Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, we compare in vitro the relative sensitivities of rabbit and human alpha- and beta-globin mRNAs to translational inhibition by cap analogues. We find that rabbit beta-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit alpha-globin mRNA, while in contrast, human beta-globin mRNA is more sensitive to cap analogue inhibition than human alpha-globin mRNA. This opposite pattern of translational inhibition by cap analogues of the rabbit and human alpha- and beta-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the alpha- and beta-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation.     

Interaction between the synthesis of alpha and beta globin.

We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.     

Separation of alpha and beta chains of rabbit globin using SDS-polyacrylamide gel electrophoresis

A procedure to separate the α and β globin chains of rabbit hemoglobin, denatured with sodium dodecyl sulfate in the presence of mercaptoethanol, on a column of polyacrylamide gel was developed. The identity of the two separated chains was verified by (a) differences in distribution of radioactivity between the chains when the hemoglobin samples were labeled uniformly with various ³H- or ¹⁴C-labeled amino acids; (b) the analysis of the chain distribution of radioactivity in purified hemoglobin isolated from rabbit reticulocytes, pulse-labeled with [³H] leucine; and (c) the separation pattern of a mixture of authentic [α-³H]- and [β-¹⁴C]-labeled globin chains. The globin chains of human hemoglobin A also could be separated in a similar manner. This procedure is particularly useful when only microgram quantities of hemoglobin are available for study.     

alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition.

alpha and beta-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12: the observed melting temperatures are 27 degrees C for beta-oligodeoxyribonucleotide/RNA hybrid and 53 degrees C for alpha-oligodeoxyribonucleotide/RNA. alpha-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5'-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:alpha- or beta-d(G2T12G2) hybrids or on mRNA:alpha- or beta-oligonucleotides hybrids. Specific hybridization protects RNA strech when using alpha-oligonucleotides but not beta-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in alpha-oligodeoxyribonucleotides:RNA duplexes.     

PPAR-gamma-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.     

Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.     

Convenient synthesis and evaluation of glycosidase inhibitory activity of alpha- and beta-galactose-type valienamines, and some N-alkyl derivatives

Valienamine analogues having alpha- and beta-galactose-type structures were synthesized by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol. Four N-alkyl derivatives of the beta-anomer were readily prepared selectively by treatment of key intermediate 2,6-di-O-acetyl-3,4-O-isopropylidene-5a-carba-alpha- and beta-l-arabino-hex-5(5a)-enopyranosyl bromides with alkyl amines. All compounds were assayed for inhibitory activity against six glycosidases, and the N-dodecyl derivative was shown to be a very strong inhibitor of beta-galactosidase (IC(50) 0.01 microM, bovine liver).     

Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.     

Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

Polyhalogenobenzimidazoles: synthesis and their inhibitory activity against casein kinases

A series of novel polyhalogenated benzimidazoles have been prepared by exhaustive bromination of a variety of 2-substituted benzimidazoles. The efficacy of both new compounds and a number of their previously described cognates as inhibitors of casein kinases CK1, CK2 and G-CK was investigated. The type of N-1 alkyl substituent as well as introduction of a polyfluoroalkyl moiety at position 2 did not markedly influence the inhibitory efficacy toward CK2 of the respective 4,5,6,7-tetrabromobenzimidazole derivatives which conversely were almost ineffective toward CK1 and G-CK. However, 4,5,6,7-tetrabromobenzimidazoles substituted at position 2 with either chlorine, bromine or sulfur atom, while manifesting a still considerable inhibitory activity against CK2 (IC(50) in the 0.49-0.93 microM range) proved to be potentially powerful inhibitors also against CK1 (IC(50) in the 18.4-2.2 microM range).     

4-Alkyliden-beta-lactams conjugated to polyphenols: synthesis and inhibitory activity

A series of compounds combining the beta-lactam and polyphenol scaffold have been prepared and evaluated for inhibition of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on the 'overlapping-type' strategy where two pharmacophores are linked in a single molecule. The most powerful compound against elastase was an N-galloyl-4-alkyliden beta-lactam, [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene]-acetic acid ethylester, with an IC50 of 0.5 microM; while the most powerful against MMP-2 was a 4-alkyliden beta-lactam arylated on the C-3 hydroxy side chain (3,5-bis-benzyloxy-4-hydroxy-benzoic acid 1-(2-benzyloxycarbonylmethylene-4-oxo-azetidin-3-yl)-ethyl ester) with an IC50 of 4 microM. Of the total 35 compounds tested, high levels of inhibition of elastase and of MMPs were separately exerted by distinct molecules.     

Effect of structural variations in cholesteryl-conjugated oligonucleotides on inhibitory activity toward HIV-1.

A number of oligonucleotide analogues containing internucleoside phosphorothioate linkages and a covalently attached cholesteryl residue was synthesized and tested for activity against HIV-1 in cultures of Molt3 cells. Structural features important for high antiviral activity are the presence of a cholesteryl moiety, a run of terminal phosphorothioate groups, and the presence of nucleoside residues. An increase in length of the tether between cholesteryl and phosphorus from six to 14 atoms has no significant effect on antiviral activity, and up to one-half of the internucleoside links in a cholesteryl-conjugated phosphorothioate oligomer and one-third of the internucleoside links in a nonconjugated phosphorothioate can be replaced with phosphodiester links without much change in antiviral activity. However, replacement of nucleoside units in the oligomers by a simple analogue (-OCH2CH2CH2O-) yields inactive or very weakly active compounds, even in the presence of a cholesteryl group. Dose-response patterns for assays in which cholesteryl-conjugated oligomers are added to test cells either simultaneously or subsequently to viral infection are similar for homooligomer derivatives and for oligomers containing "antisense" sequences, suggesting a similarity in mode of action for the two classes of oligomers in this system.     

Antisense inhibitory effect: a comparison between 3'-partial and full phosphorothioate antisense oligonucleotides.

Phosphorothioate (PS) antisense oligonucleotides are currently used to inhibit many cell functions both in vivo and in vitro. However, these modified oligos provide reasonable sequence specificity only within a narrow concentration range. To overcome such a limitation we synthesized antisense oligomers, partially phosphorothioated, targeted against the human N-myc mRNA. We utilized such modified oligomers in a human neuroblastoma cell line where the N-myc gene expression was very high, and compared them to full phosphorothioate oligonucleotides. Both full PS and partial PS antisense oligos produced a maximum reduction in target mRNA after 6 h of treatment. They were able to maintain a good level of inhibition for 20 h only at high concentration. While partial PS oligos produced a dose dependent and sequence specific inhibition of N-myc mRNA, full PS molecules suffer from some disadvantages at the highest concentration used. Our results showed that partial PS molecules were capable of reducing gene expression showing a greater sequence specificity over a far broader concentration range. For this reason we conclude that partial PS antisense oligos, with respect to full PS antisense oligos, might be particularly useful for studying gene function.     

Cellular delivery and biological activity of antisense oligonucleotides conjugated to a targeted protein carrier

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here, we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with poly(ethylene glycol) (PEG) chains that terminate in an RGD ligand that selectively binds the alphavbeta3 integrin. As a test system, we utilized human melanoma cells that express the alphavbeta3 integrin and that also contain a luciferase reporter gene that can be induced by delivery of SSOs to the cell nucleus. The RGD-PEG-SSO-albumin conjugates were endocytosed by the cells in an RGD-dependent manner; using confocal fluorescence microscopy, evidence was obtained that the SSOs accumulate in the nucleus. The conjugates were able to robustly induce luciferase expression at concentrations in the 25-200 nM range. At these levels, little short-term or long-term toxicity was observed. Thus, the RGD-PEG-albumin conjugates may provide an effective tool for targeted delivery of oligonucleotides to certain cells and tissues.