Quantitative Struktur-Funktions-Korrelation an BiomembranenQuantitative Correlation of Structure and Function on Biomembranes

Microscopical studies on cells or tissues vitally stained with Neutral red (NR) were hitherto almost invariably confined to a few objects that could be investigated without tissue sectioning, for instance tissue culture. For NR, a cationic dye (molecular weight 289, PK 6,75) is easily soluble in water and organic solvents and diffuses during histological preparation for paraffin sectioning and even from cryostat sections. Thus comprehensive studies of vital staining of laboratory animals such as mouse and rat don't exist yet.This explains why it still remains doubtful, wether NR stains ‘preexisting’ or ‘newly formed’ granules, ‘paraplasmic’ cytoplasmic inclusion bodies or - complete or only partially - lysosomes and why it does so. We tried to solve these problems. Moreover we found that after intravital injection of the dye NR ‘stained’ the cells of the APUD-series (PEARSE) rather selectively as do catecholamines or catecholamine precursors. So it was to follow up wether NR could serve as a model for distribution studies of biogenic amines, too.The histological method that allows the localization of intravitally injected NR in tissue sections is freeze-drying. We applied freeze-drying to cryostat sections. The main advantages of this modiftcation are both the short time needed for the drying procedure and the large number of different tissues that can be cut and frozen-dried in the same time. In this way nearly all organs and tissues of the rat could be investigated.1 min after the intravenous injection of the dye NR has disappeared from the blood - at least in concentrations that are demonstrable in tissue sections. By using fluorescence microscopy the dye instead can be localized in all tissues being investigated mainly intracellularly. Apart from the diffuse staining of the cytoplasm in some tissues stained cell nuclei are observed. Intensely coloured nuclei together with a diffuse to scattered staining of the cytoplasm are signs of cell death.In some endocrine cells - mostly belonging to the APUD-series - a strong, often granular, reddening of the cytoplasm is seen; the nuclei are not stained. While the dosis and the form of application necessary to stain these endocrine cells, the intracellular localization and even the reactive groups (presumably carboxylic groups of the granule matrix) to which NR and catecholamines and their precursors are bound seem to be rather identic, some essential differences do exist: NR is easily and rapidly taken up and stored for a relatively short period, whereas biogenic amines accumulate in APUD - cells by active transport in a time - consuming process; their precursors are only stored following decarboxylation. - Secretory granules of some exocrine gland cells, too, may be vitally stained by NR.NR-staining of lysosomes is a well-known fact. We can add some details: a) There are-very characteristic for each of the tissues investigateddifferences in the time needed to stain lysosomes as well as in the duration of lysosomal staining: For instance lysosomes in the kidney proximal convolution are stained very rapidly directly after the injection of the dye and are destained in about 24 h, lysosomes in the thyroid epithelium are rapidly stained and destained, lysosomes in other organs need 45 to 60 min to get stained. c) There is no correlation between the vital staining of lysosomes and the characteristics lysosomes exhibit when stained by histologicalhistochemical methods in tissue sections. This may be due to the extraction from the tissue section of that particular component that, intravitally, binds the dye to lysosomes. - Differences in the composition of the lysosomal matrix in various organs are discussed as one major point of the heterogeneity of lysosomal vital staining with cationic dyes. d) NR vital staining of lysosomes of younger animals is less, that of older animals much more pronounced; lipopigment may, but must not neccessarily do so, exhibit a strong binding capacity for the cationic dye. e) In dehydration experiments the binding capacity oflysosomes is stronger, after premedication with reserpine less pronounced than normally. Desmethylimipramin has no effect at all.The so-called NR-crinom appears only in a few organs, resulting from autophagic as well as from heterophagic cell activity.Following albumin injection enlarged lysesomes are stained vitally in the renal proximal convolution. ‘Vacuoles’ induced by premedication with Macrodex® and glycerol remain unstained.NR is concentrated in the urine of the distal nephron and in the gastric lumen. Reabsorbtion occurs in the distal intestine.Essentially two factors are believed to be responsible for the pattern of NR vital staining: a) Its solubility that explains the distribution of the dye all over the organism, its diffusion through cell membranes and its elimination from the organism by ‘Non-ionic diffusion’. b) Its qualities as a light cationic dye that cause its - electrostatical - binding (‘storage’) to anionic sites of the tissue, for instance to carboxylic groups of the secretory granules of the APUDcells and carboxylic and/or phosphate groups of the lysosomal matrix.     

Charges fixes du protoplasme des fibres musculaires de balaneMyoplasmic fixed charges of the barnacle muscle fiber

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and øX, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061–1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced øX, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100g KCl concentration in the external medium equal to 0.15 M, øX was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in øX of 20–50% depending on [KCl] in the external solutions. The addition of ATP, Mg²⁺ and Ca²⁺ also causes a reduction of øX by 30–50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when KCl is replaced by NaCl. It also suggests a reduction of øX during muscular contraction.     

Biosynthèse de l'udpglucose par les microsomes des hépatocytes de ratBiosynthesis of UDPglucose by rat liver microsomes

Evidence is presented to show that all enzymes and all intermediary metabolites of a UDPglucose biosynthesis pathway are present in the microsomal membranes of rat liver. Glucose 6-phosphate, glucose 1-phosphate and UDPglucose are characterized by chromatography.The properties of phosphoglucomutase and UTP: D-Glucose-1-phosphate uridyltransferase are studied. The K_m values of phosphoglucomutase at pH 7.2 and 42°C were 0.26 · 10^?3 mM for glucose 1,6-diphosphate and 80 · 10^?3 mM for glucose 1-phosphate. The K_m values of UTP: D-glucose-1-phosphate uridyltransferase at pH 8.5 and 37°C were 220 · 10^?3 mM for UTP and 166 · 10^?3 mM for glucose 1-phosphate. These values are compared to the given values for enzymes from different species, and to those found for soluble enzymes. The significance of this membranous pathway is discussed.     

Membranes des globules lipidiques du laithumain. Preparation,etude morphologique et composition chimiqueHuman milk fat globule membranes. Preparation,morphological studies and chemical composition

Two procedures for the preparation of the human milk fat globule membranes have been used, and morphological and enzymatic comparisons were made. Membranes obtained by the churning procedure were investigated for their protein, lipid, carbohydrate and amino acid contents. Analysis of the proteins by acrylamide gel electrophoresis in sodium dodecyl sulfate were also performed. Several bands are observed: two of these represent glycoproteins. Nucleic acids were analysed on a sucrose density gradient.     

Presence de glycosyltransferases a accepteurs chromatiniens dans les membranes nucleaires d'hepatocytes de singeThe presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100 000 × g. The yield was 2.2 · 10⁷ nuclei per g of liver, and 70% of the homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively.The nuclei are hydrolysed by DNAase I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100 000 × g, under a buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.     

On the simultaneous presence of synapticulae and tabulae in regular ArchaeocyathidsSur la presence de synapticules et de planchers dans les Archaeocyatides reguliers

The association tabulae-synapticulae is uncommon in the Regulares. As this pair is now known in different families, it is possible to propose the use of it in systematic definition at generic level.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.