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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 109 records · Page 6Linked to original sources
PubMed1994

Crystal structure of a four-stranded intercalated DNA: d(C4).

The crystal structure of d(C4) solved at 2.3-A resolution reveals a four-stranded molecule composed of two interdigitated or intercalated duplexes. The duplexes are held together by hemiprotonated cytosine-cytosine base pairs and are parallel stranded, but the two duplexes point in opposite directions. The molecule has a slow right-handed twist of 12.4 degrees between covalently linked cytosine base pairs, and the base stacking distance is 3.1 A. This is in general agreement with the NMR studies. A biological role for DNA in this conformation is suggested.

Computer Graphics↗
PubMed1994

Crystal structure of intercalated four-stranded d(C3T) at 1.4 A resolution.

The crystal structure of d(C3T), solved at 1.4 A resolution, reveals that the molecule forms a four-stranded intercalated complex. It consists of two parallel-stranded duplexes, each of which is held together by cytosine-protonated cytosine base pairs. The two duplexes are intercalated with each other and have opposite strand orientation. The molecule has a flat, lath-like appearance, and the covalently bonded cytosines have a slow right-handed twist of 17.1 degrees. However, there is considerable asymmetry. On one of the flat sides, the phosphate groups are rotated away from the center of the molecule. They are held in this orientation by bridging water molecules that bind the NH of cytosine and a phosphate group of an opposite chain. There is also considerable microheterogeneity in the structure. The cytosine hemiprotonation occurs even at pH 7 where stable crystals form.

Crystallography, X-Ray↗
PubMed1994

Three-dimensional crystal structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor-groove-binding drug Hoechst 33258.

The molecular structure of the DNA A-tract dodecamer d(CGCAAATTTGCG) complexed with the drug Hoechst 33258 has been determined by X-ray diffraction analysis. The Hoechst molecule binds in the DNA minor groove covering the sequence AATTT of the central A-tract, with the piperazine group close to one of the GC regions. The drug molecule makes two three-centered hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and O2 atoms of the DNA bases. Although a high propeller twist is observed in the A-tract, only one unsymmetrical three-centered hydrogen bond is present in the DNA major groove. The structure is compared with other minor-groove-binding drug complexes and the influence of these drugs on DNA A-tracts is discussed.

Base Sequence↗
PubMed1994

Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.

We dissected the domain of HAP2 that mediates subunit association in the heteromeric CCAAT-binding complex, first by genetic mutational analysis and then by structural studies. The mutational data suggest that a very short region in HAP2 mediates protein-protein association and that the structure of this domain is likely to be an alpha-helix. The CD analyses of a 15-residue synthetic oligopeptide covering this region confirm this surmise. The oligopeptide indeed formed an unusually thermal stable alpha-helix in aqueous solution. Eight amino acids that lie along one face of this helix, including three arginines, are found to be critical for protein-protein association. The partner that interacts with this helical motif is likely to be another subunit in the HAP complex, since the CCAAT-binding factor is shown to contain one molecule of HAP2. Our results suggest that very short regions in proteins can encode precise structures and mediate stable and specific protein-protein recognition and interactions.

Amino Acid Sequence↗
PubMed1994

The gene for biotin synthase from Saccharomyces cerevisiae: cloning, sequencing, and complementation of Escherichia coli strains lacking biotin synthase.

Biotin synthase catalyzes the insertion of a sulfur atom between two carbon atoms of dethiobiotin to form biotin in the last step of the biotin biosynthesis pathway. In Escherichia coli, biotin synthase is coded for by bioB gene. We report here cloning, sequencing, and initial functional characterization of the yeast gene for biotin synthase in Saccharomyces cerevisiae. We have named this gene BIO2. It consists of a 355-codon open reading frame near the ZUO1 gene. Analysis of the yeast protein encoded by the BIO2 gene reveals that it shares extensive homology with biotin synthases of E. coli and Bacillus sphaericus. The yeast and the two bacterial biotin synthase proteins have similar molecular weights, amino acid compositions, and hydropathies. The plasmid pUCBIO2 containing the yeast BIO2 gene completely complements E. coli bioB- and delta bio mutants and enables these mutants to grow on dethiobiotin. Although BIO2 is physically linked to ZUO1, which encodes the putative left-handed Z-DNA binding protein zuotin, it appears to be regulated independently from it. The yeast BIO2 and ZUO1 genes reside near ADE3 gene on chromosome VII. BIO2 is the first eukaryotic gene reported from the biotin biosynthetic pathway.

Amino Acid Sequence↗
PubMed1994

The low-temperature crystal structure of the pure-spermine form of Z-DNA reveals binding of a spermine molecule in the minor groove.

The X-ray crystal structure of the pure-spermine form of the left-handed Z-DNA duplex [d(CGCGCG)]2 has been determined at a temperature of -110 degrees C. Whereas the previously described room temperature structure of the pure-spermine form showed only the presence of a single "interhelix" spermine molecule, mediating contacts between neighboring duplexes (Egli et al., 1991), a second "intrahelix" spermine molecule as well as two hydrated sodium ions were found in the structure determined at low temperature. This second spermine molecule binds primarily within the minor groove of two hexamer duplexes that are stacked in an end-to-end fashion in the crystal lattice. Thus, the intrahelix spermine molecule interacts with a single infinite helix. The spine of hydration observed in other structures of Z-DNA hexamers is partially replaced and partially displaced by the intrahelix spermine molecule. In Z-DNA, phosphate groups are relatively closely spaced across the minor groove compared to the right-handed double-helical conformation of B-DNA. The intrahelix spermine molecule decreases cross-groove electrostatic repulsion within the Z-DNA helix, thereby increasing its relative stability. This structure may therefore provide an explanation for the role of spermine as a very effective inducer of the conformational B-DNA to Z-DNA transition with alternating dG-dC sequences in solution.

Binding Sites↗
PubMed1994

Unusually stable beta-sheet formation in an ionic self-complementary oligopeptide.

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH2) is able to form an unusually stable beta-sheet structure. The beta-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the beta-sheet structure. The beta-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide beta-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of beta-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research.

Amino Acid Sequence↗
PubMed1994

Ascending myogenic autoregulation: interactions between tubuloglomerular feedback and myogenic mechanisms.

A mathematical model of the renal vascular and tubular systems was used to examine the possibility that synergistic interactions might occur between the tubuloglomerular feedback (TGF) and myogenic autoregulatory mechanisms in the kidney. To simulate the myogenic mechanism, the renal vasculature was modelled with a resistance network where the total preglomerular resistance varies with intravascular pressure. In addition, a steady-state model of glomerular filtration, proximal and Henle's loop reabsorption, and TGF-modulation of afferent arteriolar resistance was derived. The results show that, if TGF acts on the distal portion of the preglomerular vasculature, then any TGF-induced vasoconstriction should raise upstream intravascular pressure and, thereby, trigger a myogenic (AMYO) response. The model further predicts that the magnitude of the AMYO response can be similar in magnitude to the TGF-induced increment in afferent resistance. Hence, the effects of TGF excitation on whole kidney hemodynamics may be much greater than predicted from measurements in single nephrons. Moreover, a significant fraction of the intrinsic myogenic autoregulatory response to increased renal perfusion pressure may result from a synergistic interaction between the TGF and myogenic mechanisms.

Arterioles↗
PubMed1994

Carbon monoxide stimulates a potassium-selective current in rabbit corneal epithelial cells.

The effects of CO on ion currents in freshly dispersed rabbit corneal epithelial cells were assessed using the perforated patch whole cell voltage-clamp technique. Bath perfusion with 1% CO resulted in a 84 +/- 18% (mean +/- SE, n = 14) increase in potassium current (IK) and a membrane hyperpolarization from -42 +/- 4 to -51 +/- 4 mV. The CO-stimulated current reversed at -64 +/- 7 mV [reverse potential (EK) = -87 mV]. The stimulated current was blocked by 1 mM quinidine or 1 mM diltiazem, agents that inhibit IK in rabbit corneal epithelial cells. Single potassium-channel currents measured in the cell-attached configuration showed that exogenous CO increased the steady-state open probability from 0.003 to 0.156 at a holding potential of -40 mV. CO did not affect open probability in excised patches. The single-channel conductance measured from -40 to +40 mV was unaffected. Intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration measured with radioimmunoassay techniques was found to increase from 0.41 +/- 0.24 to 0.55 +/- 0.27 pmol/10(6) cells after the addition of 1% CO (P < 0.05). The data show that bath perfusion with exogenous CO activates IK and hyperpolarizes the resting membrane potential; the data also suggest that CO modulates intracellular cGMP concentration.

Animals↗
PubMed1994

Acoustic rhinometry in the evaluation of children with nasal or oral respiration.

The purpose of this study was to determine if there are any differences in the nasal geometry of children with nasal respiration or predominantly oral respiration (mouth breathing). The technique utilized was the acoustic reflection technique or acoustic rhinometry. Acoustic rhinometry is a non-invasive method, which can be easily applied to young children. Twenty children participated and both nostrils were evaluated. Statistically significant differences were found in the total nasal volumes between the two groups, with the mouth breathers presenting with reduced nasal volumes. No differences were observed in the nasal cross sectional areas.

Acoustics↗
PubMed1993

DNA comes in many forms.

Forty years ago, we learned of the major double helical structure adopted by DNA. It combined both elegance and simplicity in its design. Since then we have learned that DNA can also adopt other conformations. We now know that it can exist in a variety of triple-stranded and quadruple-stranded forms, as well as forms that are left-handed. The list of alternative conformations that can be adopted by this molecule is still growing. These conformations represent a major biological challenge to understand their role in biological systems. This type of work represents an active frontier in molecular biology today.

Animals↗
PubMed1993

Molecular structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor groove binding drug netropsin.

The molecular structure of the complex between the minor groove binding drug netropsin and the dodecamer d(CGCAAATTTGCG) has been solved and refined by X-ray diffraction analysis to an R-factor of 19.8% and 2.2-A resolution. The drug lies in the narrow minor groove of the B-DNA fragment, covering five of the six A.T base pairs (from A5.T20 to T9.A16). The long six A.T base pair tract allows the drug to bind in a position that optimizes its contacts with the DNA, establishing hydrogen bonds with O2 of thymines and N3 of adenines. The DNA molecule shows a high propeller twist only at the A6.T19 step of the A-tract. Two three-centered hydrogen bonds are observed in the major groove at half of the A-tract.

Base Sequence↗
PubMed1993

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

Animals↗
PubMed1993

Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.

A 16-residue peptide [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] has a characteristic beta-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes. Scanning EM reveals a network of interwoven filaments approximately 10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.

Amino Acid Sequence↗
PubMed1993

Z-DNA binding protein from chicken blood nuclei.

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

Animals↗