Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study.

Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher Km and Vmax levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both Km and Vmax between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between Km and Vmax were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products.

Reflectance enzyme histochemistry (REH): visualization of cerium-based and DAB primary reaction products of phosphatases and oxidases in cryostat sections by confocal laser scanning microscopy.

In the present study the reflectance mode of confocal laser scanning microscopy was adapted to detect and to assess semiquantitatively cerium-based primary reaction products of oxidases [Ce(IV) perhydroxide] and phosphatases [Ce(III) hydroxyphosphate converted into Ce(IV) perhydroxyphosphate] as well as of the 3,3'-diaminobenzidine (DAB)-based primary reaction product of cytochrome c oxidase in cryostat sections. Confocal laser scanning microscopy offers a unique way of making visible histochemical reaction products which are weakly absorbant but sufficiently reflective. It was easily possible to record simultaneously the reflectance signals at the wavelength of the exciting laser (preferentially 488 nm) and the autofluorescence signals ( > 580 nm in our set-up) of glutaraldehyde-fixed tissue. The results of an imbibition study of cerium-containing model precipitates indicate that the cerium, generally, should be oxidized prior to observation because the index of refraction of Ce(IV) compounds is considerably higher than that of the corresponding Ce(III) compounds. An attempt at comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is presented. The proposed technique may open new possibilities in enzyme- and immunohistochemistry.

Quantitative screening for fluconazole-amphotericin B antagonism in several Candida albicans strains by a comparative agar diffusion assay.

Antagonism between fluconazole (FCZ) and amphotericin B (AMB) was determined with an agar diffusion technique using series of agar plates containing no or 10 mgl-1 FCZ (comparative diffusion assay). Serial dilutions of AMB produced concentration-dependent inhibition zones that varied between the two agar plate series. This technique served as screening method to determine FCZ-AMB interactions in 18 Candida albicans strains. The critical concentrations of AMB were enhanced 1.33- to 7.0-fold by FCZ. The critical time, T0, was reduced by half by FCZ.

Post-antibiotic effect and post-expositional polyene antagonism of azole antifungal agents in Candida albicans: dependence on substance lipophilia.

The lipophilic azoles itraconazole (ICZ), ketoconazole (KCZ) and miconazole (MCZ) have two things in common regarding their effect on Candida albicans. First, these azoles cause a growth inhibition that persists for at least 24 h after exposure (post-antibiotic effect), although this is only occasionally observed for ICZ. Secondly, these substances cause a decrease in the fungicidal activity of amphotericin B (AMB, 1 mg l-1) upon subsequent exposure to this drug. In contrast, fluconazole (FCZ) exhibits neither of these two effects. Further tests suggest that both of these phenomena observed may be related to the non-covalent binding of the three lipophilic azoles to lipophilic cytoplasmic components of yeast cells. With fluconazole, such bonds seem to be much weaker. The amount of relatively hydrophilic fluconazole that is bound non-specifically to the fungal cell is evidently too low to produce long-lasting post-exposure effects like those caused by lipophilic azoles.

Dysmorphic erythrocytes in glomerulonephritis. 1. Electron microscopical and histochemical investigation.

Dysmorphic erythrocyte malformation in urine is characteristic of glomerulonephritis. The mechanisms leading to this phenomenon are still unknown. To obtain evidence of the site as well as of the process of erythrocyte damage, electron microscopical and histochemical investigations of renal biopsy materials from 19 patients with histologically defined glomerulonephritis were performed. The results suggest that the initial damage of erythrocytes in the glomerular area is reasoned by enzymatic glycocalyx destruction. On passage through the tubular system the osmotically sensitized surface altered cells undergo rapid hemolysis and losses of membrane skeletal proteins leading to dysmorphic shape transformations.

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.

[Preclinical biological testing of veneer composite materials]. / Präklinische biologische Prüfung von Verblendkompositen.

The toxicity of commercial (Dentacolor, Visio-Gem) and other light reactive veneer composite materials under development (DG) was investigated by the hemolysis test. In contrast to a standard composite filling material (Evicrol) the veneer composite materials caused only small hemolysis rates.

[Ektacytometry: a method for characterizing erythrocyte deformability]. / Ektazytometrie--ein Verfahren zur Charakterisierung der Deformierbarkeit von Erythrozyten.

In principle, the ektacytometer consists of a combination of a Laser-illuminated diffractometer with a circular viscometric fluid flow, analogous to that of a rotation viscometer. In this device erythrocytes are deformed to ellipsoid-like shapes and produce a diffraction pattern which, depending on the flow conditions, is to some degree elliptically distorted. From the shape of this pattern the extent of cell deformation can be deduced. In this survey an introduction into the mode of operation of the ektacytometer as well as an overview on methodical variants are given on the basis of literature. Since its publication in 1974 ektacytometry has found many applications, mainly in basic research. Presumably, it will receive still broader interest also as a routine method in haematological or pharmaceutical laboratories.

Preservation of red cell concentrates. Dependence on storage time of IgG binding, osmotic fragility, MCV, and surface area index.

In a comparative study of red cells from concentrates preserved in SAG medium without and with 30 mM sucrose, mannitol or sorbitol, resp., we determined the variation of IgG binding, osmotic fragility, MCV and the surface area index with storage time. IgG binding gave no conclusive results. Osmotic fragility turned out to be increased in simple SAG in comparison to the sugar-supplemented media. From measurements of microhematocrit and pH, the mean cellular volume (MCV), standardized to the initial pH value, turned out to decrease in all the media tested by not more than about 5 per cent after 3 weeks, and 10 per cent after 6 weeks. This is in advantageous contrast to the strong decrease in microhematocrit formerly observed in red cell concentrates in CDS-AG medium. Cells resuspended in simple SAG medium exhibited the smallest decrease in MCV. However, as inferred from data on hemolysis and vesiculation (D. Stibenz, accompanying paper), in these cells the loss of surface area proved to be maximal.

[Quantitative histochemical studies of islands of Langerhans in the (Psammomys obesus) in the development of diabetes mellitus. II. Surface measurement of the islands]. / Quantitativ-histochemische Untersuchungen an Langerhansschen Inseln von Sandratten (Psammomys obesus) während der Ausbildung eines Diabetes mellitus. II. Flächenmessung der Inseln.

Measurement of the islet-area are pointed out in 30 sand rats. The animals are divided in 3 groups: 8 sand rats are the reference group, 11 animals are the IGT-group (impaired glucose tolerance), and the diabetic group consists also of 11 sand rats. The islet-area is in the IGT-group increased in 173.2%. It is concluded that the islet-volume is also increased. The diabetic group is characterized by the maximal increase of the islet area (431.4%). Hyperplasia of Langerhans' islets is detectable histologically. The sand rat is a biological model of the human non-insulin-dependent diabetes mellitus (NIDDM).

[Quantitative histochemical study of pancreatic islets of sand rats (Psammomys obesus) in diabetes mellitus I. Cytophotometric measurement of insulin content]. / Quantitativ-histochemische Untersuchungen an Langerhansschen Inseln von Sandratten (Psammomys obesus) während der Ausbildung eines Diabetes mellitus. I. Cytophotometrische Messung des Insulingehalts.

The insulin-content in islets of Langerhans on sand rats with disturbances of the carbon hydrate tolerance (reference-, impaired glucose tolerance-, and diabetic group) is measured cytophotometrically. In the IGT-group (impaired glucose tolerance) is a decrease of the insulin content in B-cells detectable. However in the diabetic group of sand rats is the insulin-content increased. This results demonstrate the stimulation of the B-cell function in the islets of Langerhans. The diabetes mellitus of sand rats is characterized by disturbed glucose tolerance hyperinsulinaemia, and elevated readiness to insulin secretion. That are characteristics of the human type-II-diabetes (insulin-independent). In conclusion, the sand rat is a biological model of the human type-II-diabetes (NIDDM).

[Elasticity of the erythrocyte membrane: overview and attempted explanation based on recent data on the ultrastructure of the membrane skeleton]. / Die Elastizität der Erythrocytenmembran: Ubersicht und Versuch einer Deutung auf Grund neuerer Daten zur Ultrastruktur des Membranskelets.

Membrane shear elasticity enables the mammalian erythrocyte to reassume its discoid shape after traversing narrow constrictions of the microcirculation. Both the high extensibility of the erythrocyte membrane under shear and its positive heat of deformation represent special features of its elastic response, which are poorly understood as yet. Following an introductory outline of the conceptual framework and ultrastructural basis of erythrocyte membrane shear elasticity, an attempt is made to explain qualitatively the strange deformation behaviour of this membrane in terms of present day knowledge on the molecular arrangement and performance of the major erythrocyte membrane protein, spectrin.

Deformability of erythrocytes incubated at various pH-values.

The deformability of human erythrocytes was studied at pH values ranging from 6.4 to 8.5 using micropipette aspiration of whole cells. Also the mean cell volume was measured in dependence on pH. The observed high increase of the transit pressure pt with lowering of the pH in a fairly narrow interval suggests that within the range of pH values under consideration pi is mainly controlled by the effect of the critical volume of the cells.

[Importance of the cell membrane for the sequestration of aged erythrocytes]. / Die Bedeutung der Membran für die Sequestration gealterter Erythrocyten.

The erythrocyte membrane is an important cell organelle which determines the intravital life time of the cell in a decisive way. Their mechanic properties are closely connected with the metabolism of the cells. Increasing disturbance of the cellular metabolism especially in the course of biological aging leads to enhanced cell rigidity. It is supposed that the decreased elasticity of the aged erythrocytes in critical regions of the blood circulation (capillaries with a diameter of 2.5 to 3.0 micrometers) leads to pressure induced vesiculation and spherocytosis. It is possible that also IgG-receptors are demarked by this process. After passing a threshold concentration as immunobiological signals increased irreversible binding of IgG induces the sequestration of the erythrocytes by the erythrocyte destructing system.

[Changes in shape and plasticity of preserved erythrocytes]. / Formwandel und Verformbarkeit konservierter Erythrozyten.

The present study is concerned with alterations of erythrocyte shape and deformability during banking of ACD-AG blood samples. Highly diluted blood plasma suspensions of erythrocytes previously stored for 1 and 43 days, respectively, were employed for a) the microscopic evaluation of cell shapes, and b) measurement of the negative pressure pt essential for a cell's complete transit of a cylindrical micropipette 3.2 micrometers in diameter. Each of the blood samples was studied in an air equilibrated state either prior and following the fractionation of light and heavy cells. The classification of cell shapes was accomplished by a particular index. The findings are in accord with the banking related transformation of erythrocytes as known from recent studies. Although, exept of spheroechinocytes and spherocytes, a significant decline of deformability of stored red cells was found only in the case of discocytes and type 3 echinocytes, all types of heavy erythrocytes were shown less deformable in comparison with light cells equally banked for 43 days. The relation between our pt values and published data is discussed, and a crude estimate of the trapping time of the most rigid cells by the spleen after retransfusion is presented.

[Inverse pH-dependent shape changes of erythrocytes in the presence of albumin]. / Inverser pH-abhängiger Formwandel der Erythrozyten in Gegenwart von Albumin.

A comparative study on the shape of human erythrocytes suspended in 7 different media showed, contrary to the well-known albumin-free case, an enhancement of the number of discocytes and stomatocytes for pH rising in all HSA containing media applied. At the same time, the transmembrane potential as determined by extra- and intracellular pH was lowered in all of 6 media tested. Consequently, there is no simple relationship between the pH-dependent behaviour of cell shape and corresponding changes of transmembrane potential.

Alteration by procaine of spectrin cross-links, deformability, and fluidity related properties of the erythrocyte membrane.

The study on procainized human erythrocytes revealed a) temperature dependent changes of cellshapes, b) a significant increase of deformability, c) low amounts of cross-linked membrane proteins, d) a mean cell volume identical with the volume of nonprocainized cells, e) severe reduction of induced anisotropy, f) no loss of negatively charged groups of the glycocalyx, g) decrease of electrophoretic velocity, h) a significant rise of adhesiveness to glass, and i) increase of agglutinability with antiserum and lectin. The findings are discussed in their relationships to alterations of the molecular structure of the red cell membrane.

[Measurements of storage-induced morphologic changes in preserved blood by means of light scattering]. / Untersuchungen zur Messung lagerungsbedingter morphologischer Veränderungen im Konservenblut mittels Lichtstreuund.

This study is concerned with changes in diffuse backscattering of white light caused by laminarly streaming or resting banked blood stored for periods up to 7 weeks. The most pronounced changes, i.e. a steep rise of scattering intensity, were found at the beginning of storage. Under streaming conditions such as resulting in a maximum of backscattering and effective disaggregation of rouleaux the temporal changes of scattering intensity obviously were not simply related to morphological properties of the blood. In addition, there was considerable individual variation in the amount of backscattering from various preserves. This invalidates an assessment of banked blood by means of light scattering by a single-data type measurement.