Journal of Histochemistry & Cytochemistry

CLARK, MICHAEL A.; ACKERMAN, G. ADOLPH

doi: 10.1177/19.12.727pmid: 4111148

The chemical nature of the pyroantimonate-osmium (PAO) reaction in normal human bone marrow cells has been evaluated by the application of a number of digestion and blocking procedures as well as the electron microprobe. Five foci of reactivity are localized in cells fixed directly in the PAO reagent, viz., nucleoli, heterochromatin, cytoplasmic granules, particulate glycogen and the outer plasmalemmal surface. Heterochromatin and nucleolar staining are attributable to calcium bound to nucleic acids as well as to reactive amino groups on histones. Granule staining is dissociable into acid-dialyzable, ribonuclease (RNase)-stable and RNase-labile components but sulfate groups of acid mucosubstances are probably not involved. Glycogen staining by the PAO reagent has been verified and shown to result from the formation of organometallic complexes between the pyroantimonate ion and the C2-C3-free hydroxy groups of the glucose residues. PAO reactivity has been definitely localized along the outer plasmalemmal surface, but this component of PAO staining is resistant to all control measures employed. These studies have illustrated the complexity and polyvalency of the PAO reaction and have shown that PAO staining is not exclusively associated with metallic cation localization.

MYLROIE, ROBERT; KOENIG, HAROLD

doi: 10.1177/19.12.738pmid: 4111149

Purified neurosecretory granules (NG) from bovine neurohypophysis stain metachromatically with acridine orange. NG were sonicated in 0.2% Triton X-100. The soluble protein, 83% of the total protein, was separated by ultracentrifugal flotation in KBr into two lipoprotein (LP) fractions, a high density lipoprotein fraction (HDLP, D < 1.35 g/ml), 37%, and a very high density LP fraction (VHDLP, D > 1.35 g/ml), 63% of the total. The HDLP contained 0.46 mg phospholipid and 0.13 mg cholesterol/mg protein. The VHDLP contained 0.08 mg phospholipid and 0.025 mg cholesterol/mg protein. The amino acid composition of the delipidated LPs resembled that of bovine neurophysins. Disc gel electrophoresis resolved two major and four minor components of identical mobilities in HDLP and VHDLP. The major anodic components, 90% of the protein, and one minor cathodic component were sudanophilic, reduced iodine and stained metachromatically with acridine orange. The NG lysates prepared without detergent or sonication contained 52% of the total protein and phospholipid. The insoluble residue consisted of membrane profiles and had a different lipid and amino acid composition. Gel electrophoretograms of water extracts of NG revealed the same protein components present in Triton X-100 extracts. The major anodic bands and the cathodic band were colored by Sudan black B and iodine and stained metachromatically with acridine orange. Prior extraction of gels with chloroform-methanol (2/1, v/v) abolished these staining reactions. Neurophysins prepared by extraction of acetone-dried tissue with 0.1 N HCl contained about 7% of the phospholipid present in Triton X-100 extracts. Gel electrophoretograms of neurophysins disclosed the same protein constituents seen in Triton X-100 and water extracts of NG. The major components did not stain with Sudan black B or iodine but stained metachromatically with acridine orange due to the presence of residual phospholipid. Neurophysins are essentially apoproteins of these soluble acidic LPs.

LIDBRINK, PETER; JONSSON, GÖSTA

doi: 10.1177/19.12.747pmid: 4111150

The fluorescence-concentration relationship of noradrenaline (NA) in NA nerve terminals of rat cerebral cortex and hypothalamus demonstrated with the Falck-Hillarp formaldehyde fluoresence method has been investigated. Comparisons were made between experimentally induced changes in NA concentrations as recorded by chemical-analytical assay and fluorescence intensities of NA nerve terminals as estimated subjectively in the fluorescence microscope. The results obtained show that in cerebral cortex there was a close correlation between NA concentration and fluorescence intensity up to the normal endogenous NA level, indicating a linear relationship between the two parameters. In hypothalamus, however, a correlation between fluorescence intensity and NA concentration was observed only when the nerves contained less than 50% of the endogenous NA level. Above this value, there was no further proportional increase in fluorescence intensity, probably because of a concentration-dependent quenching of the fluorescence. Essentially the same results were obtained when investigating previously emptied NA nerve terminals refilled with various amounts of 3H-NA. The data presented clearly show that a good NA quantitation of cortical nerve terminals can be made by careful subjective estimation of the fluorescence intensity.

LEEMANN, U.; WEISS, S.; SCHMUTZ, E.

doi: 10.1177/19.12.758pmid: 5143192

A centrifugation technique for microscopical preparations of cells or cell particles either unfixed or previously fixed in suspension has been developed. It allows more consistent results of cytofluorometric and interferometric measurements than the conventional smear technique.

DIAZ, MANUEL; HIERRO, JOSE; DE DIAZ, GRACIELA DEMICHELI

doi: 10.1177/19.12.761pmid: 4111151

A new method is proposed to study the secondary structure of deoxyribonucleic acid (DNA) in situ in fixed chromatin. It is based on acriflavine staining and on differentiation with a nitrous acid solution of the fixed cytologic preparation. The presence of green fluorescence after this treatment is regarded as indicative of double stranded DNA. Experiments are described with DNA-acriflavine mixtures in solution, DNA-agar models and cytologic preparations submitted to different pretreatments. The feasibility and limitations of the method are discussed in the light of the results reported upon.

YOKOYAMA, MASAO; CHANG, JEFFREY P.

doi: 10.1177/19.12.766pmid: 4111152

Ultrastructural and ultracytochemical features of the efferent duct of Chinese hamster have been studied. The ducts are composed of two main types of epithelial cells, ciliated and nonciliated. Distinct structural and cytochemical characteristics of these cells are apparent. Presence of fibrogranular complex which is supposedly related to basal body replication was demonstrated in ciliated cells for the first time in this tissue. Thiamine pyrophosphatase activity in Golgi apparatus, acid phosphatase activity in Golgi apparatus and lysosomes and alkaline phosphatase activity on basal plasma membranes of both ciliated and nonciliated cells have been localized. However, thiamine pyrophosphatase activity was seen only on the luminal surface, apical vacuole and apical tubular structure of nonciliated cells but not on the surface of ciliated cells. Similarly, horseradish peroxidase was absorbed only by nonciliated cells. The cytochemical and ultrastructural differences between the two types of cells indicate a functional specialization. The results indicate that the ciliated cells are concerned with the transportation of the sperms and that the nonciliated cells are concerned with the regulation of fluid composition in the duct since the latter are capable of both secretion and absorption.

TIXIER-VIDAL, ANDRÉE; PICART, RENÉE

doi: 10.1177/19.12.775pmid: 4111153

Structures demonstrating the presence of glycoproteins, acid phosphatase activity and OsO4 impregnation were localized by means of the electron microscope in duck and in quail pituitary cells. Two methods for the electron microscopic demonstration of glycoproteins were used: a chromic acid-phosphotungstic acid mixture on glycol-methacrylate-embedded tissues, and the periodic acid-thiocarbohydrazide-silver proteinate technique. Both methods showed glycoproteins in the following sites: (a) the secretory granules in three types of cells (A, B, C) which are part of the seven different cells of the avian pituitary; (b) the several kinds of dense bodies which are richer in reaction product than the secretory granules. A correlation with previous studies on similar species of birds is helpful in identifying each of the three positive types of cells as thyrotropic cell (A), prolactin cell (B) and gonadotropic cell (C). The presence of glycoproteins within the Golgi saccules (on condensing granules) was found with the periodic acid-thiocarbohydrazide-silver proteinate method in these gonadotropic cells only. In gonadotropic and thyrotropic cells, acid phosphatase activity is weak in the inner Golgi saccules and strong in the "Golgi Endoplasmic Reticulum Lysosomes" system, in the lysosomes, in the dense bodies and in the vacuolated dense bodies. The structures which are richest in glycoproteins are also those which have the most acid phosphatase activity. On the contrary, OsO4-stained structures in duck gonadotropic cells (nuclear pericisterna, rough endoplasmic reticulum, cisternae and outer Golgi saccules) have no glycoproteins or acid phosphatase activity.

CHOKROVERTY, S.; PARAMESWAR, K. S.; CO, C.

doi: 10.1177/19.12.798pmid: 4111154

By the use of acetyl- and butyrylthiocholine and α-naphthyl acetate as substrates and selective inhibitors, we have shown the presence of both specific and nonspecific esterases in the myoneural junction of vastus medialis and peroneus brevis muscles of normal subjects as well as in paretic muscles of hemiplegic patients. The presence of nonspecific esterases in human myoneural junction has not been recorded previously. The nonspecific esterases in our fixed tissue were noted to be predominantly E600-sensitive at 10–5M concentration although resistant at 10–6M and 5 x 10–6M concentration.

MATSUZAWA, TATSUJI; ANDERSON, H. CLARKE

doi: 10.1177/19.12.801pmid: 4335252

The presence and distribution of alkaline phosphatase, adenosine triphosphatase (ATPase) and acid phosphatase activities in the epiphyseal plates of young mice were studied by electron microscopic cytochemical methods. Alkaline phosphatase and ATPase activities were associated with the plasma membranes of chondrocytes and with the investing membranes of matrix vesicles. These vesicles contain the earliest recognized deposits of hydroxyapatite and may promote calcification through an active process. Alkaline phosphatase and ATPase activities were greatest in the hypertrophic zone of the epiphysis, which is an area of beginning calcification. Acid phosphatase activity was demonstrable in the cytoplasm of chondrocytes in association with dense bodies which were larger than matrix vesicles and were devoid of alkaline phosphatase and ATPase activities. These cytoplasmic lysosome-like bodies were slightly more numerous in the hypertrophic zone but disappeared in the underlying zone of chondrocyte degeneration and matrix calcification. Our observations do not support a previous suggestion by others that matrix vesicles represent lysosomes. The presence of ATPase and alkaline phosphatase is compatible with an enzymatic calcium- and/or phosphate-concentrating mechanism in the matrix vesicles.

SELIGMAN, ARNOLD M.

doi: 10.1177/19.12.809pmid: 5004055