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Biomedical subjects

H Engelhardt

Publications and source records attributed to H Engelhardt.

At least 37 records · Page 2Linked to original sources
PubMed1998

A function for lipoxygenase in programmed organelle degradation.

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells of the eye lens, and keratinocytes. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.

Animals↗
PubMed1998

Optical sensor arrays for odor recognition.

Optical sensor arrays containing fluorescent solvatochromatic dyes immobilized in a plurality of polymers generate information-rich responses upon exposure to organic vapors. The response profiles are used to train a variety of computational networks such that subsequent exposure of the array to the vapors enables them to be classified and/or quantified. A number of strategies can be taken to enhance sensitivity and to increase sensor diversity.

Animals↗
PubMed1998

Voltage-dependent closing of porin channels: analysis of relaxation kinetics.

The anion-selective porin Omp34 from Acidovorax delafieldii was unidirectionally reconstituted in planar lipid membranes. Pore closing was recorded particularly at low salt conditions for negative and positive membrane potentials in the range of +/-10 to +/-100 mV. Relaxation curves were fitted by exponential functions in order to describe and to analyze the voltage-dependent behavior. Omp34 exhibited the following characteristics: (i) The channels are asymmetric with respect to closing characteristics and corresponding functional parameters. (ii) Relaxation curves can be fitted by a single exponential function in the low voltage range only, at >/=40 mV combinations of two exponential functions are required. (iii) Beyond 60 to 70 mV a third exponential function is necessary to fit the fast closing components properly. The time constants differ by two to three orders of magnitude. (iv) Hysteresis in I-V-diagrams originate from slow relaxation components which are different for positive and negative voltages. The implications for models aiming at description of voltage-dependent closing are discussed.

Cell Membrane↗
PubMed1998

Pleistocene collapse of the west antarctic ice sheet

Some glacial sediment samples recovered from beneath the West Antarctic ice sheet at ice stream B contain Quaternary diatoms and up to 10(8) atoms of beryllium-10 per gram. Other samples contain no Quaternary diatoms and only background levels of beryllium-10 (less than 10(6) atoms per gram). The occurrence of young diatoms and high concentrations of beryllium-10 beneath grounded ice indicates that the Ross Embayment was an open marine environment after a late Pleistocene collapse of the marine ice sheet.

Journal Article↗
PubMed1998

Crystallization and preliminary X-ray crystallographic studies of the native and chemically modified anion-selective porin from Comamonas acidovorans.

Omp32, the strongly anion-selective porin from Comamonas acidovorans, has been crystallized. Two crystal forms were observed, both of which belong to space group R3, but exhibit different cell dimensions a = b = 106.7, c = 140.6 A (crystal form I) and a = b = 87.1, c = 135.3 A (crystal form II) with one trimer per asymmetric unit. The crystals diffract to 2.2 and 2.3 A resolution, respectively. Omp32 was chemically modified by introducing negative charges through succinylation. The number and positions of the individual modifications were determined using mass spectrometry and X-ray crystallography. Chemically modified porins yielded crystals of a third form, also of space group R3 but with cell constants of a = b = 109.3 and c = 263.2 A (crystal form III), showing a virtually doubled c axis. Crystals of form III diffract to 3.5 A resolution.

Bacterial Proteins↗
PubMed1998

A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

Adenosine Triphosphatases↗
PubMed1998

Nonlinear and asymmetric open channel characteristics of an ion-selective porin in planar membranes.

The open channel characteristics of the bacterial porin Omp32 from Comamonas acidovorans were investigated by means of conductance measurements in planar lipid bilayers of the Montal-Mueller type. Particularly at low salt conditions (< or = 30 mM KCl) Omp32 exhibited some unusual asymmetric and nonlinear functional properties. Current-voltage relationship measurements showed that conductance depends on the orientation of porin molecules and is a nonlinear function of the applied membrane potential. Conductance also depends on the salt concentration in a manner not common to porins and the salt concentration modulates the nonlinearity of conductance-voltage relationships. Omp32 is strongly anion-selective. The nonlinear and asymmetric conductance of the open channel is a new observation in porins.

Biophysical Phenomena↗
PubMed1998

Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes.

The structure of 20 S particles, consisting of NSF, SNAPs, and SNARE complexes, was analyzed by electron microscopy and fluorescence resonance energy transfer. Structural changes associated with the binding of alpha-SNAP and NSF to SNARE complexes define the contribution of each component to the 20 S particle structure. The synaptic SNARE complex forms a 2.5 x 15 nm rod. alpha-SNAP binds laterally to the rod, increasing its width but not its length. NSF binds to one end of the SNAP/SNARE complex; the resulting 20 S particles measure 22 nm in length and vary in width from 6 nm at their narrowest point to 13.5 nm at their widest. The transmembrane domains of VAMP and syntaxin emerge together at the NSF-distal end of 20 S particles, adjacent to the amino terminus of alpha-SNAP.

Adenosine Triphosphate↗
PubMed1998

The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties.

Omp21, a minor outer membrane protein of the soil bacterium Comamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of beta structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic beta strands, which includes virulence proteins, such as the neisserial opacity proteins, Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF from Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1997

Role of uterine immune cells in early pregnancy in pigs.

The immune system discriminates 'self' from 'non-self', and eliminates that which it determines to be non-self. Mammalian pregnancy appears to represent a failure of self-non-self discrimination, yet it is a highly successful reproductive strategy. We present evidence that the immune system of the female pig responds to the challenges of both mating and the presence of conceptuses. Mating induces an influx of inflammatory leukocytes into the endometrial stroma and uterine lumen. This response, while partially under endocrine control, is amplified by as yet unidentified factors in seminal plasma. In addition to preventing microbial infection, this mating-induced immune response may enhance reproductive performance. During the first month of pregnancy when intimate contact between maternal and fetal tissues is being established, the number of uterine lymphocytes decrease in the luminal epithelium and increase in the endometrial stroma at sites of conceptus attachment. The majority of these lymphocytes express the CD2 and CD8 surface markers, consistent with either T or natural killer (NK) cell lineage. Dispersed endometrial cells obtained during early gestation exhibit a pregnancy-specific increase in NK-like lytic activity. Our ongoing efforts to determine whether these NK-like lymphocytes are the ones that localize to sites of conceptus attachment are discussed. We speculate on how the reactions of pig uterine leukocytes to seminal plasma and conceptuses might contribute to successful pregnancy.

Animals↗
PubMed1996

Characterization of a porin from the outer membrane of Vibrio anguillarum.

The outer membranes of the 10 serovars of Vibrio anguillarum showed a common major protein with a size of around 40 kDa. Antibodies against the major outer membrane protein (MOMP) of V. anguillarum AO18 (serovar O1) cross-reacted with the MOMPs of all the other serovars but not with the outer membrane proteins of Escherichia coli. The MOMP of V. anguillarum serovar O1 was isolated, reconstituted to two-dimensional crystals, and structurally characterized by electron microscopy and image processing. The unit cell structure of the crystalline MOMP, as well as the secondary structure composition of the protein with a high amount of beta-structure, is strongly reminiscent of that of bacterial porins. The functional properties of the pores were investigated by conductance measurements with the MOMP reconstituted in planar lipid membranes. The V. anguillarum MOMP is characterized by a relatively weak cation selectivity and a moderate surface charge, and it shows voltage-dependent conductance effects. The MOMP is functionally similar to OmpF from E. coli, and it can be classified as a general diffusion porin.

Biological Transport↗
PubMed1996

Alkaline phosphatase activity in the theca of ovarian follicles of the hen throughout follicular development.

Within the hen's ovary, the theca tissue of small follicles that have not yet entered the preovulatory hierarchy is a major contributor to ovarian steroidogenesis. Relatively little is known about developmental stages in the theca layer within this pool of follicles and of the histological properties of this tissue. In pigs and sheep, alkaline phosphatase activity has been identified in the theca of preovulatory follicles and in theca-derived cells in the corpus luteum. The objectives of this study were to document morphological changes in the theca layer through pre- and postovulatory follicular development, and to assess alkaline phosphatase activity at these developmental stages. Ovarian tissue containing small white, large white, small yellow, large yellow, and postovulatory follicles (POF) was obtained from mature White Leghorn hens and embedded in either methacrylate for morphological assessment or paraffin for alkaline phosphatase histochemistry. Alkaline phosphatase activity was found to be specific for cells of the theca interna in preovulatory follicles. Activity was first detectable in very small white follicles, the earliest stage in which the theca interna could be distinguished from the theca externa, and highest in the yellow yolky follicles. After ovulation, activity decreased to undetectable levels by the POF4 to 5 stages in POF within 4 to 5 d after ovulation. This study has shown that in the domestic hen, alkaline phosphatase activity is present in cells adjacent to the basal lamina of the theca interna of preovulatory follicles and POF up to 4 to 5 d after ovulation. Identification of the functional significance of this histological reaction will provide new information on ovarian function in the hen.

Alkaline Phosphatase↗
PubMed1995

Tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability.

The surface (S-) layer of the hyperthermophilic archaebacterium Staphylothermus marinus was isolated, dissected into separate domains by chemical and proteolytic methods, and analyzed by spectroscopic, electron microscopic and biochemical techniques. The S-layer is formed by a poorly ordered meshwork of branched, filiform morphological subunits resembling dandelion seed-heads. A morphological subunit (christened by us tetrabrachion) consists of a 70 nm long, almost perfectly straight stalk ending in four straight arms of 24 nm length that provide lateral connectivity by end-to-end contacts. At 32 nm from the branching point, tetrabrachion carries two globular particles of 10 nm diameter that have both tryptic and chymotryptic protease activity. Tetrabrachion is built by a tetramer of M(r) 92,000 polypeptides that form a parallel, four-stranded alpha-helical rod and separate at one end into four strands. These strands interact in a 1:1 stoichiometry with polypeptides of M(r) 85,000 to form the arms. The arms are composed entirely of beta-sheets. All S-layer components contain bound carbohydrates (glucose, mannose, and glucosamine) at a ratio of 38 g/100 g protein for the complete tetrabrachion-protease complex. The unique structure of tetrabrachion is reflected in an extreme thermal stability in the presence of strong denaturants (1% (w/v) SDS of 6M guanidine): the arms, which are stabilized by intramolecular disulphide bridges, melt around 115 degrees C under non-reducing conditions, whereas the stalk sustains heating up to about 130 degrees C. Complete denaturation of the stalk domain requires treatment with 70% (v/v) sulfuric acid or with fuming trifluoromethanesulfonic acid. The globular protease can be heated to 90 degrees C in 6M guanidine and to 120 degrees C in 1% SDS and represents one of the most stable proteases characterized to date.

Archaea↗
PubMed1995

Recognition of alpha-helical peptide structures using high-performance liquid chromatographic retention data for D-amino acid analogues: influence of peptide amphipathicity and of stationary phase hydrophobicity.

The reversed-phase HPLC behaviour of double D-amino acid replacement sets of amphipathic and non-amphipathic helix-forming peptides consisting exclusively of leucine, lysine and alanine residues was studied on different polymer-encapsulated silica-based stationary phases. Plotting the retention times versus the position of D-amino acid substitution gives a characteristic pattern showing decreased retention times in the helical region. The retention time profile obtained using an amphipathic alpha-helix is caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. However, the effect is similar but less pronounced using a non-amphipathic helical peptide that is unable to interact by a preferred binding site. The results demonstrate that reversed-phase HPLC data for peptide analogues provide an indication event of a non-amphipathic helical structure in peptides.

Amino Acid Sequence↗
PubMed1995

Expression of inhibin alpha- and beta A-subunit mRNA and protein in the fetal sheep ovary throughout gestation.

In adult sheep, inhibin expression in developing follicles appears to be associated with antrum formation. Our objective was to investigate using in situ hybridization and immunohistochemistry whether antral follicles present before birth in the sheep expressed mRNA or peptide for inhibin alpha- and beta A-subunits. At days 70 and 100 when only primordial and primary follicles were present, there was no detectable mRNA or peptide for either inhibin subunit. By days 130 and 140 (term = 145 days), many secondary follicles were present, a proportion of which (approximately 50%) expressed detectable levels of alpha-subunit mRNA but not peptide. A number of antral follicles were present by this stage, all of which expressed alpha-subunit mRNA and peptide. Expression of beta A-subunit mRNA and peptide was undetectable at all stages of gestation. Our results indicates that even in non-ovulatory follicles present before birth, expression of inhibin, at least the alpha-subunit, is developmentally linked with antrum formation.

Animals↗
PubMed1995

Expression of mRNA and immunocytochemical localization of inhibin alpha- and inhibin beta A-subunits in the fetal sheep testis.

In order to investigate the ontogeny of gonadal inhibin production in the male fetal sheep, testes were collected from male fetuses at days 70, 100, 130 and 140 of gestation (term = 145 days). The expression and localization of inhibin alpha- and inhibin beta A-subunit mRNA and protein were evaluated using in situ hybridization and immunocytochemistry. The expression of inhibin alpha-subunit mRNA was localized within the seminiferous cords of the developing fetal testis and progressively increased with gestational age. Immunostaining corresponding to immunoreactive inhibin alpha-subunit was detected in Sertoli cells within the seminiferous cords at days 100, 130 and 140 of gestation. In addition, immunostaining was detectable in a small proportion of Leydig cells. No expression of inhibin beta A-subunit mRNA or immunoreactivity was detected in any testicular tissue at any stage of gestation. These data show that the Sertoli cells of the developing fetal sheep testis have the capacity to produce inhibin alpha-subunit by day 100 of gestation and that production increases during late gestation.

Animals↗