PHYSICAL AND CHEMICAL PROPERTIES OF AMYLOID FIBERS II. ISOLATION OF A UNIQUE PROTEIN CONSTITUTING THE MAJOR COMPONENT FROM HUMAN SPLENIC AMYLOID FIBRIL CONCENTRATESGLENNER, G. G.; CUATRECASAS, P.; ISERSKY, C.; BLADEN, H. A.; EANES, E. D.
doi: 10.1177/17.12.769pmid: 4983715
A major protein component (B protein) obtained from concentrated fibrils of each of two human amyloidotic spleens has been isolated by a series of fractionation methods using extraction in buffered 8 M urea, complete denaturation in buffered 6 M guanidine and column chromatography on Sepharose 4 B (Pharmacia). The major protein obtained by column chromatography was found to be also the major protein component on sodium dodecyl sulfate polyacrylamide disc electrophoresis. This protein was shown to have a molecular weight of 21,000-22,500. Immunologic identity was demonstrated for the B protein derived from the amyloid fibril concentrates of each spleen. No evidence of antibody cross-reaction to native or denatured components of normal human spleen or serum was discernible. It would appear, therefore, that the B protein derived from the two splenic amyloid fibril preparations, in addition to being the major protein component of the fibril, is not found in significant quantity in normal tissue or serum.
ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCE AND ANTIMONATE-PRECIPITABLE CATION IN HUMAN AND RABBIT PLATELETS AND MEGAKARYOCYTESSPICER, S. S.; GREENE, W. B.; HARDIN, J. H.
doi: 10.1177/17.12.781pmid: 4189436
For selective ultrastructural localization of acid mucosubstance in rabbit and human platelets and megakaryocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde or osmium tetroxide, sectioned at 40 µ and stained with the Rinehart-Abul-Haj solution of dialyzed iron. In specimens from both rabbit and man, dialyzed iron staining was observed within nucleoids of the cytoplasmic granules (α-granulomeres) of platelets and megakaryocytes, on the outer surface of the plasma membranes of platelets and megakaryocytes and on the luminal surface of demarcation membranes of megakaryocytes. These results were obtained following any of the three fixation procedures, except when nucleoids failed to stain after glutaraldehyde fixation. For ultrastructural localization of pyroantimonate-precipitable cation, bone marrow and buffy coat specimens were fixed in Komnick's solution of potassium pyroantimonate and osmium tetroxide. In specimens from both species, antimonate deposits were localized within the dense bodies (5-hydroxytryptamine organelles) of platelets and within nucleoids of cytoplasmic granules of platelets and megakaryocytes. The dense bodies were well preserved in platelets fixed in a pyrophosphate-osmium tetroxide solution but were poorly, if at all, preserved by osmium tetroxide solutions containing other buffers.
DISTRIBUTION OF ACID AND ALKALINE PHOSPHATASE ACTIVITY IN UNDEMINERALIZED SECTIONS OF THE RAT TIBIAL DIAPHYSISWERGEDAL, JON E.; BAYLINK, DAVID J.
doi: 10.1177/17.12.799pmid: 4189437
The distribution of acid and alkaline phosphatase activity in the rat tibia has been studied in transverse sections sawed from fresh undemineralized diaphyses. The endosteal resorbing surfaces had the highest acid phosphatase activity as demonstrated with 50 mM α-naphthol phosphate as the substrate. Strong activity was present not only in osteoclasts and adjacent osteocytes but extracellularly lining Howship's lacunae. In areas of bone formation, moderate acid phosphatase activity was present in osteoblasts and young osteocytes. In addition, a zone of activity was found at the mineralizing front where mineralization is initiated. This active zone (a) was separated from the active periosteal cell layer by a zone of inactive osteoid and (b) in formol-calcium-fixed demineralized sections extended into young bone. The identity of the mineralizing front was confirmed by tetracycline labeling. The activity at the mineralizing front had the properties of an enzyme. Alkaline phosphatase activity demonstrated with naphthol ASTR phosphate was largely associated with the osteoblasts and other mesenchymal cells at forming surfaces.
THE FATE OF WURSTER'S RED FREE RADICAL IN TISSUES WITH HIGH AND LOW OXYGEN UTILIZATIONS (HEART MUSCLE AND GINGIVA)PERSON, P.; FELTON, J. H.; O'CONNELL, D. J.
doi: 10.1177/17.12.807pmid: 5367016
When N,N-dimethyl p-phenylenediamine is incubated with fresh frozen sections of rat heart muscle, there is a rapid, enzymatic, single electron oxidation of the diamine to form the pink Wurster's free radical with absorption maxima at 550 and 510 nm. This is followed by radical dimerization and other interactions to form a purple color accompanied by a new peak at 660-670 nm and finally a substantive blue dye. In contrast, with frozen sections of gingiva, instead of the pink color of the Wurster's free radical, one first sees a green color which slowly changes to blue. If heart muscle is kept at room temperature for 30-60 min or in a deep freeze for several days prior to use, it also exhibits formation of the green color. Spectrophotometric studies of the above tissue systems reveal that the new green color does not represent formation of a new compound with new absorption maxima. The same absorption maxima are present in each of the above systems; however, there are differences among the systems with respect to the rates of formation of the absorption maxima in different regions of the spectrum. The latter phenomena produce the color changes seen by the eye. It is suggested that the kinetics of Wurster's Red free radical interactions in tissue may be dependent upon the metabolic (redox) status of the tissue.
ORANGE FLUORESCENCE IN THE ACOUSTIC NERVEROSS, MURIEL D.
doi: 10.1177/17.12.814pmid: 5367017
Orange fluorescent granules are numerous in the acoustic nerve in the rat and the mouse. The granules are often accumulated in masses distributed along, or within, the acoustic nerve neurons within the cochlear modiolus. They are not found more peripheralward. The orange fluorescing granules are not autofluorescent; they are absent in sections mounted directly from the cryostat, and the fluorescence disappears in sections treated with water or dilute isopropanol. The fluorescence is sensitive to ultraviolet light and largely disappears after treatment with α-methyl-m-tyrosine or reserpine (24 hr). Heat alone induces some orange fluorescence, restricted to the distal ends of the axons of the neurons. Spectrofluorometric analyses of heat-induced and vapor-induced orange fluorescence have thus far failed to demonstrate significant spectral differences. It is concluded that orange fluorescence may indicate the presence of an amine in a distinctive storage form.
A NEW FLUORESCENT METHOD WITH PHENANTHRENEQUINONE FOR THE HISTOCHEMICAL DEMONSTRATION OF ARGININE RESIDUES IN TISSUESMAGUN, BRUCE E.; KELLY, JOHN W.
doi: 10.1177/17.12.821pmid: 5367018
Phenanthrenequinone, in an alkaline ethanolic solution, is shown to be specific for the guanidino group of arginine in tissues. The fluorescence measured in chick erythrocyte nuclei is highly reproducible among areas on one slide and among different slides. Benzil and cyclohexanedione, two agents known to be specific for the guanidino group, prevent 85% of the fluorescence developed in the phenanthrenequinone reaction. Glyoxal blocks phenanthrenequinone reactivity only partially, while alkaline formaldehyde has little effect. The effects of fixation and postfixation of chick erythrocyte nuclei indicate that fluorescence is most intense after formalin fixation, and that conformational factors may prevent ethanolacetic acid-fixed nuclei from reacting completely. By means of protein precipitation and prolonged incubation in ethanol, it was shown that the fluorescent chromophore is strongly bound to phenanthrenequinone-reacted gelatin and to tissue sections. Excitation and emission spectra are presented for phenanthrenequinone-reacted arginine, gelatin and a tissue section.
THE INTERMEDIATE MUSCLE FIBER OF RATS AND GUINEA PIGSEDGERTON, V. REGGIE; SIMPSON, D. R.
doi: 10.1177/17.12.828pmid: 4312624
The soleus, plantaris and gastrocnemius muscles of 60 Sprague-Dawley rats and 70 Hartley guinea pigs were studied histochemically. In the plantaris and gastrocnemius muscles, myosin adenosine triphosphatase activity differentiated intermediate fibers from red and white fibers as determined by malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. Contrary to what is commonly reported, red fibers could not be distinguished from white fibers on the basis of myosin adenosine triphosphatase activity as is commonly reported. The intermediate fiber was characterized by minimal menadione-mediated α-glycerophosphate dehydrogenase, phosphorylase and myosin adenosine triphosphatase activity and moderate malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It is suggested that fibers with intermediate oxidative enzyme activity are physiologically slow, white fibers are fast and red fibers are moderately slow or even fast contracting fibers.
A TECHNIQUE FOR THE RADIOAUTOGRAPHY OF GERM CELLS IN WHOLE MOUNTS OF SEMINIFEROUS TUBULESHUCKINS, CLAIRE; KOPRIWA, BEATRIX MARKUS
doi: 10.1177/17.12.848pmid: 4189438
A technique has been described for preparing radioautographs of germ cells in intact segments of seminiferous tubules from rat testis. Pieces of tubules were dissected from testes, fixed in Bouin's fluid and stained in Harris hematoxylin. For radioautography, rectangular coverslips were first dipped in liquid Kodak NTB2 emulsion. The tubules were placed on the partially set emulsion, and thus became evenly coated on their viewing surface with a thin layer of emulsion. Following routine exposure and processing, the coverslips bearing these tubules were inverted onto microscope slides to give the final whole mount radioautographic preparation.