Methods of heavy metal electron microscopic histochemistry applied to frog lung surfactant

Summary Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques — ruthenium red-osmium tetroxide, osmium tetroxideferrocyanide, acidic phosphotungstic acid in chromate (Rambourg technique) — more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies from — when released — membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium.     

Methods of heavy metal electron microscopic histochemistry applied to frog lung surfactant.

Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques-ruthenium red-osmium tetroxide, osmium tetroxide-ferrocyanide, acidic phosphotungstic acid in chromatic (Rambourg technique)--more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies form--when released--membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium.     

Acid phosphatase of osteoclasts demonstrated by electron microscopic histochemistry

Summary Location of acid phosphatase outside and inside the osteoclast was studied by electron microscopic histochemistry. Osteoclasts with a ruffled border apposed to the bone showed enzyme activity in a) membrane-limited cytoplasmic bodies of different dimensions, b) some Golgi vesicles and inner Golgi cisternae, c) vacuoles and vacuole-like profiles, d) extracellular channels and channel expansions in the ruffled border, e) cell-bone interspace. The possibility of bone degradation by lysosomal enzymes both in the cytoplasmic vacuoles and in the cell-bone interspace is discussed.This research was supported by the Danish Medical Research Council. Grant. no. 512-819.I am indebted to Professor Arvid B. Maunsbach for valuable discussions and suggestions and to Mrs. Ruth Nielsen for technical assistance.     

The incubation of unfixed tissues for electron microscopic histochemistry

Summary Small tissue blocks of various organs of the rat were incubated for gradually increasing times in a neutral buffer-sucrose medium modelling the main parameters of the histochemical incubations. Following incubation the blocks were fixed in paraformaldehydeosmium and embedded in Durcupan. The electron microscopic study revealed that even after 40 minutes of incubation prior to fixation, fine structure is preserved satisfactorily from the histochemical point of view. With consideration to further advantages of such a proceeding, the incubation of unfixed tissue blocks for electron-histochemistry is recommended.     

Complex formation between polyadenylic acid and formycin B

Polyadenylic acid forms a 2:1 complex with the C-nucleoside formyein B at both pH 7.0, 0.15 m-Na⁺ and pH 6.0, 0.15 M-Na⁺. The formation of this complex has been followed by equilibrium dialysis, and by optical rotatory dispersion measurements in the range 333 to 450 nm. At pH 7, melting curves for thermal dissociation of the complex (followed by the optical rotation at 345 nm) show a strongly co-operative helix-coil transition. From the variation of T_m with the free formyein B concentration at this temperature, the partial molar enthalpy of formation of the complex, at the mid-point of the transition, has been calculated as -12.8 kcal./mol of formyein B. Viscometry and optical rotatory dispersion measurements indicate that the structure of the complex at pH 6 is the same as at pH 7, and that it may be formed in preference to the double-helical acid form of poly (A). The structure and properties of the complex are discussed.     

Complex formation equilibria between fumaric acid and lanthanides

Complexation in the lanthanidefumaric acid (H₂L) system has been studied by potentiometric titration over the pH fange of 3–4. The measurements were performed in 0.10 M NaClO₄ at 25 °C. The data indicate the formation of the species LnL⁺ and LnHL²⁺ but not of Ln₂L. The results are compared to comparable data for complexing by various carboxylic and dicarboxylic acids.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Complex formation between polyuridylic acid and adenosine using spin-labeling

2 poly(u) . 1 adenosine complex formation was studied by spin-technique in the temperature range from 7 to 25 degrees C. It is shown that adenosine binding induces the conformational transition of poly(u) structure from flexible coil into rigid helix. The contribution of hydrogen bonding and stacking of adenosine to the total change of enthalpy and entropy upon complex formation was estimated.     

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Non-freezing light- and electron microscopic enzyme histochemistry by means of polyetylene glycol embedding

Summary A technique is described, by means of which the undesirable effects by freezing and thawing necessary in cryostat histochemistry might be avoided using embedding in polyetylene glycol, which does not affect enzyme activity nor ultrastructural appearance of cellular detail. The technique permits sectioning with an ordinary sliding microtome.     

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.     

Complex formation between spin-labeled polyuridylic acid and pyrimidine nucleosides.

Complex formation between poly (U) and pyrimidine nucleosides, uridine and cytidine, was observed using spin labeling technique. The binding of these nucleosides with poly (U) takes place within a narrow range of their concentration and is characterized by a relatively strong cooperativity. It is shown, that both hydrogen bonding and stacking interaction contribute to the complex stability. Some thermodynamic parameters of the process were obtained from the binding isotherms. At 21 degrees C the equilibrium constants for nucleation were found to be 0.23 M-1 and 0.42 M-1, and those for chain growth 2.63 M-1 and 2.19 M-1 for uridine and cytidine respectively. Complex formation of poly (U) with adenosine was also studied by spin labeling method.