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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 145 records · Page 8Linked to original sources
PubMed1991

Interaction between the left-handed Z-DNA and polyamine. The crystal structure of the d(CG)3 and N-(2-aminoethyl)-1,4-diamino-butane complex.

The DNA fragment d(CG)3 was co-crystallized with N-(2-aminoethyl)-1,4-diaminobutane (PA(24], a chemically synthesized polyamine. The complex crystal contained one polyamine, 3 magnesium cations and one sodium cation per duplex of d(CG)3, and well diffracted the X-ray intensities up to 1.0 A resolution. The d(CG)3 took a left-handed Z-DNA conformation, and the PA(24) molecule electrostatically interacted with the phosphate groups of the d(CG)3 duplex.

Crystallization↗
PubMed1991

The Z-Z junction: the boundary between two out-of-phase Z-DNA regions.

The boundary between two segments of Z-DNA that differ in the phase of their syn-anti alternation about the glycosidic bond is termed a Z-Z junction. Using chemical probes and two-dimensional gel electrophoresis, we examined a Z-Z junction consisting of the sequence d[(CG)8C(CG)8] inserted into a plasmid and used energy minimization techniques to devise a three-dimensional model that is consistent with the available data. We show that both alternating CG segments undergo the B-Z transition together to form a Z-Z junction. The junction is very compact, displaying a distinctive reactivity signature at the two base pairs at the junction. In particular, the 5' cytosine of the CC dinucleotide at the junction is hyperreactive toward hydroxylamine, and the two guanines of the GG dinucleotide on the complementary strand are less reactive toward diethyl pyrocarbonate than are the surrounding Z-DNA guanines. Statistical mechanical treatment of the 2-D gel data yields a delta G for forming the Z-Z junction equal to 3.5 kcal, significantly less than the cost of a B-Z junction and approximately equal to the cost of a base out of alternation (i.e., a Z-DNA pyrimidine in the syn conformation). The computer-generated model shows little distortion of the Z helix outside of the central two base pairs, and the energy of the structure and the steric accessibility of the reactive groups are consistent with the data.

Base Sequence↗
PubMed1991

Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei.

Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.

Antibodies, Monoclonal↗
PubMed1991

Drug-induced DNA repair: X-ray structure of a DNA-ditercalinium complex.

Ditercalinium is a synthetic anticancer drug that binds to DNA by bis-intercalation and activates DNA repair processes. In prokaryotes, noncovalent DNA-ditercalinium complexes are incorrectly recognized by the uvrABC repair system as covalent lesions on DNA. In eukaryotes, mitochondrial DNA is degraded by excess and futile DNA repair. Using x-ray crystallography, we have determined, to 1.7 A resolution, the three-dimensional structure of a complex of ditercalinium bound to the double-stranded DNA fragment [d(CGCG)]2. The DNA in the complex with ditercalinium is kinked (by 15 degrees) and severely unwound (by 36 degrees) with exceptionally wide major and minor grooves. Recognition of the DNA-ditercalinium complex by uvrABC in prokaryotes, and by mitochondrial DNA repair systems in eukaryotes, might be related to drug-induced distortion of the DNA helix.

Base Sequence↗
PubMed1991

DNA-nogalamycin interactions.

The anthracycline antibiotic nogalamycin differs from the more common daunomycin-type anthracyclines by substitution on both ends of the intercalating chromophore, giving nogalamycin the approximate shape of a dumbbell. The chromophore of daunomycin is substituted on only one end. In nogalamycin, the positively charged amino sugar substituent of daunomycin is replaced by an uncharged nogalose sugar and a methyl ester group. The other end of nogalamycin, where daunomycin is unsubstituted, is fused to a bicyclo amino sugar with a positively charged dimethylamino group. Much larger DNA fluctuations are required for intercalative entry of nogalamycin than for entry of daunomycin. This report describes the X-ray crystal structure of the complex between nogalamycin and the self-complementary DNA hexamer d(me5CGTsAme5CG). The DNA contains cytosines methylated at the 5-positions and a phosphorothioate linkage at the TpA step. Nogalamycin intercalates at the terminal CpG steps and interacts with both strands in both grooves of the DNA. Large conformational adjustments in both nogalamycin and the DNA are necessary to form a stable, intercalative complex. The interactions of the bases with the nogalamycin substituents lead to sliding of bases relative to each other along the normal to Watson-Crick hydrogen bonds. The planarities of base pairs surrounding the intercalation site are distorted. The backbones of the two strands are distorted asymmetrically by nogalamycin with large deviations from standard B-DNA geometry. The complex between nogalamycin and DNA illustrates the conformational flexibility of DNA. The hydrogen-bonding interactions between nogalamycin and DNA do not suggest a sequence-specific binding of the drug, although additional secondary effects might lead to differences between various intercalation sites.

Crystallography↗
PubMed1991

Electrogenic bicarbonate secretion in the turtle bladder: apical membrane conductance characteristics.

We have recently shown that stimulation of electrogenic HCO3- secretion is accompanied by a simultaneous increase in short-circuit current (Isc, equivalent to HCO3- secretion rate under these conditions), apical membrane capacitance (Ca, proportional to membrane area), and apical membrane conductance (Ga, proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase in Ga and the possibility that the rate of electrogenic HCO3- secretion is regulated by changes in Ga. Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO3- secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO3- secretion with cAMP. The major conclusions are: (i) a measurable apical Cl- conductance exists in control hemibladders; (ii) the transport-associated increase in Ga includes a Cl(-)-conductive component; (iii) Ga also appears to reflect a HCO3- conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl- and HCO3- are similar; (v) 9-AA reduces Ga and Isc in cAMP-stimulated hemibladders; and (vi) alterations in Isc appear to be mediated by changes in Ga.

Animals↗
PubMed1991

Oral pemphigus vulgaris in young adults.

Three patients, aged 31, 26, and 22 years, had pemphigus vulgaris. A delay in diagnosis and appropriate treatment occurred because the initial presentation was confined to the oral mucosa.

Adult↗
PubMed1990

Ternary interactions of spermine with DNA: 4'-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.

Antibiotics, Antineoplastic↗
PubMed1990

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate.

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.

DNA↗
PubMed1990

Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts.

We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.

Adenine↗
PubMed1990

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin.

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.

Amino Sugars↗
PubMed1990

Changes in membrane conductances and areas associated with bicarbonate secretion in turtle bladder.

Transepithelial impedance-analysis studies were performed in turtle bladder epithelium in order to measure changes in the different epithelial membranes resulting from stimulation of electrogenic bicarbonate secretion. Changes in membrane conductance relate to changes in ionic permeability, whereas changes in membrane capacitance relate to changes in membrane area, since most biological membranes exhibit a specific capacitance of approximately 1 muF/cm2. The results of this investigation are summarized as follows: (i) cAMP and carbachol, agents which have been shown previously to stimulate electrogenic bicarbonate secretion, result in increases in apical-membrane conductance and capacitance; (ii) these changes occur concomitantly with the observed change in transport (measured using the short-circuit-current technique), thereby suggesting that bicarbonate secretion may be regulated in part by changes in the chloride conductance of the apical membrane; (iii) the increase in conductance does not reflect an increase in the membrane's specific conductance, thereby indicating that it results from the addition of membrane possessing similar ionic permeability as the existing apical membrane; (iv) the magnitude of the changes in capacitance indicate that a minor cell population (beta-type carbonic-anhydrase-rich cells) increase their apical-membrane area by several-fold; (v) a lack of transport-associated changes in the basolateral-membrane parameters suggest that transport is not regulated by alterations in basolateral-membrane ionic conductance or area; (vi) a lack of colchicine sensitivity, coupled with the magnitude of the changes in apical-membrane capacitance, indicate that the membrane remodeling processes are different from those involved in the regulation of proton secretion in a different cell population (alpha-type carbonic-anhydrase-rich cells).

1-Methyl-3-isobutylxanthine↗
PubMed1990

Z-DNA affinity chromatography.

In this chapter we have detailed a method that can be generalized to link virtually any DNA substrate to a chromatography matrix at its ends via an avidin-biotin linkage. We have used this technique to construct a left-handed Z-DNA column for the purpose of identification and purification of Z-DNA-binding proteins. This technique for the linkage of DNA to a column matrix by avidin-biotin technology can be modified, however, to produce linked multimeric sequences specific for regulatory or other DNA-binding proteins.

Avian Myeloblastosis Virus↗
PubMed1990

Structure of nogalamycin bound to a DNA hexamer.

The anthracycline antibiotic nogalamycin, which binds to DNA, is composed of a planar aglycone substituted on each end to form an unusual dumbbell-shaped molecule. At one end nogalamycin contains an uncharged nogalose sugar and a methyl ester. At the other end nogalamycin contains a positively charged bicyclo amino sugar. We report the crystal structure of nogalamycin bound to the self-complementary DNA hexamer d(m5CGTsAm5CG). In this complex, the cytosines are methylated at the 5 position and the DNA contains a phosphorothioate linkage at the TpA step. Two nogalamycin molecules bind to the 6-base-pair fragment of double-helical DNA. The drug has threaded between the phosphodiester backbones with three aromatic rings intercalated within the DNA. In the major groove, the bicyclo amino sugar forms two direct hydrogen bonds to span a CG base pair and interacts indirectly with the next base pair of the duplex via a water-mediated hydrogen bond. In the minor groove, a carbonyl oxygen of nogalamycin forms a hydrogen bond directly to N2 of a guanine. The DNA base pairs are severely buckled by up to 26 degrees and are also distorted in directions perpendicular to the Watson-Crick hydrogen bonds. This complex illustrates the deformable nature of DNA.

Base Sequence↗