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Biomedical subjects

W D Wilson

Publications and source records attributed to W D Wilson.

At least 109 records · Page 6Linked to original sources

The search for structure-specific nucleic acid-interactive drugs: effects of compound structure on RNA versus DNA interaction strength.

The RNA genomes of a number of pathogenic RNA viruses, such as HIV-1, have extensive folded conformations with imperfect A-form duplexes that are essential for virus function and could serve as targets for structure-specific antiviral drugs. As an initial step in the discovery of such drugs, the interactions with RNA of a wide variety of compounds, which are known to bind to DNA in the minor groove, by classical or by threading intercalation, have been evaluated by thermal melting and viscometric analyses. The corresponding sequence RNA and DNA polymers, poly(A).poly(U) and poly(dA).poly(dT), were used as test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA (e.g., netropsin, distamycin, and a zinc porphyrin derivative) do not have significant interactions with RNA. Compounds that bind in the minor grove in AT sequences of DNA but have other favorable interactions in GC sequences of DNA (e.q., Hoechst 33258, DAPI, and other aromatic diamidines) can have very strong RNA interactions. A group of classical intercalators and a group of intercalators with unfused aromatic ring systems contain compounds that intercalate and have strong interactions with RNA. At this time, no clear pattern of molecular structure that favors RNA over DNA interactions for intercalators has emerged. Compounds that bind to DNA by threading intercalation generally bind to RNA by the same mode, but none of the threading intercalators tested to date have shown selective interactions with RNA.

DNA↗

Characteristics of Escherichia coli isolated from septic foals.

Fifteen Escherichia coli isolates from the blood and tissue of foals with septicemia were compared with 15 from the feces of clinically normal horses. Comparisons were made with respect to survival in normal equine serum, production of aerobactin, and production of hemolysin. Isolates from the blood and tissues of septic foals were more likely to be resistant to equine serum than were isolates from feces of clinically normal horses. There were minimal differences between the isolates with respect to aerobactin and hemolysin production, almost all being nonhemolytic and aerobactin negative. Serum resistance is probably a virulence determinant for invasive E. coli in horses.

Animals↗

Equine influenza.

Influenza continues to be one of the most important diseases of horses despite the availability and widespread use of equine influenza vaccines for almost 30 years. In recent years, infection with the influenza A/equine/2 subtype has become endemic in the equine populations of North America, Europe, and Scandinavia. Continued antigenic drift of field virus has compromised the efficacy of vaccines, most of which contain antigens prepared from influenza viruses isolated more than 10 years ago. This article reviews the history, virology, epidemiology, pathogenesis, immunology, clinical presentation, diagnosis, treatment, control, and prevention of influenza in horses and emphasizes recent developments in diagnostic methods and vaccine technology.

Animals↗

Linkage of serum resistance, aerobactin production, and resistance to antimicrobial agents on conjugal plasmids in some strains of Escherichia coli isolated from septic foals.

Fifteen isolates of Escherichia coli obtained from the blood and tissues of septic foals had plasmid DNA of size ranging from 2.5 to 93 megadaltons. These isolates grew in normal equine serum (serum resistant), a trait previously documented to be expressed by isolates obtained from blood and tissues of septic foals, but not by isolates obtained from the feces of clinically normal horses. Of these isolates, 3 contained conjugal plasmids that encoded resistance to multiple antimicrobial agents linked to serum resistance and, in 1 isolate, to production of aerobactin as well. Serum resistance and production of aerobactin are related to virulence of septicemic E coli from non-equine sources.

Animals↗

Clostridium septicum septicemia in a neonatal foal with hemorrhagic enteritis.

Clostridium septicum was isolated by anaerobic culture of blood collected from a 3-day-old foal with hemorrhagic enteritis and signs suggestive of septicemia. The foal responded well to treatment with intravenous fluids, antibiotics, plasma, and oral gastrointestinal protectants. One month after apparent complete recovery from the septicemia and hemorrhagic enteritis, the foal was euthanized during an acute episode of colic that was caused by severe, strangulating intestinal adhesions, thought to have formed as a result of peritonitis secondary to the hemorrhagic enteritis. The value of anaerobic culture of blood in foals with signs suggestive of septicemia is emphasized by the case presented here, as is the importance of evaluating the presence and extent of peritoneal inflammation in foals with hemorrhagic enteritis. To our knowledge, Clostridium septicum has not previously been reported to cause septicemia in neonatal foals.

Animals↗

Stabilities of duplexes and triplexes of dA19 + dT19 with alternating methylphosphonate and phosphodiester linkages.

Results from UV thermal melting, polyacrylamide gel electrophoresis and circular dichroism (CD) experimetns are used to compare stabilities of complexes formed from 1:1 and 1:2 molar ratio mixtures of dA19 or dA*P19 (alternating phosphodiester and methylphosphonate linkages on the A chain) with dT19 or dT*P19 (alternating phosphodiester and methylphosphonate linkages on the T chain). Complexes formed from 1:1 and 1:2 molar ratio mixtures of dA*P19 with dT19 are more stable than the respective unmodified 'parent' complexes, but less stable than complexes of dA*19 (17 methylphosphonates on the A chain) + dT19. At 0.1 M Na+ complexes containing dT*P19 are less stable than those with dT19, yet more stable than complexes containing the highly modified dT*19 (17 methylphosphonates on the T chain). A value of 2.6 kcal/mol base triplets is calculated for delta H of dA19.2dT19-->dA19.dT19+dT19.

Circular Dichroism↗

Substituent position dictates the intercalative DNA-binding mode for anthracene-9,10-dione antitumor drugs.

Molecular modeling studies [Islam, S.A., Neidle, S., Gandecha, B.M., Partridge, M., Patterson, L.H., & Brown, J.R. (1985) J. Med. Chem. 28, 857-864] have suggested that anthracene-9,10-dione (anthraquinone) derivatives substituted at the 1,4 and 1,8 positions with-NH(CH2)2NH(CH2CH3)2+ side chains intercalate with DNA with both substituents in the same groove (classical intercalation) while a similarly substituted 1,5 derivative intercalates in a threading mode with one side chain in each groove. Modeling studies also suggested that anthracene-9,10-dione (anthraquinone) derivatives substituted at the 2,6 positions with -NHCO(CH2)R (where R is a cationic group) should bind to DNA by the threading mode, and several such derivatives have been synthesized [Agbandjie, M., Jenkins, T.C., McKenna, R., Reszka, A., & Neidle, S. (1992) J. Med. Chem. 35, 1418-1429]. We have conducted stopped-flow kinetics association and dissociation experiments on the interaction of these anthraquinones with calf thymus DNA and with DNA polymers with alternating AT and GC base pairs to experimentally determine the binding mode and how the threading mode affects intercalation rates relative to similarly substituted classical intercalators. The binding modes, determined by analysis of relative rates, energies of activation, and effects of salt concentration on association and dissociation rate constants, agree completely with the modes predicted by molecular modeling methods. Association and dissociation rate constants for the threading mode are approximately a factor of 10 lower than constants for the classical intercalation mode, and the two modes, thus, have similar binding constants. Variations in rate constants for changes in cationic substituents at the 2 and 6 positions of the anthraquinone ring were surprisingly small.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Stereoelectronic factors in the interaction with DNA of small aromatic molecules substituted with a short cationic chain: importance of the polarity of the aromatic system of the molecule.

We have performed a quantitative analysis of the interaction with DNA of several unfused aromatic compounds synthesized in our laboratory and substituted with one or two short cationic chains. These and similar literature compounds, for which DNA binding data are available, bind with DNA by partial intercalation of the aromatic system, groove interaction of the linker chain, and groove electrostatic interactions of the terminal cationic group. Several independent quantitative and qualitative approaches show consistently that the strength of the interaction of the aromatic unit of the molecule with DNA binding sites depends on the direction and magnitude of polarity of the aromatic system. The phenomenon is explained in terms of the greatest negative potential in the DNA grooves, a concept extensively elaborated by Pullman and Pullman [cf. Lavery, R. and Pullman, B. [(1985) J. Biomol. Struct. Dyn. 2, 1021-1032] and references therein]. Classical, fused-ring planar intercalators do not follow the polarity-DNA affinity correlation, presumably because the intercalative forces depend more strongly on polarizability than on polarity of the aromatic system.

Animals↗

Prepurchase evaluation of horses: 134 cases (1988-1990).

To quantify some components of prepurchase evaluations in horses, records from 134 evaluations performed during a 2-year period were reviewed and the outcome was determined via telephone follow-up interview. Sixty-two percent of the prepurchase evaluations had been performed at the clinic and 38% had been performed in the field by the ambulatory service. All evaluations included physical and lameness examinations, whereas radiography (49%), endoscopy (15%), nerve blocking (5%), transrectal palpation (3%), hematologic analysis (2%), electrocardiography (2%), drug testing for analgesic agents (2%), and ultrasonography of the flexor tendons (1%) were not always performed. Fifty-nine percent of horses evaluated at the clinic were radiographed, compared with 33% of horses evaluated in the field (P less than 0.05). Thirty-seven percent of horses evaluated were judged serviceable for their intended use. Thirty-five percent of horses evaluated at the clinic were assessed to be serviceable, compared with 41% of those evaluated in the field (P less than 0.05). Horses used for pleasure riding (48%) tended to be considered serviceable more often than horses used for more athletic endeavors (3-day eventing, 33%; hunter/jumper, 24%; show, 31%; dressage, 30%). The most common basis for finding a horse unserviceable was lameness (88%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites.

The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.

Adenine Nucleotides↗

A molecular mechanics investigation of RNA complexes. I. Ethidium intercalation in an HIV-1 TAR RNA sequence with an unpaired adenosine.

Nucleic acid complexes with ethidium intercalated into different sites in a segment of HIV-1 TAR RNA with an unpaired A base, along with corresponding complexes with a normal RNA sequence without an unpaired base were studied by molecular mechanics energy minimization methods. Different intercalation geometries as well as different orientations of the ethidium molecule in the intercalation sites were tested. A general binding affinity enhancement for the ethidium binding to the bulge sequence compared with the normal RNA segment was obtained. With the unpaired adenosine base stacked in the duplex, the binding site adjacent to the 3' side of the bulge was found to be the most energetically favorable binding site, and the intercalation site 5' to the bulge in the same sequence is much less favorable. Unique correlated backbone conformational changes on binding of ethidium to the intercalation site 3' to the bulge were found to relieve backbone strains caused by the stacking of the unpaired base into the helix. These backbone conformational changes present a plausible molecular basis for the experimentally observed ethidium binding preference in this bulge RNA segment (L.S. Ratmeyer, R. Vinayak, G. Zon and W.D. Wilson, J. Med. Chem. 35, 966, 1992).

Base Sequence↗

Structures of 1-(3,3-dimethylamino)propyl naphtho[2,1-b]thiophene-4- carboxylate and N-(3,3-dimethylamino)propyl-8-methoxynaphtho[2,1- b]thiophene-4-carboxamide, intercalators into double-helical DNA.

(1) C18H19NO2S, Mr = 313.42, monoclinic, P2(1)/c, a = 11.503 (3), b = 15.932 (2), c = 9.133 (2) A, beta = 102.17 (2) degrees, V = 1636.1 A3, lambda(Cu K alpha) = 1.54178 A, mu = 1.762 mm-1, F(000) = 664, T = 293 (1) K, R = 0.047 for 2044 significant reflections. (2) C19H21NO3S, Mr = 343.45, triclinic, Pl, a = 9.873 (2), b = 13.163 (3), c = 14.065 (3) A, alpha = 101.33, beta = 94.30 (3), gamma = 91.35 (3) degrees, V = 1785.8 A3, Z = 4, Dx = 1.28 Mg m-3, lambda(Cu K alpha) = 1.54178 A, mu = 1.699 mm-1, F(000) = 728, T = 293 (1) K, R = 0.052 for 1787 significant reflections. The torsion angle between the naphthothiophene ring and the carbonyl O atom of the side chain is 5.5 (4) degrees in structure (1) and 25 (2) and -32 (2) degrees in the two independent molecules of (2). This difference is due to out-of-plane distortions in (2) that arise from steric hindrance between H atoms on the amide and the ring system, at position 2.

Intercalating Agents↗

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations.

Base Sequence↗

Kinetic and equilibrium analysis of a threading intercalation mode: DNA sequence and ion effects.

The interaction of a symmetric naphthalene diimide with alkylamino substituents at each imide position was investigated with the alternating sequence polymers, poly[d(A-T)]2 and poly[d(G-C)]2. Spectrophotometric binding studies indicate strong binding of the diimide to both sequences although the GC binding constant is 20-25 times larger than the AT binding constant. Analysis of the effects of salt concentration on the binding equilibria shows that the diimide forms two ion pairs in its complex with both polymers as expected for a simple dication. Stopped-flow kinetics experiments demonstrate that the diimide both associates and dissociates from DNA more slowly than classical intercalators with similar binding constants. Analysis of salt concentration effects on dissociation kinetics rate constants (kd) reveals that slopes in log kd versus log [Na+] plots are only approximately half the value obtained for classical dicationic intercalators that have both charged groups in the same groove. These kinetics results support a threading intercalation model, with one charged diimide substituent in each of the DNA grooves rather than with both side chains in the same groove, for the diimide complex with DNA. In the rate-determining step of the mechanism for dissociation of a threading complex only one ion pair is broken; the free side chain can then slide between base pairs to put both diimide side chains in the same groove, and this is followed by rapid full dissociation of the diimide. This sequential release of ion pairs makes the dissociation slope for dicationic threading intercalators more similar to the slope for classical monocationic intercalating ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Hairpin formation within the enhancer region of the human enkephalin gene.

The 3',5'-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephalin gene is located within an imperfect palindrome of 23 base pairs. We have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays an active role in cAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.

Animals↗