Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production,Isolation and Pharmacological Studies of AI-77s

Two potent gastroprotective substances against experimental gastric ulcers in rats induced by stress and five of their analogues were isolated from the culture broth of bacterial strain AI-77 which was classified taxonomically as Bacillus pumilus. Physico-chemical properties and pharmacological activities of the seven compounds were examined and compared with each other.     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When -adrenoceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)