Identification of a highly conserved 3′ UTR in the translationally regulated mRNA for prostaglandin synthase

Prostaglandin H synthase (E.C. 1.14.99.1) is induced by growth factors and lymphokines such as EGF and IL-1, and is suppressed by anti-inflammatory glucocorticoids. Inhibition of enzyme synthesis by glucocorticoids is mediated by a novel translational control that appears to involve conversion of the PG synthase mRNA into a cryptic non-hybridizable form.

In order to understand expression of the enzyme in more detail, a full length 2.8 Kb cDNA was cloned from a human embryonic lung cell cDNA library and the complete mRNA including the 3′ untranslated region (3′ UTR), was sequenced. The coding sequence for the human PG synthase shows greater than 90% homology with the sheep and mouse enzymes. A high degree of conservation (70%), however, was also observed in the approximately 750 nucleotide sequence that comprises the 3′ non-coding domain of both sheep and human PG synthase mRNA's and with the approximately 900 nucleotide 3′ UTR of the mouse RNA (68% sheep vs mouse; 47% human vs mouse). Extensive microregions of 10–30 nucleotides are distributed throughout the 3′ UTR where homology between species is 95–100%.

This high degree of conservation in a non-coding region and recent evidence from other genes suggests that these 3′ UTR sequences have important regulatory functions possibly related to translational control of this mRNA by growth factors and glucocorticoids.     

Molecular Recognition Theory of the complementary (antisense) peptide interactions

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203–207; Nature Med. 1 (1995) 876–878; Biochem. J. 234 (1986) 679–683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5′→3′ direction) and antisense peptides coded in 3′→5′ direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide–receptor systems with verified ligand–receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an “in vivo” experiment. It was shown that 3′→5′ peptide antisense of -MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.     

Detection of an abasic site in RNA with stem-loop DNA beacons: application to an activity assay for Ricin Toxin A-Chain

The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the GAGA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “GAGA” sequence can be discriminated from the product containing an abasic “GabGA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Pyridoxal 5′-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5′-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5′-phosphate pools in the nuclei: a “mobile” pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5′-phosphate), and a “stable” pool, which was independent of cytoplasmic fluctuations of pyridoxal 5′-phosphate (about 9 pmol pyridoxal 5′-phosphate/mg DNA). Reduction in pyridoxal 5′-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 ± 19.7 vs 762 ± 291 fmol/mg DNA, mean ± SEM). The degree of 1,25-dihydrovitamin D accumulation in the nuclei appeared to be an exponential function of the “mobile” nuclear pyridoxal 5′-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5′-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r=−0.95). These data suggest that pyridoxal 5′-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5′-phosphate with the vitamin D receptor protein in the nuclei.     

On the discovery of interferon-inducible, double-stranded RNA activated enzymes: the 2'-5'oligoadenylate synthetases and the protein kinase PKR

The demonstration that double-stranded (ds) RNA inhibits protein synthesis in cell-free systems prepared from interferon-treated cells, lead to the discovery of the two interferon-induced, dsRNA-dependent enzymes: the serine/threonine protein kinase that is referred to as PKR and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which converts ATP to 2′,5′-linked oligoadenylates with the unusual 2′-5′ instead of 3′-5′ phosphodiesterase bond. We raised monoclonal and polyclonal antibodies against human PKR and the two larger forms of the 2′,5′-OAS. Such specific antibodies proved to be indispensable for the detailed characterization of these enzyme and the cloning of cDNAs corresponding to the human PKR and the 69–71 and 100 kDa forms of the 2′,5′-OAS. When activated by dsRNA, PKR becomes autophosphorylated and catalyzes phosphorylation of the protein synthesis initiation factor eIF2, whereas the 2′-5′OAS forms 2′,5′-oligoadenylates that activate the latent endoribonuclease, the RNAse L. By inhibiting initiation of protein synthesis or by degrading RNA, these enzymes play key roles in two independent pathways that regulate overall protein synthesis and the mechanism of the antiviral action of interferon. In addition, these enzymes are now shown to regulate other cellular events, such as gene induction, normal control of cell growth, differentiation and apoptosis.     

Copper-induced peroxidation of phosphatidylserine-containing liposomes is inhibited by nanomolar concentrations of specific antioxidants

Copper-induced peroxidation of liposomal palmitoyllinoleoyl-phosphatidylcholine (PLPC) is inhibited by -tocopherol at micromolar concentrations. In our previous study we found that when the liposomes contain phosphatidylserine (PS), nanomolar concentrations of Toc were sufficient to inhibit peroxidation. In an attempt to gain understanding of the origin of this extreme antioxidative potency, we tested the antioxidative potency of 36 additional antioxidants and the dependence of their potency on the presence of PS in the liposomes. The results of these studies reveal that only 11 of the tested antioxidants possess similar antioxidative potency to that of Toc. These include trolox, butylated hydroxytoluene (BHT), curcumin, nordihydroguaiaretic acid (NDGA), diethylstilbestrol (DES), 2 of the 13 tested flavonoids (luteolin and 7,3′,4′-trihydroxyflavone; T-414), -naphthol, 1,5-, 1,6- and 1,7-dihydroxynaphthalenes (DHNs). Propyl gallate (PG), methyl syringate, rosmarinic acid, resveratrol, other flavonoids, as well as β-naphthol, 1,2-, 1,3-, 1,4-, 2,3-, 2,6-, and 2,7-DHNs were either moderately antioxidative or pro-oxidative. For liposomes made of PLPC (250 μM) and PS (25 μM) the “lag” preceding copper-induced peroxidation (5 μM copper) was doubled upon addition of 30–130 nM of the “super-active” antioxidants.

We propose that the mechanism responsible for the extreme antioxidative potency against copper-induced peroxidation in PS-containing liposomes involves replenishment of the antioxidant in a ternary PS–copper-antioxidant complex. Based on structure–activity relationship of the 37 tested antioxidants, the “super-antioxidative potency” is attributed to the recycling of relatively stable semiquinone or semiquinone-like radicals.     

3′-Fluoro-3′-deoxyribonucleoside 5′-triphosphates: Synthesis and use as terminators of RNA biosynthesis

3′-Fluoro-3′-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5′- triphosphates, NTP(3′-F). It was shown that NTP(3′-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.     

DNA sequence of Nodulin-45 from Lupinus angustifolius

A partial cDNA clone encoding Lupinus angustifolius Nodulin-45 was isolated by differential hybridisation. A genomic clone was also isolated, from which the DNA sequence was obtained for the 5′ end of the gene (including 1.2 kb of 5′ upstream region). The upstream region includes putative cis-elements, found upstream of other nodulin genes. Southern analysis indicates the presence of several Nodulin-45-like sequences in the lupin genome. The Nodulin-45 protein has a putative N-terminal endoplasmic reticulum-type signal sequence and also contains a large glycine-rich repeat sequence. The cDNA sequence is highly homologous to a Nodulin-45 cDNA sequence from Lupinus luteus (Szczyglowski et al., Plant Sci., 65 (1989) 87–95), although major sequence rearrangements are apparent between the L. luteus and L. angustifolius cDNAs.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).     

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5′-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D′ boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2′-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2–3.     

A new species of Acaenodera (Digenea: Acanthocolpidae) parasitizing Conger orbignyanus (Pisces: Congridae) from the coasts of Argentina

A new species, Acaenodera spinosior, is described based on specimens collected from the intestine of Conger orbignyanus Valenciennes (Pisces: Congridae) caught in waters off Mar del Plata (38°08′S, 57°32′W), Argentina. Five of 36 congers examined were parasitized (prevalence: 13.9%, mean intensity: 2.6 ± 1.52, range: 1–5). The new species is distinguished from its only congener, Acaenodera placophora Manter and Pritchard, 1960, a parasite of congers off Hawaii, by its larger size, lacking eyespots, and exhibiting a greater number of circumoral, mid-ventral and lateral spines. This is the first record of Acaenodera Manter and Pritchard, 1960 in Atlantic waters.     

Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Cytokines regulate the affinity of soluble CD44 for hyaluronan

DNA enzymes are RNA-cleaving single-stranded DNA molecules. We designed DNA enzymes targeting the PB2 mRNA translation initiation (AUG) region of the influenza A virus (A/PR/8/34). The modified DNA enzymes have one or two N3′-P5′ phosphoramidate bonds at both the 3′- and 5′-termini of the oligonucleotides, which significantly enhanced their nuclease resistance. These modified DNA enzymes had the same cleavage activity as the unmodified DNA enzymes, determined by kinetic analyses, and reduced influenza A virus replication by more than 99%, determined by plaque formation. These DNA enzymes are highly specific; their protective effect was not observed in influenza B virus (B/Ibaraki)-infected Madin–Darby canine kidney cells.