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V Toome

Publications and source records attributed to V Toome.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA.

Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.

Cell Line↗
PubMed1992

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.

Amino Acid Sequence↗
PubMed1992

Stereoselective synthesis of (24R and 24S) 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols and (25R and 25S) 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentols using a modified osmium-catalyzed Sharpless asymmetric dihydroxylation process.

Described herein are the stereoselective syntheses of the (24R, 24S) and (25R, 25S) isomers of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentols by using a modified osmium-catalyzed Sharpless asymmetric dihydroxylation process. Also presented herein are the results of lanthanide-induced CD Cotton effect measurements and 1H- and 13C-nuclear magnetic resonance studies of (24R, 24S) and (25R, 25S)-5 beta-cholestanepentols and their derivatives. These compounds were required to study the biosynthesis of cholic acid from cholesterol.

Cholestanols↗
PubMed1990

aS,7S-absolute configuration of natural (-)-colchicine and allo-congeners.

The aS,7S-absolute configuration of (-)-colchicine (1) and (-)-N-acetylcolchinol methyl ether (3, NCME) suggested on the basis of 1H NMR data and negative Cotton effects at about 260 nm (EtOH) is firmly established by an X-ray analysis of urea 5, a compound derived from 3. Binding of these compounds to tubulin requires an aS-configuration of the biaryl system.

Colchicine↗
PubMed1990

Purification and characterization of recombinant Rev protein of human immunodeficiency virus type 1.

Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.

Chromatography, Gel↗
PubMed1989

Solution structure of an analogue of vasoactive intestinal peptide as determined by two-dimensional NMR and circular dichroism spectroscopies and constrained molecular dynamics.

Structures have been determined for a potent analogue of vasoactive intestinal peptide (VIP), Ac-[Lys12, Lys14, Nle17, Val26, Thr28]VIP (VIP'), in methanol/water solutions. In CD studies, both VIP and VIP' were helical in methanol/water, with the percentage of alpha-helix increasing with percentage methanol. The pH had little effect on the structure. Complete 1H NMR assignments were made for VIP' in 25% methanol at pH 4 and 6 and in 50% methanol at pH 6, using two-dimensional COSY, NOESY, and relay-COSY experiments. There were no widespread changes in chemical shifts between the samples at pH 4 and 6; however, widespread changes were observed between the samples in 25% and 50% methanol. Complete sets of NOEs were obtained for VIP' in 25% methanol, pH 4, and in 50% methanol, pH 6. These NOEs were converted into distance constraints and applied in molecular dynamics and energy minimization calculations using the program CHARMM. A set of low-energy structures was obtained for VIP' in each solvent system. In 25% methanol, VIP' has two helical segments at residues 9-17 and 23-28. The remainder of the structure is not well determined. In 50% methanol, residues 8-26 form a regular, well-defined alpha-helix and residues 5-8 form a type III beta-turn. The remaining residues are not ordered. These structural assessments agree with the CD data. In the lowest energy structure in 50% methanol, the side chains of Asp3, Phe6, Thr7, and Tyr10 are clustered together--these residues are conserved throughout the family of peptide hormones homologous to VIP.

Circular Dichroism↗
PubMed1988

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.

Binding, Competitive↗
PubMed1988

Synthesis, biological activity and conformational analysis of cyclic GRF analogs.

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.

Animals↗
PubMed1986

Precursors of the mammalian synthesis of morphine: (+)-salutaridine and (-)-thebaine from (+)-6-demethylsalutaridine, and (-)-N-13CH3-thebaine from (-)-northebaine.

Standard samples of pure (+)-salutaridine and (-)-thebaine required to study the mammalian origin of morphine, were prepared from (+)-6-demethylsalutaridine by published procedures and were characterized by CD spectra and physical data. Reductive N-methylation of (-)-northebaine afforded (-)-thebaine, and when 13C-labeled formalin was used, (-)-thebaine with a 13C label on the N-methyl carbon atom resulted. The latter represents a model procedure to prepare ultimately N-14CH3-labeled (-)-thebaine and 14C-labeled congeners.

Animals↗
PubMed1986

Configuration at C-25 in 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol excreted by patients with cerebrotendinous xanthomatosis: circular dichroism and 13C-NMR studies.

The configuration at C-25 in 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol isolated from the bile and feces of patients with cerebrotendinous xanthomtosis (CTX) was determined from the lanthanide-induced circular dichroism (CD) Cotton effects and 13C-NMR measurements. Under anhydrous conditions, CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol in the presence of Eu(fod)3 exhibited a large induced negative Cotton effect at 320 nm. On the basis of the empirical rule (primary-tertiary-alpha-diols) in which R compounds have positive Cotton effects and S compounds have negative Cotton effects at 320 nm, it was concluded that 25,26-pentol has the 1,2,glycol structure with C-25 having the S-configuration. This assignment was based upon comparison with model compounds, 25(R and S),26-dihydroxy cholesterols and 25(R and S),26-dihydroxy cholecalciferols whose single-crystal X-ray structure and 13C-NMR studies have been performed. It is suggested that these data may be helpful to clarify the stereospecificity of the hydroxylation of the terminal methyl group of the cholesterol side chain in CTX.

Brain Diseases↗
PubMed1985

Synthesis and structure of 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol isolated from the urine and feces of a patient with sitosterolemia and xanthomatosis.

The urine and feces of a patient with the rare inherited lipid storage disease, sitosterolemia and xanthomatosis, were analyzed. Substantial quantities of C26-bile alcohol, 26 (or 27)-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24S,25 xi-pentol along with 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24R,25-pentol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol were found. The structure of the C26-bile alcohol was confirmed by direct comparison (gas-liquid chromatography-mass spectrometry and thin-layer chromatography) with a standard sample synthesized from cholic acid. The configurational assignment at C-24 was determined by lanthanide-induced circular dichroism Cotton effect measurements. The increased excretion of these C26- and C27-bile alcohols suggests an abnormality of bile acid biosynthesis in this disease.

Adolescent↗
PubMed1983

Application of MDPF and fluorescamine XV chiroptical properties of MDPF condensation compounds with dipeptides in situ.

2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) reacts readily with the free amino group of a dipeptide to form a pyrrolinone-type chromophore with absorption maxima at 275-285 and 370-390 nm. A simple test tube procedure is described which allows in situ correlation of the absolute configuration of the NH2-terminal amino acid of a dipeptide with the chiroptical properties of its chromophoric derivative. In several cases, unexpected deviation of the chiroptical characteristics from previously established empirical rules is observed.

Circular Dichroism↗
PubMed1983

Ro 22-5417, a new clavam antibiotic from Streptomyces clavuligerus. III. Absolute stereochemistry.

The complete stereostructure of the new antibiotic Ro 22-5417 has been established as 3-[(3S,5S)-7-oxo-1-aza-4-oxabicyclo[3.2.O]hept-3-yl]-L-alanine. This result together with the synthesis of an (3R,5R)-L-analog allowed us to postulate that clavams require the R-configuration at the ring juncture for beta-lactamase inhibitory activity, while the opposite S-stereochemistry is essential for antifungal activity.

Anti-Bacterial Agents↗
PubMed1983

Metabolites of an antipsychotic chiral pyrroloisoquinoline and their effect on prolactin secretion in the rat.

Racemic-2, 6-dimethyl-3-ethyl- 4,4a,5,6,7,8,8a, 9-octahydro-4a,8a-trans-1H-pyrrolo[2, 3-g]isoquinoline-4-one hydrochloride (rac-l HCl) appeared to be equipotent to haloperidol in increasing serum prolactin levels in rats although only about one-twentieth as potent as haloperidol in reversing dopamine-inhibited prolactin release by rat anterior pituitary cells in vitro. The metabolism of 14C-labeled rac-l HCl was studied in rats and the activity of metabolites was evaluated in the in vitro prolactin release assay. Four metabolites, two C11-monohydroxy diastereomers and one C10-and one C12-monohydroxy metabolite, plus parent drug were isolated from the urine of rats administered [14C]rac-l HCl. These were shown to be optically active, indicating that the racemic drug was stereoselectively metabolized. All of the identified metabolites plus those in extracts of urine and feces proved inactive in the in vitro prolactin release assay. In addition, the brains of three rats removed 1 hr after single 3.6 mg/kg oral doses of [14C]rac-l HCl contained essentially only unchanged drug. We conclude that the potency of rac-l HCl in raising serum prolactin levels is not due to the formation of active metabolite(s) in vivo.

Animals↗
PubMed1980

Circular dichroism studies of keto-bile acids.

Circular dichroism curves for various saturated bile acid derivatives with carbonyl groups at carbon atoms 3,7, and 12 are presented and it is demonstrated that this method offers a convenient solution for the location of mono and diketo carbonyl groups in the bile acid molecule. From the shapes (n leads to II transition) of these CD curves the precise localization of the carbonyl groups and the stereochemistry of the A/B ring juncture can be determined.

Bile Acids and Salts↗
PubMed1978

Preparation and characterization of (24R and 24S)-5beta-cholestane-3alpha,7alpha,24,25-tetrols and (24R and 24S)-5beta-cholestane-3alpha,24,25-triols.

(24R and 24S)-5beta-cholestane-3alpha,7alpha,24,25-tetrols were prepared by osmium tetroxide oxidation of 5beta-cholest-24-ene-3alpha,7alpha-diol. The resulting diastereomeric tetrols were separated by thin-layer chromatography, their purity ascertained by melting point, gas-liquid chromatography and mass spectra and their structural configurations were assigned by molecular rotation measurement and circular dichroism studies. In a similar fashion, the (24R and 24S)-5beta-cholestane-3alpha,24,25-triols were prepared and their structures identified.

Cholestanols↗