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Biomedical subjects

S Ebel

Publications and source records attributed to S Ebel.

At least 19 recordsLinked to original sources
PubMed2004

Analysis of atropine, its degradation products and related substances of natural origin by means of reversed-phase high-performance liquid chromatography.

Chromatographic separation and quantification methods of tropa alkaloids were often described. In order to separate atropine from its degradation products ion-pair chromatography (IPC) has been frequently applied. Beside long equilibration times IPC often suffers from poor robustness. The aim of this study was to develop robust and simple HPLC methods for both stability testing of atropine solutions and limitation of related substances in atropine from plant material. Using a hydrophilic embedded RP18 column and a gradient elution gave baseline separation of all components.

Atropine↗
PubMed1994

Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme.

Two series of hammerhead-derived ribozymes, or 'minizymes', in which helix II has been replaced by linkers of non-nucleotidic moieties, have been synthesised by solid-phase methods. In the first series, the minizymes had linkers containing one, two, three, four or five repeated units of phosphopropanediol, so that the number of atoms in the chain connecting the 3'O of the conserved A9 to the 5'O of the conserved G12 varied from 7 to 31. In the second, more-limited series, the minizymes contained linkers of either tetra- or hexa-ethyleneglycol. The rates at which these minizymes cleaved their cognate 13-nucleotide substrate were determined at 30 degrees C, and compared with the rates of cleavage by an analogous series of minizymes containing from two to six repeated units of thymine deoxyribonucleotide in place of helix II. In all three series, the cleavage rates increased with increasing linker length, with a plateau being reached at the longer lengths tested. Relative cleavage rates within the phosphopropanediol and the thymidine series depended strongly on linker length, but maximal activity was achieved in both series with 25 atoms in the chain joining A9 and G12. The lengths of linkers required to achieve maximal activity of the minizymes are considerably greater than the linkers of 13 atoms which are sufficient to stabilise the ends of double-helices of DNA or RNA.

Base Sequence↗
PubMed1994

Crystal and molecular structure of r(CGCGAAUUAGCG): an RNA duplex containing two G(anti).A(anti) base pairs.

BACKGROUND: Non-Watson-Crick base pair associations contribute significantly to the stabilization of RNA tertiary structure. The conformation adopted by such pairs appears to be a function of both the sequence and the secondary structure of the RNA molecule. G.A mispairs adopt G(anti).A(anti) configurations in some circumstances, such as the ends of helical regions of rRNAs, but in other circumstances probably adopt an unusual configuration in which the inter-base hydrogen bonds involve functional groups from other bases. We investigated the structure of G.A pairs in a synthetic RNA dodecamer, r(CGCGAAUUAGCG), which forms a duplex containing two such mismatches. RESULTS: The structure of the RNA duplex was determined by single crystal X-ray diffraction techniques to a resolution in the range 7.0-1.8A, and found to be an A-type helical structure with 10 Watson-Crick pairs and two G.A mispairs. The mispairs adopt the G(anti).A(anti) conformation, held together by two obvious hydrogen bonds. Unlike analogous base pairs seen in a DNA duplex, they do not exhibit a high propeller twist and may therefore be further stabilized by weak, reverse, three-center hydrogen bonds. CONCLUSIONS: G(anti).A(anti) mispairs are held together by two hydrogen of guanine and the N6 and N1 of adenine. If the mispairs do not exhibit high propeller twist they may be further stabilized by inter-base reverse three-centre hydrogen bonds. These interactions, and other hydrogen bonds seen in our study, may be important in modelling the structure of RNA molecules and their interactions with other molecules.

Animals↗
PubMed1994

Thermodynamic stability and solution conformation of tandem G.A mismatches in RNA and RNA.DNA hybrid duplexes.

G.A mismatches form a variety of hydrogen-bonded structures in DNA, most of which destabilise the duplex. Tandem G.A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon. G. & Wilson, W.D. (1991) Proc. Natl Acad. Sci. USA 88, 26-30], which permits extensive base-base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. & Brown, T. (1992) Biochemistry 31, 12083-12086]. We have measured the thermodynamic stability of several RNA and RNA.DNA hybrid duplexes containing tandem G.A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases. NOE and coupling-constant data show that all of the sugars are in the C3'-endo range of conformations, and glycosidic torsion angles are in the range -160 degrees to -180 degrees in [sequence: see text]. Both sequential NOE intensities and circular-dichroism measurements indicate that the global conformation of the mismatched RNA is A-like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino-pairing scheme. Determination of the structure using 'loose' NMR-derived constraints shows that the potential energies of the imino-paired and amino-paired forms are similar, but substantially higher than energy-minimised RNA. Using tighter constraints derived from more extensive analysis of one-dimensional and two-dimensional NOE data showed that the amino-paired structure agrees with the constraint data better than the imino-paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino-paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B-DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.

Base Sequence↗
PubMed1994

Neutral and anionic cyclodextrins in capillary zone electrophoresis: enantiomeric separation of ephedrine and related compounds.

Two methods for the enantiomeric separation of chiral ephedrine alkaloids (ephedrine, methylephedrine, methylpseudoephedrine and norephedrine) by capillary zone electrophoresis in uncoated capillaries were developed. Both methods were optimized to more than 100,000 theoretical plate numbers. The first method used a neutral cyclodextrin (CD) derivative: heptakis (2,6-di-O-methyl)-beta-cyclodextrin at an acidic pH of 2.5 (20 mM phosphate buffer) at an 18 mM concentration. The second used a newly developed acidic CD derivative, the tetrakis[6-O-(4-sulfobutyl)]-beta-cyclodextrin sodium salt. The benefits of this new reagent for the chiral separation are a wide range of basic pH available for the enantiomeric resolution. It is also useful for other cases, e.g. preventing adsorption without additives.

Anions↗
PubMed1993

NMR assignments and solution conformation of the DNA.RNA hybrid duplex d(GTGAACTT).r(AAGUUCAC).

Nearly complete assignments of the 1H-NMR spectrum of the DNA.RNA hybrid d(GTGAACTT).r(AAGUUCAC) have been obtained by two-dimensional methods. Three-bond coupling constants measured from cross-sections of two-dimensional NOE spectra and double quantum-filtered correlation spectra showed that the sugars in the DNA strand are predominantly in the S domain of puckers, whereas the ribofuranoses are mainly C3'-endo. Analysis of time-dependent NOE intensities from one- and two-dimensional experiments showed that the glycosidic torsion angles in the DNA strand are near -120 degrees, whereas those in the RNA strand are in the range- 140 degrees to -160 degrees. These nucleotide conformations correspond to those typically found in B-DNA and A-RNA, respectively. The circular dichroism of the duplex is similar to that of A-form RNA, consistent with a global A-like conformation. A large number of duplex structures was generated in which the nucleotides were fixed in the experimentally determined conformations. A subset of these structures was found that satisfied the internucleotide NOE intensities, backbone constraints and had acceptable Lennard-Jones energies. The base pairs in the duplex were found to have positive inclinations, a translation (Dx) of about 0.4 nm from the helix axis, and more than 10 bp/turn on average. This implies a helical structure in the A family of conformations.

Base Sequence↗
PubMed1993

Thermodynamic stability and drug-binding properties of oligodeoxyribonucleotide duplexes containing 3-deazaadenine:thymine base pairs.

We have used ultraviolet melting techniques to study the effect on stability of incorporating the nucleoside analogue 2'-deoxy-3-deazaadenosine (d3cA) into the duplex 5'-d(CGCAATCG)-3'-d(GCGTTAGC). Our results demonstrate that the successive replacement of dA by d3CA increasingly destabilises the duplex. The destabilising effect of this analogue is considerably enhanced as the pH is lowered and the results are consistent with protonation of 3-deazaadenine (probably at N-1) contributing to duplex destablisation. Surprisingly, the incorporation of d3CA does not significantly affect the binding of distamycin-A.

Adenine↗
PubMed1992

Very stable mismatch duplexes: structural and thermodynamic studies on tandem G.A mismatches in DNA.

We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.

Adenine↗
PubMed1992

Solution conformation of a deoxynucleotide containing tandem G.A mismatched base pairs and 3'-overhanging ends in d(GTGAACTT)2.

We have used 31P and 1H NMR spectroscopy and circular dichroism to define the solution conformation of d(GTGAACTT)2 which contains tandem G.A mismatched base pairs and 3'-overhanging TT ends. Measurements of coupling constants and NOE intensities show that the sugar puckers of the nucleotides are predominantly in the south domain (i.e., near C2'-endo) and that the glycosidic torsion angles are anti. The sequential NOE intensities indicate the presence of a right-handed helix. Analysis of the 31P and 1H NMR spectra of the duplex shows that the tandem mismatch forms a block in which there are unusual backbone torsion angles (i.e., in the BII state), within an otherwise B-like structure. The chemical shift of the N1H of the mismatched guanosine and NOEs between the mismatched base pairs and their nearest neighbors are inconsistent with the imino pairing present in single A.G mismatches or in the X-ray structure of a tandem mismatch [Privé, G. G., et al. (1987) Science 238, 498-503] but the data are consistent with the amino pairing found by Li et al. (1991) [Li, Y., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The strong base-base stacking both within the tandem G.A block and between the G.A mismatches and their other nearest neighbors offsets the intrinsic destabilizing effects of the mismatch. Further, the 3'-TT overhangs stack onto the ends of the helix and stabilize the duplex against fraying, which accounts for the observed increase in the melting temperature compared with the flush-ended duplex.

Adenine↗
PubMed1992

Barnase has subsites that give rise to large rate enhancements.

Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.

Bacillus↗
PubMed1992

[Hand disinfection as the central factor in prevention of puerperal mastitis. Clinical study and results of a survey].

The incidence of puerperal mastitis was evaluated in the course of two 12-month periods with and without additional hand disinfection at the bedside. Disinfection was available throughout both periods for all patients, mounted at the door of each room. Without additional disinfection at the bedside, 32 patients developed mastitis. With reference to the birth rate of 1095 during this period, the incidence of mastitis was 2.9%. When additional bedside disinfection was available, mastitis dropped to 8 cases per 12 months (1212 births = 0.66%) (p less than 0.001). A telephone survey, at 55 obstetrical units revealed that 41.8% of maternity clinics do not provide hand disinfectants at the bedside. These results indicate to the breast-feeding mother, that in the pathogenesis of puerperal mastitis, the essential recommendation emphasises, that puerperal women practise strict hygiene in maternity.

Breast Feeding↗
PubMed1991

Pore-forming peptide of pathogenic Entamoeba histolytica.

A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.

Amino Acid Sequence↗
PubMed1990

[Analytical methods for stability testing of metronidazole infusion solutions].

UV-spectroscopy, thin layer- and high pressure liquid-chromatography as well as voltammetry (polarography) are suggested and compared as methods for use in stability testing of solutions of metronidazol. If some conditions are fulfilled UV-spectroscopy is the method of highest precision. TLC/HPTLC has advantages compared to HPLC, for more polar degradation products are visible at the starting point, whereas in HPLC these substances may be irreversibly adsorbed on the columne. Combination of HPLC and voltammetric (polarographic) techniques may lead to the optimal solution of this analytical problem with respect to best accuracy and precision (reproducibility) as compared in this paper.

Chemistry, Pharmaceutical↗
PubMed1989

Analytical oxidation of reserpine.

Electrochemical (anodic) oxidation of reserpine in an acidic medium results in the formation of 3,4-dehydroreserpine instead of the reported 10-hydroxy-reserpine. Electrochemical and chemical oxidation by sodium nitrite lead quantitatively to the same product; few by-products or consecutive products are formed if protected from light. The validity of the conditions of the spectrophotometric assay of the European Pharmacopoeia were confirmed by following the reaction using HPLC with a photodiode-array detector.

Chromatography, High Pressure Liquid↗
PubMed1989

The llama industry in the United States.

This chapter provides an overview of the llama in the United States. It covers the animal and its history, the people who own llamas, and the business that is developing around them and describes the industry's needs from the veterinary profession in terms of health care.

Animal Husbandry↗