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Biomedical subjects

H J Vogel

Publications and source records attributed to H J Vogel.

At least 19 recordsLinked to original sources
PubMed1992

Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Proton NMR studies of angiotensin II and its analogs in aqueous solution.

The 1H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H2O/10% D2O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population below pH 5.5 and increases to 12-20% around pH 7.0. The two sets of signals result from a cis-trans isomerization of the His-Pro peptide bond with the major resonances arising from the trans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants JNH-alpha CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-alpha CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY and pH-titration experiments. Moreover, pH effects and the unusual amide exchange behavior of the Arg epsilon NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH2 protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteins.

Amino Acid Sequence↗
PubMed1991

In vivo regulation of virulence in Pseudomonas aeruginosa associated with genetic rearrangement.

A chronic pulmonary infection model was used to induce conversion to the mucoid phenotype by Pseudomonas aeruginosa PAO. At 6 months after initial inoculation, organisms isolated from infected lungs demonstrated the mucoid phenotype. Significant decreases (P less than .01) were seen in the levels of exotoxin A, exoenzyme S, phospholipase C, and pyochelin produced by the mucoid P. aeruginosa PAO rat lung isolates that returned to parental levels after reversion to the nonmucoid phenotype. In addition, lipopolysaccharide of the mucoid PAO lung isolates failed to react with serotype B-specific antibody in contrast to the original PAO and the revertant PAO organisms. Digestion of chromosomal DNA and hybridization with P. aeruginosa virulence factor-specific probes demonstrated that conversion to the mucoid phenotype was associated with rearrangement of chromosomal DNA upstream of the exotoxin A gene. Analysis of DNA from revertant organisms revealed hybridization patterns identical to the original PAO organism.

Animals↗
PubMed1991

Normal anorectum: dynamic MR imaging anatomy.

In this study, the anatomy of the anorectum in relation to the surrounding structures and the anorectal angle were analyzed with magnetic resonance (MR) imaging at rest, during perineal contraction, and during straining in 10 asymptomatic subjects. The intra- and inter-observer and intra- and interpatient variations in the measurements of the anorectal angle, position of the anorectal junction, and position of the plica of Kohlrausch in the rectum were established at rest, during perineal contraction, and during straining. The values for the anorectal angle and position of the anorectal junction obtained with MR imaging were compared with standard radiography defecography findings. It was shown that MR imaging has the potential for measuring these parameters in a more precise and more patient-friendly way than defecography. Unlike dynamic defecography, MR imaging is able to depict the mobility of the posterior rectal wall. A descent of over 20 mm from rest to straining should be considered pathologic. This finding might play a role in patient selection for operation.

Adult↗
PubMed1990

Structure of the oligosaccharide of hen phosvitin as determined by two-dimensional 1H NMR of the intact glycoprotein.

The major form of the oligosaccharide of hen phosvitin was studied with two-dimensional 1H NMR of the intact glycoprotein. Its structure was determined from an analysis of the chemical shifts of the structural reporter groups, and it was further confirmed by comparison to several related model oligosaccharides. The oligosaccharide is N-linked and is present in a 1:1 stoichiometry to the protein. It has a complex type 1 triantennary structure with two NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,2 arms linked to the Man-4 and Man-4' and a third Gal beta 1, 4GlcNAc beta 1,4 arm attached to the Man-4. The oligosaccharide contains the common core sequence which is present in all N-linked glycoproteins [Man alpha 1,3(Man alpha 1,6)-Man beta 1,4GlcNAc beta 1,4GlcNAc beta 1,N]. In the course of this study, we have found that unique spin systems for the GlcNAc and NeuAc are obtained for spectra recorded in 90% H2O. Their NH peaks were assigned at low pH, and these assignments proved useful for confirming the identity of cross-peaks in the anomeric region. In addition, the protons of GlcNAc-1 could be correlated to the NH of the asparagine link. The cross-peak patterns determined in phase-sensitive 2D experiments for the H1,H2 protons have a different appearance for each type of monosaccharide, and this information was also used for making first-order assignments. A comparison with model compounds suggests that the solution conformation of the oligosaccharide is not affected by its attachment to the protein.

Animals↗
PubMed1990

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

DNA↗
PubMed1990

Abdominal ultrasonography: improved image quality with the combined use of a diet and laxatives.

Intestinal contents (gas and food particles) are well known to cause problems in abdominal ultrasonography by inducing confusing and inconclusive images. We prospectively studied the effect on the combination of a 2-day low-calorie, low gas-forming diet with laxatives on abdominal sonographic image quality. For a total group of 100 consecutive outpatients, 50 patients (diet-laxative or DL group) were randomly assigned to follow the preparation of a diet and laxatives, and 50 patients (control or C group) served as a control group without any special preparation. The sonographic imaging quality of abdominal organs and retroperitoneal structures was assessed without knowledge of which group the patients were assigned to. The DL group patients showed a significant improvement in visibility of the extra-hepatic common bile duct (p = 0.009), head and body of the pancreas (p = 0.015 and 0.004, respectively), para-aortic region (p = 0.001), and the iliac vessels (p = 0.0005). The combined use of a low-calorie, low gas-forming diet and laxatives improves the abdominal sonographic image quality of the above-mentioned structures to such an extent that the sonographer will be able to make more pertinent statements about the presence or absence of pathology.

Abdomen↗
PubMed1989

Characterization of a parallel-stranded DNA hairpin.

Recently we have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present study, we have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T)8C4(A)8-3' and the related antiparallel (APS) hairpin 5'-d(T)8C4(A)8-3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. However, the small chemical shift dispersion observed in proton NMR spectra of the PS hairpin suggests that the stem of this hairpin is more regular than that of the APS hairpin. On the basis of NOESY measurements, we find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1988

Binding of a dihydropyridine felodipine-analogue to calmodulin and related calcium-binding proteins.

A dihydropyridine-affinity column was prepared by coupling a physiologically active and vasoselective amino-derivative of felodipine to divinylsulfone-activated Trisacryl GF2000. Calmodulin (CaM) as well as the homologous calcium-binding proteins skeletal and cardiac Troponin C (sTnC and cTnC) and S100b bound to this resin in a calcium-dependent manner. In contrast, other homologous proteins such as parvalbumin and the intestinal calcium-binding protein did not bind. Competition studies showed that CaM had a higher affinity for the felodipine-column than sTnC or cTnC. Through studies with a series of proteolytic fragments of CaM and sTnC, it was found that the felodipine binding site is located in the amino-terminal domain of the protein. These results illustrate the utility of affinity-chromatography for the study of dihydropyridine-binding proteins.

Binding, Competitive↗
PubMed1988

Structural comparison between oxidized and reduced Escherichia coli thioredoxin. Proton NMR and CD studies.

Escherichia coli thioredoxin (Mr 11,700) usually functions as a hydrogen carrier protein that undergoes reversible oxidation/reduction reactions of its active-site disulfide linkage. By use of a number of assigned and identified resonances in one- and two-dimensional 1H NMR spectra, the two forms of the protein have been compared. Only groups that are relatively close to the active-site Cys-32, Cys-35 linkage such as Trp-28, Trp-31, Phe-27, Ala-29, and Val-25 undergo substantial changes in their 1H NMR chemical shift upon reduction. Various residues that are further removed from the active site, like Tyr-49, Tyr-70, His-6, Phe-12, Phe-81, and Phe-102, appear to be little affected (less than 0.02 ppm) by the reduction, suggesting that the rest of the protein structure is not much affected. Thus, the structural changes that occur upon reduction appear to be localized to the disulfide-containing turn and the central strand of the twisted beta-sheet that directly leads to this turn. Notwithstanding the apparent similarity in the secondary and tertiary structures of the oxidized and reduced forms of the protein, the thermal stability of the protein decreases by 10 degrees C upon the reduction of the single disulfide. This was found by both 1H NMR and near- and far-ultraviolet circular dichroism studies. Oxidized thioredoxin was also more resistant to alkaline denaturation. Furthermore, the exchange rate of the relatively stable slow-exchanging backbone amide protons that are part of the core of the twisted five-stranded beta-sheet of thioredoxin increases substantially after reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1988

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein.

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.

Animals↗
PubMed1988

Purification of rabbit skeletal muscle troponin C.

Troponin C binds to phenyl-Sepharose in the presence of Ca2+ and can be eluted with EDTA. This property was used as an essential step in the purification of this protein from rabbit skeletal muscle. Troponin C was extracted with 6M urea from extensively washed ground muscle. The protein was bound to and eluted from DEAE-Sephadex, fractionated by size on Sephadex G75, and in a final step purified from UV-absorbing non-protein impurities on phenyl-Sepharose. The total yield of electrophoretically pure protein was 60 mg per 100 g of muscle, which is considerably higher than that previously obtained.

Animals↗
PubMed1987

The binding of Ca2+, Mg2+ and Cd2+ to tryptic fragments of skeletal muscle troponin C. Cadmium-113 and proton NMR studies.

The structure of skeletal troponin C (sTnC) comprises two independently folded domains connected by an alpha-helical loop (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659). Both the NH2-terminal domain TR1C (9-84) and the COOH-terminal domain TR2C (89-159) can be obtained by limited tryptic cleavage. Here we report on proton and cadmium-113 NMR studies of Ca2+, Mg2+, and Cd2+ binding to these two separated domains. Our studies indicate that both halves retain a structure in the apo- and Ca2+-forms which resembles that of the intact protein. The events accompanying Ca2+ binding to these fragments are consistent with a biphasic binding pattern for sTnC, where the two sites in the COOH-terminal half are filled before those in the NH2-terminal half. Mg2+ only binds strongly to the two calcium-binding sites in the COOH-terminal half. The binding of this metal ion gives rise to a similar conformation for TR2C as that obtained with Ca2+. Whereas the binding of Ca2+ to the COOH-terminal part of sTnC takes place in a positive cooperative manner, the binding of this metal ion to TR2C occurs sequentially. Nevertheless, both Ca2+ ions bound to TR2C are in slow exchange (koff less than 10s-1). The binding of Mg24 both to sTnC and TR2C follows a sequential pattern with one site in slow exchange (koff less than 20 s-1) and one site in fast exchange (koff greater than 800 s-1). Binding of Cd2+ to TR2C occurs in a positive cooperative manner and results in the same conformation as observed with Ca2+. Cadmium-113 NMR spectra obtained at 5 degrees C confirm that two strong Cd2+-binding sites are localized in the COOH-terminal half and two weaker ones in the NH2-terminal half. It is concluded that these two proteolytic fragments comprise reasonable structural models for intact sTnC.

Animals↗