Preliminary attempts at ultrastructural sulfhydryl demonstration

Summary Trials to visualize tissue-bound sulfhydryl (SH) groups were made by means of incubating thin slices of mammalian liver and pancreas specimens at pH 7.4 with the specific SH-reagents p-chloromercuribenzoate or mercury orange, the former followed by brief fixation in glutaraldehyde and/or inert dehydration, the latter used after dehydration of unfixed tissue. Uncontrasted thin sections showed no obvious increase in electron density over that of non-incubated controls. After contrasting with uranyl acetate, however, some lysosomes showed small, comma-like precipitates with both reagents.Trials to obtain a better visualization of precipitated mercaptides by subsequent application on the incubated sections of the sulfide-silver procedure for heavy metals gave, so far, only equivocal results.Read in part (Pihl, Gustafson and Falkmer, 1967) at the Annual Meeting of the Scandinavian Society for Electron Microscopy in Åbo, Finland, June 1–2, 1967.This work was supported by grants from the Swedish Medical Research Council (Project No. K68-12X-718-03).     

Some aspects of the Feulgen reaction in situ

Summary Cytological observations combined with studies on absorption spectra of Feulgen stained normal and lipid — extractet HeLa and ehrlich-Lettré mouse ascites cells were performed after fixation of the cells as well in neutral formaldehyde as in Serra fixative. The effects of formaldehyde treatment of the stained cells to substitute all the free amino groups of DNA bond pararosaniline molecules, were also studied. The results obtained by using DNA samples containing 2% protein and relatively free from protein, led to the conclusion that after acid hydrolysis for a short period purines in DNA become splitted and these released aldehydes react with one or two amino groups of pararosaniline, a triphenylmethane dye (according to the arrangement of purines and pyrimidines in the helices). Some protein molecules also take part in the reaction and substitute some of the free amino groups of DNA bound pararosaniline. Peulgen stained cells fixed in Serra fixative show an absorption maximum at 546–550 m. Under appropriate conditions, as in cells fixed in formaldehyde, other substances e.g. phospholipids and lipoproteins interfere with the reaction by substituting most of the free amino groups of DNA bound pararosaniline molecules. It has been argued that in histochemical reactions monosubstituted pararosaniline molecules should be coloured and further substitution of free amino groups of pararosaniline, bound in DNA helices, does not change the intensity of the colour, but gives a shift in the wavelength of the absorption spectra.It has been suggested that the differential response of the nucleoli to the Feulgen-reaction, depending on whether the cells were fixed in formaldehyde or in Serra fixative, may be due to the formation of a protecting shield around the finely distributed intranucleolar chromatin strands, when formaldehyde is being used. After this fixation lipoproteins and other lipids, present in a relatively high percentage and closely associated with the intranucleolar chromatin strands, are especially well preserved.Evidences have been put foreward in support of the amino alkylsulfonic acid theory of Rumpf (1935) and Hörmann et al. (1958) whereas the amino sulfinic acid theory to explain the Schiffs reaction (Wieland and Scheuing, 1921) was shown not to be in agreement with our results.On leave from the Department of Botany, Calcutta University, 35, Ballygunge Circular Road, Calcutta-19, India; on a fellowship from the German Academic Exchange Service.     

Some effects of electrical stimulation and exogenous metabolites on the contractile activity and the ultrastructure of the radula-muscle of Buccinum undatum

Summary The effect of various metabolites on the contractile pattern of the radulamuscle of Buccinum undatum was recorded. Its ultrastructure was studied after varying periods of spontaneous and electrically induced work.The effect of added substrates on the contractile behaviour of the radula-muscle was most pronounced after 12 hours of work or more. Addition of succinate caused a relaxation of the muscle, and its contractions were inhibited. Addition of glutamate had the opposite effect and caused a marked contraction of the muscle. The contractile pattern was not influenced by -ketoglutarate. These results indicate that factors other than energy supply influenced the response of the radula-muscle.The ultrastructure of the control muscles and of the electrically stimulated muscles showed some changes referable to the isolation of the muscle. Contrary to the controls, those muscles that had been stimulated for 4 and 12 hours respectively showed a more regular organisation. The filaments were more parallel and electron dense dots occurred arranged in transverse or oblique bands. The distance between the bands was around 1 . This organisation of the radula-muscle was discussed and compared with that of banded muscles of various kinds. Attention was drawn to the similarities in appearance between the bands of the radula-muscle and of Z bands of developing striated vertebrate muscles. The mitochondria showed changes partly referred to the isolation of the muscle and partly to the experimental conditions.Our sincere thanks are due to Professor I. Agrell, Head of the Zoophysiological Institute, Lund, for his advice and support during the work.Thanks are also due to Dr. B. Swedmark and his staff of the Zoological Station, Kristineberg for kind help with animal material and to Professor C. G. Ahlström, Pathological Institute, Lund, for electron microscopic facilities. We thank Mrs. M. Carleson and Mrs. I. Hedström for most valuable technical assistance during the electron microscopic work. The work was supported by grants from Swedish Natural Science Research Council and the Royal Physiographic Society of Lund.     

The comparative ultrastructure and possible function of eyespots: Euglena granulata and Chlamydomonas eugametos

Summary The structure of Euglena granulata and Chlamydomonas eugametos has been studied using polarization and electron microscopy, cinematography, and chemical extraction procedures, with the main focus on the structure of the eyespot.The 50–60 granules which form the extrachloroplastic eyespot of E. granulata are large bodies, up to 1200 m in diameter. They are found in the cytoplasm near the base of the reservoir and are associated with the parabasal body which contains a large crystal. The eyespot granules are contained within membranes having a unit membrane structure; 2 or 3 are usually present in a single eyespot packet; microtubules are also contained within the packet. The eyespot granules have the structure of a positive spherite and clearly exhibit birefringence; this structure is modified by fixation.The granules of the chloroplastic eyespot of C. eugametos are about 75 m in diameter and are contained within the chloroplast in an ordered array. Occasionally, the eyespot contains elongate or helical bodies mixed with the granules. Extraction with organic solvents caused the removal of materials which formed the eyespot granules as well as that of the osmophilic globules in the chloroplasts.Several hypotheses which concern the function of the eyespots in these and other species are discussed in the light of our results. the possible origin and demise of the eyespot granules are also discussed.Supported in part by NSF Grant GB-313. We thank Dr. Harold C. Bold and Dr. W. Gordon Whaley for their support and encouragement, Dr. R.M. Brown, jr. and Dr. Tom Kantz for aid in cinephotography, Dr. Peter Sitte for his help with polarization microscopy, and Mrs. Virginia Stork for her excellent technical assistance.A preliminary report of this research was presented at the Annual Meeting, Phycological Society of America, American Institute of Biological Sciences, College Park, Maryland, August, 1966.Contribution No. 286 from the Department of Botany, The University of Tennessee, Knoxville.     

Vergleichende methodische Untersuchungen über die Aktivität der sauren Phosphatase und 5′-Nucleotidase im Rückenmark normaler Kaninchen und nach Durchschneidung des Plexus brachialis

Zusammenfassung Bei normalen erwachsenen Kaninchen und nach Durchschneidung des Plexus brachialis wurden im Rückenmark die Reaktionen für saure Phosphatase nach Gomori, Burstone und Barka-Anderson (nur im Normalzustand) und für 5-Nucleotidase nach Wachstein und Meisel bei pH 4,0 untersucht. Sowohl beim Normaltier als auch im Experiment zeigen alle vier Reaktionen gewisse Ähnlichkeiten in ihrer Lokalisation und ihrem Charakter. Es werden jedoch Unterschiede zwischen den drei sauren Phosphatase-reaktionen nach Gomori, Burstone und Barka-Anderson einerseits, sowie der bei saurem pH nachgewiesenen 5-Nucleotidase andererseits festgestellt. Die Resultate unterstützen die These von der Substratspezifität der untersuchten Enzyme und der Heterogenität der sauren Phosphatase.

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Studies on the activity of acid phosphatase and 5-nucleotidase in the spinal cord of normal and plexus brachialis-sectioned rabbits by different histochemical methods

Summary The spinal cord (normal and after brachial plexus section) of adult rabbits was examined using histochemical reactions for acid phosphatase according to Gomori, Burstone, Barka and Anderson (only for normal rabbits), and 5-Nucleotidase according to Wachstein and Meisel at pH 4.0. The reactions show similarity in their specificity and localization in the normal and experimental animals, but they are some differences between Gomori, Burstone and Barka-Anderson reactions for acid phosphatase on one hand, and 5-Nucleotidase at acid pH on the other. The results give some evidence to support the thesis concerning the substrate specificity of the examined enzymes and the heterogeneity of acid phosphatase.

     

“Self-tanning”—a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants

A Feulgen-densitometric comparison of nuclear DNA contents (C-values) was performed in various plant species (a fern, four gymnosperms, 16 woody and herbaceous angiosperms) after two types of fixation, additive (neutral formaldehyde) and non-additive (methanol-acetic acid, 3:1, MAA). Nuclei from tissues containing a significant amount of polyphenols (of the hydrolysable and non-hydrolysable tannin type) always showed reduced stainability and distorted spectral absorbance curves after MAA-fixation, while after formaldehyde-fixation no evidence for distorted staining was found. No fixation-dependent differences in Feulgen-DNA contents were stated in nuclei from tissues having no polyphenols. Distorted Feulgen-staining is a consequence of cellular self-tanning during fixation. Tanning is impaired by formaldehyde which binds to tannins and inactivates them. The rationale for using formaldehyde as a fixative in Feulgen-cytophotometry can be mainly seen in its capability of eliminating the self-tanning error. Standardization in plant DNA cytophotometry, and recent reports on unorthodox nuclear DNA variation in conifers are critically discussed.The author dedicates this paper, with emotions of respect and gratitude, to emer. O. Prof. DrElisabeth Tschermak-Woess on the occasion of the 70th anniversary of her birthday. She guided his Ph.D. Thesis in the years 1968 to 1972.     

Fixation and tissue preservation for antibody studies

Synopsis The methods of fixation and preparation of lymphoid tissues for the immuno-enzyme technique are reviewed. For this technique an enzyme is used first as an antigen and then as a marker to demonstrate its specific antibody. A variety of commonly employed fixatives satisfactorily conserve tissues for the light microscopic detection of antibody but, for electron microscopy, glutaraldehyde or formaldehyde or both are the fixatives of choice. The main technical problem for electron microscopy is to reduce the size of the tissue fragments sufficiently so that the enzymes and their substrates permeate through the fixed tissues. The merits and short-comings of the different preparative techniques are examined and it is shown that the most reproducible results are obtained with 40 m frozen sections. Some of the problems of non-specific staining arising from fixation procedures, as well as endogenous enzyme activity, are discussed. The evidence for and against antibody inactivation by fixation and enzyme inactivation by interaction with its specific antibody is reviewed.     

Electron microscopic observations of the influence of different fixatives on the appearance of cellular ultrastructure

Summary The cells of the proximal convoluted tubules of the rat kidney, mouse hepatic parenchymal cells, and the juxtaglomerular cells (J. G. cells) of the afferent arteriole in rabbit kidney served as test tissues for a study of the modifications of structure produced by 14 different fixation procedures using osmium tetroxide, a chrome-osmium solution, glutaraldehyde, and formaldehyde as fixatives. The fixative solutions containing various buffers were used either alone (osmium tetroxide-containing compounds) or in various combinations (aldehyde fixation with postfixation in osmium tetroxide). The most conspicuous differences in appearance following different fixation procedures were noted in the cytosomes of renal proximal tubular cells and the granules in the J. G. cells. However, virtually all cytoplasmic organelles showed some modification of structure with different fixatives. The evidence indicated that the chemical composition of the buffer used with OsO₄ was an important factor which particularly affected the density of the cytosomal matrix in renal proximal tubular and J. G. cells. The density of the matrix of the cytosomes was, however, also influenced by the fixative itself as indicated by studies of aldehyde-fixed tissues. With these fixatives the ultrastructural appearance was not modified by the buffer. Clumping of nuclear chromatin along the nuclear membrane and around the nucleolus occurred following fixation in aldehyde and Dalton's chrome-osmium solution, whereas the chromatin was evenly distributed in the nucleus when buffered osmium tetroxide was utilized for fixation. A specific reaction of dichromate with the granule of the J. G. cells seemed to take place rendering these granules electron opaque, thereby facilitating their identification in the light and electron microscope. The findings were discussed with particular attention to the interaction of fixatives and buffers with various tissue components.Supported by grants from the Board of Swedish Life Insurance Companies, the Stiftelsen Therese och Johan Anderssons Minne, and grant No. AM-7919 from the National Institutes of Health, Bethesda, Maryland (USA).     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

d-ephedrinephosphate,DEP: A new substrate with specificity for prostatic acid phosphatase (PAP)

Summary This report describes the synthesis and physical (including spectral) properties of a new substrate, d-ephedrinephosphate, DEP¹, which is histochemically highly specific for a secreted non-lysosomal prostatic acid phosphatase, PAP. This specificity is in contrast to other substrates which are nonspecific, i.e., which demonstrate acid phosphatases that originate from various cell types and are mainly lysosomal. When this substrate is used for light and electron microscopic histochemistry in a modified Gomori medium, PAP is demonstrated mainly in secretory granules and in the Golgi apparatus (and its related vacuoles) of prostatic epithelial cells of several species of mammals including man. This corroborates our previous suggestion that PAP is not a lysosomal enzyme as are many of the other acid phosphatases. This high degree of specificity of DEP for PAP supports the usefulness of this compound in the histochemical and biochemical characterization of PAP, and in the diagnosis of localized or disseminated prostatic disease.This investigation was supported by research grants CA-02478 from the National Cancer Institute, NIH, U.S. Public Health Service, Bethesda, MD and CA-16077 the National Prostatic Cancer Project, NCI, Buffalo, NYDedicated to the memory of the late Arnold M. Seligman, M.D., American Cancer Society Professor of Research Oncology and Cell Biology, who pioneered the development of modern enzyme cytochemistry and under whose direction part of this work was initiated     

Transforming growth factor-β-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy

We have previously shown that treatment of mice bearing a large MOPC-315 plasmacytoma with a low dose of the anticancer drug melphalan (l-phenylalanine mustard;l-PAM) results in the acquisition of a potent CD8⁺ T-cell-mediated anti-MOPC-315 cytotoxic T lymphocyte (CTL) activity by the hitherto immunosuppressed tumor bearers, and this immunity contributes to complete tumor eradication. In the studies presented here, we sought to determine how the acquisition of this antitumor immunity following low-dose chemotherapy is possible, in light of the report that MOPC-315 tumor cells produce transforming growth factor- (TGF-), an immunosuppressive cytokine that can down-regulate the generation of CTL responses. We found that the acquisition of CTL activity following low-dosel-PAM therapy is not due to a chemotherapy-induced decrease in the sensitivity of MOPC-315 tumor bearer spleen cells to TGF--mediated inhibition of CTL-generation. Moreover, even spleen cells from MOPC-315 tumor-bearing mice, which had receivedl-PAM therapy 7 days earlier and had acquired CTL activity in vivo, were sensitive to the inhibitory activity of TGF- upon culture for as little as 1 day, with or without stimulator tumor cells. However, the production of TGF- by MOPC-315 tumors decreased drastically as a consequence of the low-dose chemotherapy. Thus, the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor-bearing mice may be due, at least in part, to a reduction in TGF- production that enables the development of tumor-eradicating immunity.Work was supported by research grant IM-435 from the American Cancer Society and research grant CA54413 from the National Cancer Institute.In partial fulfillment of the requirements for the Doctor of Philosophy DegreeSupported in part by the Dorothea Fleming Cancer Research Fellowship Award     

Ultrastructural studies of normal skin and epidermal papillomas of the flathead sole,Hippoglossoides elassodon

Summary Epidermal cells of normal fin web and of the epidermal papilloma of the flathead sole, Hippoglossoides elassodon, were compared by light and electron microscopy. The predominant cells of normal epidermis are squamous and are characterized by complex microvillous interdigitations between adjacent cells, numerous desmosome-like structures, terminal bars, prominent cytoplasmic filaments, and blunt, broad-based microvilli on the outer (free) surfaces of the superficial cells. The less numerous mucous cells of normal epidermis possess abundant ergastoplasm and mucus-filled vacuoles, but lack desmosome-like structures and cytoplasmic filaments.In contrast, in the epidermal papilloma, mucous cells are absent; and the tumor cells are without microvilli, desmosome-like structures, cytoplasmic filaments, and conspicuous ergastoplasm. The papilloma cells are in addition characterized by certain features not seen in normal epidermis, including prominent nuclear pores, very large nucleoli, nuclear dense bodies, vesicular mitochondria with few cristae and dense internal granules, and three types of distinctive cytoplasmic particles of possible viral nature.This investigation was supported by Public Health Service research grant CA 08158 from the National Cancer Institute, and American Cancer Society Institutional Grant No. IN-53 E.We are indebted to Miss Mary Bens for her indispensable technical assistance.     

Special somatic spine synapses in the ciliary ganglion of the chick

Summary Somatic spine synapses modified with postsynaptic electron opaque materials were found in the axo-somatic ciliary ganglion synapse of the chick.A part of the postsynaptic cell body protrudes into the presynaptic calyciform ending as a somatic spine with about 1 in length and 0.15 in diameter, and forms the so-called synaptic complex with presynaptic process. Moreover, conspicuous electron opaque materials can be seen in the central axis of the spine, except for its end portion. Sometimes, these opaque materials are seen as arrayed dots.The morphological characteristics of the somatic spine synapses in this study are quite similar to that found in the habenula and interpeduncular nuclei of the cat (Milhaud and Pappas, 1966). the biological significance of which is obscure at present.This work was supported in part by grant from the Education Ministry of Japan.     

Penetration of substances into tumor tissue—A methodological study on cellular spheroids

Summary The penetration of [³H]thymidine, [³H]d-leucine, [¹²⁵I]albumin, and the drugs [³H]5-fluorouracil and [³H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine andd-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. the concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited. The work was supported financially by Lennanders Foundation, OE and Edla Johanssons Foundation, and the Swedish Cancer Society.     

The effect of actinomycin D on RNA synthesis and nucleolar ultrastructure in the liver of rats treated with fluorouracil

Summary Rats were given cytidine-³H and 10 min later 50 mg fluorouracil. They were killed after 25 hours. Actinomycin D was given at various times before sacrifice. The collapse of the nucleolus and the segregation of its components, seen in rats sacrificed one hour after administration of actinomycin D only, was prevented by prior treatment with fluorouracil. In rats treated with fluorouracil and given actinomycin 12 or 20 hours prior to death, there was a more or less pronounced collapse of the nucleolus but no typical segregation of its components. Radioautographs of livers from untreated rats or rats given actinomycin only at the times mentioned, and killed 25 hours after administration of cytidine-³H, were labelled mainly over the cytoplasm. Radioautographs from rats, treated with fluorouracil only, or fluorouracil plus actinomycin, showed labelling over the nucleoli, but depressed labelling over the cytoplasm. Biochemical analysis of RNA labelling showed high ribosomal peaks in untreated rats and rats treated with actinomycin only. Rats treated with fluorouracil, or fluorouracil plus actinomycin showed no labelling of the 29S and 18S ribosomal peaks. The results indicate that fluorouracil blocks or delays the formation of ribosomal RNA and that the inhibition, at least in part, takes place in the nucleolus.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-04), the Swedish Cancer Society (Project 6831), the Medical Faculty of Uppsala and the Swedish Society for Medical Research.     

Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

Localization of acid phosphatase in the differentiating root meristem

The localization of acid phosphatase was studied by Gomori’s newer technique and by azo-coupling methods (α-naphthyl phosphate + fast red ITR or fast garnet GBC; AS or AS D phosphate + fast blue B or fast red violet LB) in the root tips ofVicia faba L. on paraffin sections (fixation with Wolman’s acidified ethanol) and on frozen sections (fixation with Baker’s calcium formol). Analogous results were obtained on the material treated in various ways and using different methods. In the broad bean, the reaction is most intense in the cap. In the meristematic zone, the primary core is more intensely stained than the ground parenchyma of the central cylinder. The phloem and xylem poles are usually strongly positive. Using both types of methods on Wolman fixed paraffin embedded material, essentially the same localization of acid phosphatase was found in the root tips ofRicinus communis L.,Lupinus luteus L.,Sinapis alba L.,Allium cepa L. as in the broad bean. InZea mays L. the rhizodermis and the hypodermic layers of the primary core were found to be most active. On sections of Wolman fixed paraffin embedded broad bean the most intense reaction was observed at pH 4.2–4.8. In the same material, both the azo-coupling and the Gomori reaction is inhibited by 10⁼² M NaF, but 10^?2 M tartaric acid only inhibits the Gomori reaction.     

Electron microscopy of the neurohypophysis in normal and histamine-treated rats

Summary The fine structure of the neurohypophysis has been studied in normal and histamine-treated rats with particular reference to capillary relationships and to the neurosecretory vesicles. Certain new information on the pericapillary space has been developed and is discussed with reference to the existing literature on the posterior hypophysis and other endocrine organs. The membrane-bounded pericapillary space penetrates deeply between surrounding nerve terminals and pituicyte processes, seemingly forming a pervasive metabolic lake which undoubtedly is of physiological significance for metabolic and secretory exchange.Following histamine treatment, the neurosecretory vesicles lose their electrondense centers, the mitochondria in the nerve terminals become swollen, and the capillary endothelium shows evidence of increased pinocytosis. In one rat subjected to painful stimuli, only the first and last of the above alterations are prominent. The experimental results are interpreted in the light of previous work done by other authors, as additional evidence for the identity of the stainable neurosecretion of light microscopy and the neurosecretory vesicles of electron microscopy.The author is greatly indebted to Prof. Dr. med. W. Bargmann for the use of electron microscope facilities and for other invaluable help during the course of the work. To Frau Dr. A. Knoop of the Anatomical Institute in Kiel, and to Frl. Dr. Weichan of Siemens & Halske AG in Berlin, for aid in obtaining the micrographs, a grateful acknowledgement is extended.Presented in part at the 55th meeting of the Anatomische Gesellschaft in Frankfurt/Main, April 9–12, 1958.United States Public Health Service Special Research Fellow, on leave from Department of Anatomy, University of Minnesota Medical School, Minneapolis, Minnesota, USA.     

Glycoconjugates in the secretory epithelium of the chicken mandibular gland

Summary In the secretory epithelium of the chicken mandibular gland, glycoconjugates have been studied by means of histochemical methods of light and electron microscopy. In light microscopy, a series of histochemical procedures have been employed which included lectin—peroxidase—diaminobenzidine methods and a digestion technique with neuraminidase or-amylase. In electron microscopy, a battery of methods were used that corresponded to those employed in light microscopy. In the secretory cells of the chicken mandibular gland, vicinal diol- and sulphate-containing glycoconjugates with sialic acid,-d-mannose,-d-glucose and-d-galactose residues were visualized and the possible histophysiological significances of such glycoconjugates were discussed with special reference to the functions of the salivary gland.     

Electron microscopic evidence for the existence of an intercellular substance in rat cerebral cortex

Summary The intercellular spaces of rat cerebral cortex are filled with a dense material, demonstrable by electron microscopy. This intercellular substance is in part preserved by chemical fixation with formaldehyde and osmium tetroxide but is solubilized and largely lost during subsequent dehydration with ethyl alcohol. Dehydration with acetone or Durcupan favors the preservation of the intercellular substance, which is preserved also by freezing and drying. Whether the intercellular substance demonstrated here is part of the outer leaflets of apposing plasma membranes (glycocalyx) or truly an intercellular substance similar to connective tissue ground substance is not known. The probability of the latter is discussed with regard to proposed physiological mechanisms.This work was supported by USPHS Research Grants NB 05175 and AM 06998.