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H Engelhardt

Publications and source records attributed to H Engelhardt.

At least 37 records · Page 2Linked to original sources

On the measurement of shear elastic moduli and viscosities of erythrocyte plasma membranes by transient deformation in high frequency electric fields.

We present a new method to measure the shear elastic moduli and viscosities of erythrocyte membranes which is based on the fixation and transient deformation of cells in a high-frequency electric field. A frequency domain of constant force (arising by Maxwell Wagner polarization) is selected to minimize dissipative effects. The electric force is thus calculated by electrostatic principles by considering the cell as a conducting body in a dielectric fluid and neglecting membrane polarization effects. The elongation A of the cells perpendicular to their rotational axis exhibits a linear regime (A proportional to Maxwell tension or to square of the electric field E2) at small, and a nonlinear regime (A proportional to square root of Maxwell tension or to the electric field E) at large extensions with a cross-over at A approximately 0.5 micron. The nonlinearity leads to amplitude-dependent response times and to differences of the viscoelastic response and relaxation functions. The cells exhibit pronounced yet completely reversible tip formations at large extensions. Absolute values of the shear elastic modulus, mu, and membrane viscosity, eta, are determined by assuming that field-induced stretching of the biconcave cell may be approximately described in terms of a sphere to ellipsoid deformation. The (nonlinear) elongation-vs.-force relationship calculated by the elastic theory of shells agress well with the experimentally observed curves and the values of mu = 6.1 x 10(-6) N/m and eta = 3.4 x 10(-7) Ns/m are in good agreement with the micropipette results of Evans and co-workers. The effect of physical, biochemical, and disease-induced structural changes on the viscoelastic parameters is studied. The variability of mu and eta of a cell population of a healthy donor is +/- 45%, which is mainly due to differences in the cell age. The average mu value of cells of different healthy donors scatters by +/- 18%. Osmotic deflation of the cells leads to a fivefold increase of mu and 10-fold increase of eta at 500 mosm. The shear modulus mu increases with temperature showing that the cytoskeleton does not behave as a network of entropy elastic springs. Elliptic cells of patients suffering from elliptocytosis of the Leach phenotype exhibit a threefold larger value of mu than normal discocytes of control donors. Cross-linking of the spectrin by the divalent S-H agents diamide (1 mM, 15 min incubation) leads to an eightfold increase of mu whereas eta is essentially constant. The effect of diamide is reversed after treatment with S-S bond splitting agents.

Diamide

Structure of the porin from a bacterial stalk.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coli porins OmpC and OmpF. The natural two-dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three-dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coli porin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

Bacterial Outer Membrane Proteins

Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.

Azotobacter

Three-dimensional structure of the tetragonal surface layer of Sporosarcina ureae.

The three-dimensional structure of the regular surface layer of Sporosarcina ureae has been determined to a resolution of 1.7 nm by electron microscopy and image reconstruction. The S-layer has p4 symmetry, a lattice constant of 12.9 nm, and a minimum thickness of 6.6 nm. The reconstruction reveals a distinct domain structure: a massive core, arms connecting adjacent unit cells, and spurs which make contact at the subsidiary fourfold symmetry axes. In the z-direction the domains appear to be arranged in three planes, creating two entirely different surface reliefs. The S-layer has a complex pattern of pores and gaps that are 2 to 3 nm wide. In addition, the secondary-structure composition has been determined by infrared spectroscopy: about 35% of the polypeptide appears to have a beta-structure conformation.

Bacterial Proteins

Simulation of physiological ureteral peristalsis.

Occlusive ureteral peristalsis is simulated in an enlarged technical model of the ureter with a lobe-shaped lumen. The local pressure pulse is measured with catheters of different caliber and the influence of the catheters size and position on the uromanometrogram (UMG) is examined. The measurements are compared with UMGs at pathologically changed conditions (stagnation, residual urine depot etc.) and suggestions are given concerning size and shape of catheters to be used for urodynamic measurements.

Humans

The tetragonal surface layer of Clostridium aceticum: three-dimensional structure and comparison with the hexagonal layer of Clostridium thermohydrosulfuricum.

The regular surface layer (S-layer) of Clostridium aceticum has been isolated and the three-dimensional structure determined to a resolution of 2.0 nm from tilt series of negatively stained preparations. It has tetragonal symmetry with a lattice constant of 12 nm and a thickness of 6 nm; there are probably 4 protein monomers per unit cell. A large proportion of the protein is concentrated in massive "cores" at the major four-fold axes which are situated towards the inner surface of the layer. From these cores, delicate arms extend towards the minor four-fold axes, where secondary connectivity is established near the exterior surface. When viewed from the outside, each of the cores appears to have a large central depression, rather than a true "pore". Since this general pattern of mass distribution is shared by the hexagonal S-layer of Clostridium thermohydrosulfuricum, some consideration has been given to the possible evolutionary steps leading to changes in symmetry. From modelling experiments, it is evident that the change from four-fold to six-fold symmetry in this instance could be accomplished simply by the loss of a structural "domain" from the protomer.

Bacterial Proteins

Bloodpumps.

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Assisted Circulation

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.

Clostridium tetani

BCG vaccination of neonates, infants, schoolchildren and adolescents. Part I: Dose finding studies with BCG strain 1331 Copenhagen.

A BCG vaccine prepared from strain 1331 Copenhagen was given to newborns and infants in four logarithmically decreasing doses (expressed in VU = viable units): 25 X 10(4), 11 X 10(4), 5 X 10 (4), and 2.2 X 10(4). The efficacy of the vaccine was determined through the conversion rate (tuberculin skin test) with 2 and 10 T. U. of RT 23, AND 10 AND 50 I. U. of GT (Hoechst). The results indicated that the conversion rate is a function of the dose of VU of the vaccine, as well as of the units of tuberculin used for testing.

Adolescent

BCG vaccination of neonates, infants, schoolchildren and adolescents. Part II: Safety of a vaccine with strain 1331 Copenhagen.

The safety of the vaccine could be assessed by three different investigations: a) During the dose finding study (part I) the vaccine showed good safety in all concentrations employed concerning reactions at the site of injection; palpable lymph node enlargement was common. There was one case of suppurative lymphadenitis amond the 262 children who were given the vaccine in the highest concentration (25 X 10(4) VU). b) Subsequent trials in the course of 21 months, when high risk persons could be vaccinated, revealed a rate of this complication in the 1:1,000 range, no case was reported among older persons. c) After the official approval of the vaccine (10 - 30 X 10(4) VU/ml) in the FRG 1 supp. lymphadenitis among 5,000 vaccinated newborns can be calculated.

Adolescent

[Investigations carried out to ascertain the dose-effect relationship of a BCG vaccine, strain 1331 Copenhagen, in neonates and young infants (author's transl)].

1405 neonates and infants were vaccinated with BCG Vaccine (strain 1331 Copenhagen) at five clinics in the Federal Republic of Germany. Doses in logarithmic increments from 22000 to 250000 VU (viable units)/0.1 ml were given by strictly intradermal injection. Carrying out the post-vaccinal tuberculin test by the MENDEL-MANTOUX technique, the dose-effect relationship could be demonstrated (Fig. 3). Conversion rates raised from 43% to 76% (Tab. 1); they are furthermore depending from the tuberculin dose and the assessment of the skin reaction. Tests with up to 50 I.U. of purified tuberculin were resulting in conversion rates over 90% for vaccination doses of 100 000 VU and more, any palpable infiltration regarding as a positive result (Fig. 4). The vaccine showed good safety in all concentrations employed concerning reactions at the site of injection. Lympnode enlargement, palable even 12 weeks postvacc., was common. In the course of the trial there was one case of suppurative lymphadenitis among the 262 children who were given the vaccine in the highest concentration (250000 VU). Subsequent trials revealed a rate of this complication in the 1:1000 range. The approval for the vaccine with 100000-300000 VU/dose has subsequently been given by the Federal Bureau for Sera and Vaccines.

BCG Vaccine

Field trials with killed oral typhoid vaccines.

The article gives the results of the use of two types of killed oral typhus vaccine administered to students 10 to 19 years of age in two urban areas of the Province of Santiago. Vaccine from the German Democratic Republic (GDR) was administered to 37,517 students and placebo to an equivalent control group. The period of observation was 18 months (November 1973-April 1975). The other product, from the German Federal Republic (GFR) was administered to 37,566 students, with placebo given to an equivalent control group. In this case the observation period was 7 months (October 1974-April 1975). The antigenic composition of the two vaccines is qualitatively and quantitatively different as are the characteristics of their preparation. In the field trial with the product from the GDR, the morbidity for typhoid fever in the vaccinated and control groups showed statistically significant differences, even though on the border line level. Satisfactory and highly significant results had been obtained with the product in earlier field trials. The difference observed between morbidity in the two groups in the field trial with GFR vaccine was not significant. It is emphasized in this regard that the observation period was shorter in this case, which precludes definitive conclusions. A high proportion of reliable confirmation of diagnosis of cases is evident for both field trials. The findings show the need for subjecting this type of vaccine to further and better field trials.

Administration, Oral