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H Engelhardt

Publications and source records attributed to H Engelhardt.

At least 19 recordsLinked to original sources
PubMed2001

Application of carboxymethyl-beta-cyclodextrin as a chiral selector in capillary electrophoresis for enantiomer separation of selected neurotransmitters.

The aim of this work was to optimize conditions for capillary electrophoresis separation of different neurotransmitters (serotonin, phenylalanine, dopamine, adrenaline, ephedrine, propranolol and DOPA) in a single run, including separation of existing enantiomers. As chiral selectors added to the borate background, electrolyte unsubstituted alpha-, beta- and -gamma-cyclodextrins (CDs), methyl-, dimethyl-, and trimethyl-substituted beta-CDs, and hydroxypropyl-substituted alpha-, beta- and gamma-CDs were examined. Also carboxymethyl-beta-CD and succinyl-beta-CD were used for this purpose. In addition to the kind and concentration of chiral selector, some other experimental factors also have been optimized, such as concentration of borate buffer, content of methanol, pH of electrolyte, method of sample introduction into the capillary and washing procedure between consecutive runs. The best results were obtained using 20 mM carboxymethyl-beta-CD in borate buffer of pH 7.5 as running electrolyte and hydrostatic injection. The obtained sensitivity of response (peak height) varied from 0.4 for adrenalines to 2.3 mAU mM(-1) for propranolols. The concentration detection limits (S/N=3) were in the range from 0.04 mM for propranolols to 0.2 mM for adrenalines. The resolution obtained in optimized conditions in a single run was from 0.75 for adrenalins and 1.0 for propranolols up to 2.0 for ephedrines. The developed method was employed for determination of these analytes in brain tissue extracts.

Cyclodextrins↗
PubMed2001

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Animals↗
PubMed2001

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

Animals↗
PubMed2001

Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria.

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

Fungal Proteins↗
PubMed2001

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis.

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.

Amino Acid Sequence↗
PubMed2001

The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum.

In this study, a flagella-related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1-3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs): htpIX, a member of the halobacterial transducer protein gene family, and the genes flaD-K. The genes flaD, E, G, H, I and J share high homologies with genes from other Archaea. Interestingly, flaK shows similarities to bacterial genes involved in the regulation of flagellar synthesis. The ORFs of flaH, flaI and flaK contain sequences coding for nucleotide binding sites. Furthermore, flaI contains a motif called the bacterial type II secretion protein E signature, indicating a functional relation to members of the bacterial pili type IV-type II secretion protein superfamily. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the genes flaE to flaK are transcribed into one polycistronic message. In frame deletion mutants of flaI were generated by gene replacement. The deletion strain lacks motility and belongs to the fla(-) mutant class, indicating that it is deficient in flagellar biogenesis. The overall amount of flagellin protein in Delta flaI cells is reduced, although transcription of the flagellin genes is unaffected. Therefore, the flaI gene product is involved in the biosynthesis, transport or assembly of flagella in H. salinarum.

Amino Acid Sequence↗
PubMed2000

Simultaneous determination of alkali, alkaline earth and transition metal ions by capillary electrophoresis with indirect UV detection.

Determination of metal ions in aqueous samples using capillary electrophoresis can be accomplished with indirect UV detection. For optimal determination of alkali, alkaline earth, and transition metal ions, several electrolyte systems were studied. Detection at 214 nm was performed with a background electrolyte containing reagents with inherent absorbance in the UV range: imidazole, 4-methylbenzylamine and 4-aminopyridine. Glycolic acid and alpha-hydroxyisobutyric acid were used as complexing reagents. A mixture of 16 metal ions was successfully separated. The detection limits were between 92 ppb for Ca and 454 ppb for Cu with hydrostatic injection. All peaks were completely resolved and well separated. A separation efficiency of about 650,000 theoretical plates per meter was achieved for the Mg ion. The described methods can be used successfully in routine analysis of real samples. One of the methods was applied to an environmental water sample from the Georgian river Kasretula.

Calibration↗
PubMed2000

Characterization of synthetic polyelectrolytes by capillary electrophoresis.

Capillary electrophoresis in entangled polymer solutions was applied to determine the molecular mass and polydispersity of polyelectrolytes. The separation selectivities of different polyethylene glycols as buffer additive can be correlated to their average molecular mass. A universal curve correlating the selectivity and the molecular mass could be obtained by using the instrinsic viscosity of the polyethylene glycol. The separation of poly(2-vinylpyridine) standards was compared to the separation of poly(4-vinylpyridine) standards. An indirect detection system was developed to characterize the cationic polyelectrolyte polydiallyldimethyl ammonium chloride. Various polymers with oppositely charged groups (polycarboxybetaines) were investigated with respect to structure dependence, pH dependence and molecular mass dependence of interand intramolecular association.

Buffers↗
PubMed2000

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 A resolution.

BACKGROUND: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. RESULTS: The crystal structure of Omp32 was determined to a resolution of 2.1 A using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded beta barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within beta-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with beta-strand 1 of the porin. CONCLUSIONS: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.

Amino Acid Sequence↗
PubMed2000

Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans.

The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules. After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin. The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification). Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis. Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography. Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA.

Bacterial Outer Membrane Proteins↗
PubMed1999

K

Tetrahedral framework structures with topologies that show close relationships to the feldspar group or to gismondine are formed by the zincoborophosphates A[ZnBP(2)O(8)] (see picture). The compounds are readily accessible under mild hydrothermal conditions (T(max)=170 degrees C) and are thermally remarkably stable (for example, 940 degrees C for A=Cs). Metalloborophosphates are a new class of compounds, which are expected to provide access to zeolite-like microporous systems.

Journal Article↗
PubMed1999

Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin.

Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin.

Animals↗
PubMed1999

Properties of a cyclodextrin-specific, unusual porin from Klebsiella oxytoca.

The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptide-containing precursor, and possesses a high content of antiparallel beta-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of alpha-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K(S), of 28 microM for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a = 16.1 nm, b = 3.8 nm, gamma = 93 degrees. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.

Amino Acid Sequence↗
PubMed1999

How to achieve higher repeatability and reproducibility in capillary electrophoresis.

The influence of experimental parameters on precision of migration times and accuracy of integration has been studied. The repeatability of migration times strongly depends on the proper selection of the rinse steps between each run of a CE system. The rinse steps have to be optimized additionally with each separation system. This is especially important for systems providing an anodic electroosmotic flow. The errors introduced by the integration software were studied by transferring the same data sets to different commercial available integration softwares. A strategy for the transfer of raw data files between several softwares is described. By using systems with identical peak areas or with identical peak heights it could be shown that the newly introduced softwares can cope better with the leading or tailing peaks. The RSD of quantitation strongly depends on the signal-to-noise ratio. At S/N ratios larger than 35 no differences between the various softwares studied could be observed. At lower S/N ratios the newly released softwares are to be preferred.

Electrophoresis, Capillary↗
PubMed1999

Micellar electrokinetic capillary chromatography of psoralen and isopsoralen.

In this paper, a micellar electrokinetic capillary chromatography (MEKC) method to analyze the active components of psoralen and isopsoralen in the Chinese herb Psoralea corylifolia L. was developed. It was found that the optimum separation condition for the analysis of these compounds was a buffer solution which contained phosphate, borate and sodium dodecyl sulfate (pH 9.2). Through optimization of the experimental condition, the two isomers could be baseline-separated and their contents in the methanol-extracted sample were quantitatively analyzed.

Buffers↗
PubMed1999

Direct effects of ovine follicular fluid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep.

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.

Analysis of Variance↗
PubMed1998

Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer-cell wall interactions.

Surface layers (S-layers) from Bacteria and Archaea are built from protein molecules arrayed in a two-dimensional lattice, forming the outermost cell wall layer in many prokaryotes. In almost half a century of S-layer research a wealth of structural, biochemical, and genetic data have accumulated, but it has not been possible to correlate sequence data with the tertiary structure of S-layer proteins to date. In this paper, some highlights of structural aspects of archaeal and bacterial S-layers that allow us to draw some conclusions on molecular properties are reviewed. We focus on the structural requirements for the extraordinary stability of many S-layer proteins, the structural and functional aspects of the S-layer homology domain found in S-layers, extracellular enzymes and related functional proteins, and outer membrane proteins, and the molecular interactions of S-layer proteins with other cell wall components. Finally, the perspectives and requirements for structural research on S-layers, which indicate that the investigation of isolated protein domains will be a prerequisite for solving S-layer structures at atomic resolution, are discussed.

Amino Acid Sequence↗