affected by alpha-sarcin. The alpha-fragment was cleaved from 28 S rRNA in 80 S ribosomes when the concentration of alpha-sarcin was 3 x 10(-8) M and the toxin retained its specificity even when the concentration was 3 x 10(-5) M. The turnover number (kcat) for the reaction of alpha-sarcin with ribosomes was 55 min-1, establishing that the toxin acts catalytically. When total rRNA or 28 S rRNA was the substrate, alpha-sarcin caused extensive progressive digestion of the nucleic acids; however, no formation of the alpha-fragment occurred. The extent of the digestion of 28 S rRNA was related to the concentration of alpha-sarcin, but the amount of the toxin required was somewhat greater than that needed with ribosomes. Digestion of homopolynucleotides with alpha-sarcin indicated that the protein is specific for purines. When [32P]5 S rRNA was the substrate, alpha-sarcin cleaved on the 3' side of purines in both single- and double-stranded regions of the molecule. The results suggest that the unusual specificity of alpha-sarcin, in that it cleaves only one of more than 7000 phosphodiester bonds in the ribosome, is a property both of the cytotoxin and of the ribosome.