Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Oral shark cartilage does not abolish carcinogenesis but delays tumor progression in a murine model.

BACKGROUND: Shark cartilage and shark cartilage extracts have been reported to have anti-angiogenic and anti-neoplastic properties. This study reports the effects of oral administration of powdered shark cartilage on tumor progression in a murine renal tumor model. MATERIALS AND METHODS: Renal tumors were induced in CBA female mice by a single bolus of IV streptozotocin. 57 mice were fed shark cartilage and the numbers and rate of development of dysplastic convoluted tubules, papillary and solid renal epithelial tumors was compared with 57 control mice over an 88 week follow-up period. RESULTS: In the shark cartilage fed group dysplasia was first observed after 23 weeks (control 19 weeks), papillary tumors after 24 weeks (control 23 weeks) and solid tumors after 55 weeks (control 19 weeks). There was no significant difference in the rate of development of dysplastic tubules between test and control animals. The development of papillary and solid tumors was significantly delayed in the test group. CONCLUSIONS: In this tumor model oral shark cartilage delays, but does not abolish, tumor progression.

[Mycoplasma pneumoniae meningoencephalitis]. / Méningoencéphalite à Mycoplasma pneumoniae.

BACKGROUND: Severe central nervous system diseases, such as encephalitis, have been reported in association with Mycoplasma pneumoniae infections. CASE REPORT: After an ENT infection, a 9-year-old boy with Down's syndrome developed encephalitis revealed by an acute alteration in consciousness. Head computed tomography showed, after 2 weeks, an infiltration in the basal ganglia region. The diagnosis of Mycoplasma pneumoniae encephalitis was made; recovery was complete in a few weeks. CONCLUSION: Mycoplasma pneumoniae infection should be considered in all cases of acute encephalopathy; yet the pathogenesis of the disorder is unknown and the treatment uncertain.

Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.

The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage.

A series of 2 stranded hammerhead ribozymes has been synthesized in which single conserved adenosine residues have been replaced by nebularine or single guanosine residues by inosine. Comparison of the rates of trans-cleavage for the modified structures suggests the presence of interstrand non-Watson-Crick hydrogen bonding interactions including a GA:AG double mismatch. The exocyclic amino group of one of the guanosine residues is essential for cleavage.

Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors.

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.

Synthesis of site-specifically modified oligoribonucleotides for studies of the recognition of TAR RNA by HIV-1 tat protein and studies of hammerhead ribozymes.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.

Solid-phase synthesis of oligoribonucleotides.

An efficient method is described for solid-phase synthesis of oligoribonucleotides that involves use of the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection, 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection and a phosphoramidite coupling procedure.